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# DENGUE

Ewy Ortega (a, b), Pachka Hammami (a), Elena Arsevska (a), Maria Bellenghi (c), Claudia Cataldo (c), Francesca Dagostin (d), Marco Di Luca (e), Claudia Fortuna (e), Flavia Riccardo (e), William Wint (f), Busani Luca (c)

(a) UMR Animals, Health, Territories, Risks, and Ecosystems (Astre), Department of Biological Systems (Bios), French Agricultural Research and International Cooperation Organization for Development (CIRAD), Campus International de Baillarguet, Montpellier

(b) Ministère de l'agriculture et de l'alimentation, Direction générale de l'alimentation (DGAL), Paris

(c) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(d) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(e) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

(f) Department of Biology, Environmental Research Group Oxford Ltd, Oxford

## Biological, ecological and molecular features of the causative agent

### Disease name

Dengue (DEN).

### Disease agent

Dengue virus (DENV).

### Common, scientific and Latin name

Dengue virus (DENV) is the causal agent responsible for infections of dengue (DEN) a disease with a wide spectrum of symptoms, from extremely mild (unnoticeable) to severe cases and fatalities. DEN is an arthropod-borne viral disease transmitted by mosquito species of the *Aedes* genus, predominantly by *Ae. aegypti* and to a lesser extent by *Ae. albopictus*. Also known as 'break-bone fever', dengue fever was initially identified as a "water poison" associated with flying insects. The name 'dengue' derives from the Swahili phrase Ka-dinga pepo, meaning "cramp-like seizure" and came into general use only after the 1828 epidemic in Cuba (before that, it was also named Dunga) (1).

### Taxonomy

DENV belongs to the genus *Flavivirus* and family *Flaviridae*. The dengue virus complex includes four genetically and antigenically related but distinct antigenic groups (serotypes) labelled DENV 1 to 4. For each serotype, four to six genetic groups (genotypes) were identified (2,3). DENV-1 includes five genotypes (I, II, III, IV, and V), DENV-2 six (Asian I, Asian II, Cosmopolitan, American, American/Asian and Sylvatic), DENV-3 four (I, II, III, and V) and DENV-4 four (I, II, III, Sylvatic) (4). Based on phylogenetic analysis of the envelope (*E*) gene sequences, each genotype can further be subdivided into multiple lineages (5). In 2013 a fifth variant (DENV-5) was isolated in a human. However, this serotype was found only in Sarawak forests in Malaysia where it mostly circulates among non-human primates (6).
## Disease agent characteristics

DENV is an enveloped virus. Its genome is composed of a positive-sense, single-stranded RNA of approximately 10-11 kbp, which encodes seven non-structural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) and three structural genes: capsid (C, 100 amino acids), precursor membrane (prM, 75 amino acids), and envelope (E, 495 amino acids). All of them are translated as a single polypeptide chain embedded in endoplasmic reticulum membranes and flanked by two untranslated (non-coding) regions at the 3' and 5' ends (7-9).

## Physiochemical properties

Similar to other Flaviviruses, DENVs are stable at slightly alkaline pH (8.0) and low temperatures (especially at -60°C or below) and for at least 6 h in liquid aerosol suspension at room temperature and 23-80% humidity. On the other hand, ultra-low temperatures preserve infectivity almost indefinitely and once freeze-dried they survive almost indefinitely at room temperature (10).

DENV can be effectively inactivated by temperatures higher than 56°C for at least 30 minutes, and ultraviolet light irradiation of at least 45 minutes at 75 cm distance from the source (11). Similar to other Flaviviruses, DENV is readily inactivated by gamma-irradiation, and organic solvents and detergents, including 3-8% formaldehyde, 2% glutaraldehyde, 2-3% hydrogen peroxide, 500-5000 ppm available chlorine, alcohol, 1% iodine, and phenol iodophors (12). Non-ionic detergents, such as Triton X, inactivates DENV in plasma at 31°C within 10 min limiting the risk of transmission of all DENV with transfusion (13).

## Priority level for EU

The risk of introduction of DENV in the European Union (EU) is high, especially because of population susceptibility, effect of climate change in temperate areas, presence of *Ae. albopictus* mosquito vector and the increased international trade and air travel, enhancing both the risk of other competent mosquito vectors, such as *Ae. aegypti*, being introduced and the importation of infected travellers (particularly around the Mediterranean coast) (14). These covariates favouring the coexistence of the three components of the epidemiological triangle necessary for local dengue transmission: host, vector, and virus.

DENV is currently not endemic in continental Europe and most of the DEN cases are travellers infected outside of mainland Europe. Nevertheless, the DEN expansion and the number of local outbreaks increased over the last decades with more than 200 autochthonous cases reported in Europe over the past 13 years (15,16). Considering the frequency of travellers coming from high-incidence areas, as well as the past experiences of local outbreaks in Croatia, Israel, France, Italy and Spain, the occurrence of DENF outbreaks in continental Europe is a rare, sporadic but not unexpected event (15).

Vigilance must be maintained, and prevention and control strategies should be carefully thought out, as the risk of DEN outbreaks in continental Europe is present in regions with well-established competent mosquito vector populations such as around the Mediterranean and in some continental regions (see section below: 'Distribution of the pathogen'). With the further spread of the competent vectors in Europe, more regions could become at risk.
**Distribution of the pathogen**

DENV circulation is endemic in more than 128 tropical and subtropical countries, mainly in Central and South America, Southeast Asia and the Western Pacific, and establishing epidemic cycles in parts of Africa, the Eastern Mediterranean and North America. Lately, its geographic expansion has spread to non-endemic countries (15).

The fast and large-scale spread of the disease seems to be related to demographic and social changes, including global population growth, urbanization, lack of effective mosquito control and increased travel between endemic and dengue-free areas.

International movements, initially for military and war purposes and then for trade and tourism, enable the virus and its vectors to be transported from endemic to dengue-free zones (17). The increase in these movements plays a vital role in the spread of DENV (18).

The occurrence of DEN outbreaks in Europe depends on the presence of competent vectors (Figure 1 and Figure 2). To date, *Ae. albopictus* is considered the main DENV vector in Europe. It is currently present in 13 countries and 337 regions across all the Mediterranean regions of Europe and in some continental regions in Spain, France, Italy, Switzerland, Slovenia, Greece, Croatia, Bosnia-Herzegovina, Montenegro, Albania, Monaco, Bulgaria and the Netherlands. *Ae. aegypti* was eradicated from the European continent in the 1950s; in recent decades it has established in Madeira, and areas bordering the Black Sea (Georgia, Southern Russia and Turkey); in 2010 it was sporadically detected in the Netherlands, but promptly eradicated and more recently, in 2022, it was introduced in the Canary Islands and Cyprus (19).

<figure><img src="image_23.png" /><figcaption>**Figure 1.** Current 1-km probability of presence of *Ae. albopictus* across Europe, produced using random forest and boosted regression trees analyses (source: updated by ERGO for E4Warning Project).</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>Figure 2. Current 1-km probability of presence of *Ae. aegypti* across Europe, produced using random forest and boosted regression trees analyses (source: updated by ERGO for E4Warning Project)</figcaption></figure>

## Ecology and transmission routes

DENV is mainly transmitted by horizontal transmission between infected and susceptible vector (*Aedes* spp.) and the host. However, vertical transmission within the vector population (from an infected female mosquito to her offspring) has also been reported for *Ae. aegypti*, both experimentally and under natural conditions (20, 21). Within the human population, other modes of transmission from the bite of infected mosquitoes, although rare, have already been described or suggested, namely from mother to child (vertical transmission), blood transfusion, organ transplantation and exposure via needle stick or in the laboratory. Recently, sexual transmission was considered in few cases, pointing out also this possible but apparently rare mode of transmission CIT: a DENV transmission is maintained in the sylvatic (enzootic) cycle and urban endemic cycle which involve non-human primates in sylvatic habitat and humans in urban settings, respectively.

There is evidence that suggests other animals than non-human primates may play a role in both the sylvatic and urban endemic cycles as potential secondary hosts. DENV and/or DENV antibodies have been detected in different animal species including bats, birds, bovids, dogs, horses, pigs, rodents, marsupials and other small animals, however, their role as amplifying reservoirs is not confirmed yet (26) In vector, the incubation period lasts approximately two weeks. Once the virus is disseminated to the salivary glands, it can be injected into susceptible hosts during blood meals. Immediate mechanical transmission was also suggested, e.g., the transfer of the viruses from an infected host to a susceptible one occurring in a short period between the two feeding events (17).
# Drivers of the disease emergence and spread

## Ecological drivers

DENV spread is affected by multiple factors including the distribution of the competent vectors. Mosquito population dynamics (spatial and temporal densities) is itself driven by multiple components which partly depend on ecological drivers, such as temperature, precipitation or relative humidity.

Environmental conditions including land cover and urbanization also play a critical role in *Aedes* spp. establishment, favouring the vector settlement and proliferation after its introduction. Indeed, some urban environments, such as gardens, terraces and green spaces serving are conducive to their establishment and breeding near the human population.

Due to its high level of eco-physiological plasticity, *Ae. albopictus* has adapted to temperate environments following the evolution of eggs towards the diapause process (24). Moreover, the temperature can also affect vector competence accelerating the DENV replication in the vector at high temperatures (25).

# Natural history of disease in humans and animals,
including symptoms, morbidity and mortality

## Brief history of the pathogen and disease

While clinical reports referring to dengue-like symptoms were published since the Jin Dynasty (265 to 420 A.D.), the first isolations of DENV occurred in 1943 and 1945 in Japan and Hawaii respectively. Current knowledge suggests that the common ancestor of the four DENV serotypes appeared around 1,000 years ago, and that transmission to humans occurred much later, only a few hundred years ago.

From the 1940s to date, DENV largely spread all over the world mainly to tropical Asia, the Pacific Islands and the Americas, but also in many parts of Africa. Each year 100-400 million DENV infections are reported worldwide (27). It became hyperendemic in tropical and subtropical regions worldwide, mostly in urban and semi-urban areas (17, 28, 29). In recent decades, the number of DENV infections has increased. Since 2010, several outbreaks of autochthonous infections have been reported in Europe, mostly involving DENV-1 and 2 (30).

## Disease in humans

In the human host, the four serotypes can cause a wide range of symptoms from asymptomatic or paucisymptomatic (50-90%) to severe infections possibly leading to death. Following the bite of an infectious mosquito and an incubation period, an individual may or may not develop symptoms of DEN. Viremia begins toward the end of a 3 to 14 day incubation period and persists until around the time fever abates, which is typically 4 to 7 day and can go up to 10 days (2, 31).

The clinical pattern of the disease is characterized by 3 phases: febrile (3 to 7 days), critical (2 to 3 days) and recovery (2 to 3 days). The febrile phase is characterized by high fever accompanied by a range of symptoms that differ from individual to individual, from headaches, malaise, nausea, vomiting, myalgia, and abdominal pain to febrile convulsions. The critical phase is characterized by various complications. Haemorrhagic manifestations and haematological abnormalities can
occur in severe forms. Finally, the recovery phase is characterized by the loss of symptoms with the recovery of vascular permeability, although some organ dysfunctions may persist for several weeks (2, 4, 32).

In 2009 the World Health Organization (WHO) proposed a classification of DENV infection according to levels of severity: DEN and severe DEN. The clinical signs of DEN vary from asymptomatic infection (the majority of cases) to classical dengue fever, with high temperature, headaches, malaise, nausea, vomiting, myalgia, and abdominal pain. n. Dengue haemorrhagic fever and dengue shock syndrome are associated with the severe form and, as mentioned, can cause death (33). The clinical manifestations and severity of DENV infection depend on various factors such as the genetics of the virus and the host. For instance, some studies have shown that individuals with African ancestry are less likely to develop a severe form of DEN than those of European and Asian ancestry (34). Potential previous infection with a different DENV serotype or ZIKV (Zika Virus) seems to be one of the major risk factors for severe disease (22-24). An increased risk of severe forms was even highlighted in infants when anti-DENV antibodies were acquired by the mother (2). Age is also considered a risk factor, with children having a higher risk of severe forms (2,4). Finally, the serotypes DENV-2 and DENV-4 are associated with more severe or even fatal forms of the disease, even so, all 4 serotypes are capable of causing it (2).

Following the infection, the host's immune system produces antibodies specific to the serotype. These antibodies confer long-term immunity against that serotype, however, subsequent infections with new serotypes can result in cross-reactivity, leading to more serious infections, it is known as Antibody-Dependent Enhancement (ADE) (4, 32, 35). In a primary infection, anti-DEN IgM levels rise after 3 to 5 days of fever. Over the following 2 to 3 months, IgM levels decrease and are replaced by IgG, which are maintained throughout life and offered a life-long protection against repeated infection with this serotype, but not against reinfection with a different serotype. In secondary infection, the increase in IgM is much less marked than in primary infection and IgG levels rise rapidly to a high peak (2). Adults may experience profound fatigue for several weeks after recovery. The neutralizing antibodies inducted by the first infection can protect against other infections from the same serotype, but cross-protection from other serotypes is not long-term (22, 23).

# Availability of preventive, therapeutic and control measures, including licensed or pipelines vaccines

## Therapy in humans

The WHO provides guidelines on the clinical management of DEN from febrile phase to recovery. Depending on the disease severity, treatment generally includes rehydration and antipyretics and/or analgesics. In the critical phase and severe haemorrhagic forms, the therapy often includes meticulous fluid resuscitation and platelet transfusions. N-acetylcysteine and antibiotics may be indicated in specific cases. Finally, the benefit of steroids or immunoglobulins is not yet unanimous (36, 37). While there is no licensed targeted therapy or specific antiviral drugs for DENV infection, treatment in the early stage of infection with aspecific antivirals has also been tested to reduce viremia and the likelihood of developing a severe form of DENV infection (32, 38) Antiviral peptide inhibitors have also been used, with an aim to interrupt the DENV life cycle by affecting the functions of viral proteins, thus reducing viremia. Similarly, the use of neutralizing antibodies has been shown to play an important role in inhibiting the functional site of the DENV envelope (E) protein from interacting with the host cells (32). According to
some hypotheses, individuals with high viremia are more at risk of developing severe forms, thus all the above-mentioned treatments aim at preventing the progression of the disease into severe forms (38).

## Licensed or pipelined vaccines

Partly due to the cross-reaction/antibody dependent enhancement occurring in case of secondary infection with a different serotype, the development of vaccines for DENV has been very challenging.

In 2015, Dengvaxia® was the first authorized licensed vaccine, but following an increase in hospitalization in the vaccinated cohort 3 years after the vaccination, it is now recommended to individuals with previous dengue fever infection and is not available in non-endemic countries (22).

Recently, in October 2022, the Qdenga® vaccine has been approved by the European Medicines Agency (EMA) (39). Inducing antibody responses against all four serotypes but with higher vaccine efficacy documented against DENV 2 followed by DENV 1. Other vaccines are in development (40-43).

## Other prevention measures

The circulation of DENV is mainly linked to the vector, vector control is essential for both the prevention and control of DENV infection.

Effective vector control can include epidemiological and entomological surveillance, educational actions to eliminate mosquito breeding sites and to encourage the use of personal protective equipment such as appropriate clothing, chemical vector control using repellents and insecticides, monitoring vector resistance, and biological control of eggs, larvae and adults.

Other strategies, such as Sterile Insect Technique (SIT) or *Wolbachia*-infected mosquitoes (*Wolbachia*-mediated suppression and *Wolbachia* replacement method), can also be explored to reduce vector density (44,45). In certain countries such as Germany, Italy, and Spain, tools or apps have been provided to citizens for reporting mosquito presence and biting incidents.

## Disease specific recommendations

Individuals living in or travelling to endemic regions should take protective measures against mosquito bites, especially during the day, when the mosquitoes are active.

The tetravalent live, attenuated vaccine Qdenga® is recommended for seropositive travellers before travel to an endemic country, for dengue naïve travellers aged from 4 to 16 years old, and for naïve travellers aged from 17 to 60 years old only for long trips in endemic areas.

Due to a lack of studies, Qdenga® is not recommended for individuals older than 60 years old (39). The dengue tetravalent vaccine (CYD-TDV; Dengvaxia®, Sanofi-Pasteur) is recommended using the three-dose series scheduling for individuals from 9 to 45 years old with previous dengue infection in endemic countries.

The trials for the live attenuated tetravalent dengue vaccine, Qdenga® (TAK-003), are still being processed, and it is currently only approved for use in Indonesia for individuals within the indicated age range of 6 to 45 years (42).
## Epidemiological situation, at different spatial scales: past and current trends

DENV transmission is not endemic in mainland Europe and the majority of the cases are travellers infected in endemic regions. The epidemiology of locally transmitted DENV infection in Europe is characterized by relatively small outbreaks following the introduction of an imported index case. The first European epidemic was reported in Spain in 1793. From 1793 to 1945, other outbreaks were reported in different European countries (Spain, Cyprus, Gibraltar, Greece, Portugal, Austria, etc.) (47). In 2010, the first autochthonous DENV infections were diagnosed in France and Croatia with low numbers of cases (48,49). From 2008 to 2021, 25,755 cases, about 90% travel-associated, were reported in 28 different countries to the ECDC. During the summer of 2022, 65 autochthonous cases of dengue fever were identified in mainland France, across nine outbreaks (16). From August to Novembre 2023, at least four local transmission events of DENV were documented in different parts of Italy. During the same year local DENV transmission events were also reported in France and Spain.

In recent years the number of reported locally acquired DENV infections in Europe has increased. This could be due to many factors, including, among others, improved sensitivity of surveillance systems, increased climate suitability for DENV transmission, widespread establishment of *Ae. albopictus*, post-COVID-increase of international human mobility (with patterns that differ from country to country due to different preferred holiday destinations and privileged exchanges with overseas territories). The presence of sporadic local transmission events of DENV in continental Europe highlights the importance of continuous integrated surveillance of imported DENV infections, the timely activation of targeted vector control to reduce the risk of infection of local competent mosquito vectors present in Europe and subsequent onward local transmission (50).

## Sociological and demographic dimension affecting susceptibility and exposure, including gender

Human behaviour influences the introduction of both dengue virus and vectors and the local exposure of the susceptible populations.

Tourism in endemic areas increases the risk of importation and emergence in EU/EEA (European Union/European Economic Area).

The exposure to mosquito bites and, consequently, the risk of DENV infection, are driven by multiple factors.

In Europe, the influence of sociological components remains understudied, and to date, no clear correlation was identified between the risk of *Ae. albopictus* exposure and age or gender (51). Environmental covariates, such as mosquito densities or changes in land use, like increase of green areas in urban settings, were highlighted as important drivers of bite exposure.
# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

DENV infection can cause a wide range of clinical presentations, from asymptomatic and mild disease to life-threatening haemorrhagic fever and shock. Given the wide range of symptoms, clinical diagnosis for DENV infection is not straightforward and early and accurate laboratory diagnosis are essential for appropriate patient management. However, cross-reactivity of antibody responses occurring in case of co-infections with different flaviviruses such as ZIKV and chikungunya virus or different DENV serotypes makes virus detection, serological conversion and establishment of differential diagnosis particularly challenging. To date, laboratory diagnosis includes virus isolation, molecular amplification of DENV RNA with reverse transcriptase polymerase chain reaction (RT-PCR), immunoassays to capture the DENV viral protein NS1 and serological approaches (52). In 2009, DENF case definition criteria were redefined by the WHO (52).

## Infrastructure capacity to identify pathogens for each Member State

DEN is among the communicable diseases that according to the Commission Implementing Decision (EU) 2018/945 are covered by epidemiological surveillance (53). It means that EU Member States must establish national capacity for detecting and reporting human cases. The decision provides a case definition and clinical and laboratory criteria:

- *Clinical criteria*

Fever

- *Laboratory criteria*

A. Probable case:

Detection of dengue-specific IgM antibodies in a single serum sample

B. Confirmed case:

At least one of the following five:

1. Isolation of a DENV from a clinical specimen;

2. Detection of dengue viral nucleic acid from a clinical specimen;

3. Detection of dengue viral antigen from a clinical specimen;

4. Detection of dengue specific IgM antibodies in a single serum sample AND confirmation by neutralization;

5. Seroconversion or four-fold antibody titre increase of dengue-specific antibodies in paired serum samples

- *Epidemiological criteria*

History of travel to, or residence in an area with documented ongoing transmission of dengue, within the two weeks prior to the onset of symptoms

Case classification:

A. Possible case:

Not applicable.
B. Probable case:

Any person meeting the clinical and the epidemiological criteria, and the laboratory criteria for a probable case,

C. Confirmed case:

Any person meeting the laboratory criteria for a confirmed case. Regarding travel history and locally acquired case definition, an autochthonous case is defined as any case developing infection without a travel history within 15 days prior to the onset of symptoms in a given study area. In contrast to an imported case where the individual has a travel history (18).

In the EU, diagnosis is routinely made by clinical microbiology laboratories, and there is no European-wide reference laboratory network or national laboratories in most EU countries.

Estimated influence of environmental change
on the disease future trends

The major climatic covariates identified for DENV infection are temperature, rainfall, and relative humidity. Except for the temperature, which affects the extrinsic incubation of the virus, those risk factors mainly affect the vector distribution. In continental Europe, temperature increase could favour northward colonization of competent vectors *Ae. albopictus* and, possibly, the re-establishment of *Ae. aegypti* increasing the risk of dengue emergence as predicted by many predictive models (54). Inversely, the risk could decrease if climate change is negatively affecting the climatic suitability for *Ae. aegypti* (55). Modelling studies are predicting a general increase in the risk of *Aedes*-borne diseases in Europe with climate change. However, not all studies agree on the future of the areas currently most at risk. Bouzid *et al*. (56), predict an intensification of the risk in the coastal areas of the Mediterranean and Adriatic, as well as the north-eastern part of Italy (56), on the other hand, Tjaden *et al*. (57), predict that future summer droughts in northern Italy could reduce the habitat suitability for the vectors and consequently the risk of *Aedes*-borne disease (57).

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https://doi.org/10.1038/s41598-017-03566-3.
# DISEASE X

Soushieta Jagadesh (a), Claudia Cataldo (b), Annapaola Rizzoli (c), Esther van Kleef (d), Wim Van Bortel (e), Luca Busani (b)

(a) International Society of Infectious Diseases, Boston (MA)

(b) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(d) Department of Public Health, Institute of Tropical Medicine, Antwerp

(e) Department of Biomedical Sciences, Outbreak Research Team, Institute of Tropical Medicine, Antwerp

## Term definition

Disease X is a term coined by the World Health Organisation (WHO) in 2018 during the second annual review of the Blueprint list of priority diseases. The term refers to a disease caused by an unknown pathogen that could emerge as a future epidemic or pandemic (1). Disease X is caused by Pathogen X, an infectious agent that is not currently known to cause human disease, but it is an aetiologic agent of a future outbreak with epidemic or pandemic potential (2). Since the terms' introduction, COVID-19 caused by the SARS-CoV-2 virus strain was the first disease to meet the requirements of Disease X (3). Most emerging infectious diseases posing an international public health threat in the recent decades originated from wildlife reservoirs (4), as was seen with the emergence of the H1N1 influenza, the highly pathogenic H5N1 avian influenza (5), the Severe Acute Respiratory Syndrome Coronavirus (SARS) (6), Ebola Virus Disease (EVD) (7), COVID-19, and Mpox (previously known as Monkeypox) (8).

Various pathogens, including viruses, bacteria, fungi, parasites, and prions can potentially be causative agents of Disease X. Transmission characteristics, host requirements, and availability of antimicrobials have seen viruses as the predominant causative-agents of outbreaks (9-11). RNA viruses specifically make up about 94% of zoonotic viruses with their ability to spillover from non-human hosts to humans, evade host defences and reproduce across a variety of host species due to their great mutability (12-14). The high mutation rates in RNA viruses lead to their rapid evolution and environmental adaptability to reach adaptive equilibrium within their host species very quickly (15). Their ability to acquire significant resistance to antiviral treatment after a brief exposure as observed with HIV and antiretroviral treatment is also an additional cause of concern (16). Furthermore, RNA viruses are found to be the most common pathogens causing emerging infections in humans, with a rate of two to three novel viruses being discovered each year (14).

Therefore, in this report, we focus on viral families with pandemic potential characteristics rather than on a specific virus that may or may not present a future threat. Prevention and control countermeasures, including pharmacological measures and vaccines, against one member of the family could easily be adapted to another member in due course when the next threat emerges.

The pandemic potential characteristics defining the high-risk viral families are: 1) no pre-existing immunity in the global population; 2) transmittable by asymptomatic cases; 3) transmitted via airborne or direct contact; 4) zoonotic potential; and 5) no existing, effective therapeutics or vaccines (17-19). This criterion was developed following foundational assessments on viral families from Johns Hopkins University (17) and was subsequently used in the "Framework for U.S. Pandemic Preparedness Policy" (18). In Table 1, a list of high-risk viral families that could emerge to the next Disease X are presented.
<table><caption>Table 1. Description of the potential agents for Disease X</caption><thead><tr><td>Viral family</td><td>Notable viruses</td><td>Mode of transmission</td><td>Zoonotic transmission</td><td>Genetic material</td><td>Existing interventions</td></tr></thead><tbody><tr><td>Adenoviridae</td><td>Adenovirus 7, 14</td><td>Respiratory, fecal-oral</td><td>Confirmed</td><td>DNA</td><td>Vaccine available for Type 4 and 7</td></tr><tr><td>Arenaviridae</td><td>Lassa fever virus*, lymphocytic choriomeningitis</td><td>Direct contact with the blood or other body fluids of infected individuals or contaminated objects</td><td>Confirmed</td><td>RNA</td><td>None</td></tr><tr><td>Coronaviridae</td><td>SARS CoV 1 and 2*, MERS CoV*</td><td>Respiratory, fecal-oral, surface contact</td><td>Confirmed</td><td>RNA</td><td>Vaccines available SARS-CoV-2</td></tr><tr><td>Filoviridae</td><td>Ebola virus (EBOV)*, Marburg virus*</td><td>Direct contact with the blood or other body fluids of infected individuals or contaminated objects</td><td>Confirmed</td><td>RNA</td><td>None</td></tr><tr><td>Orthomyxoviridae</td><td>Influenza</td><td>Respiratory, water</td><td>Confirmed</td><td>RNA</td><td>Influenza (including seasonal H1N1, seasonal Influenza B, and H5N1)</td></tr><tr><td>Paramyxoviridae</td><td>Nipah virus*, Hendra virus*, Mumps virus, Rubeola virus</td><td>Respiratory, Direct contact with the blood or other body fluids of infected individuals or contaminated objects</td><td>Confirmed</td><td>RNA</td><td>Vaccines for Rubeola and mumps</td></tr><tr><td>Picornaviridae</td><td>Poliovirus, foot-and-mouth disease virus</td><td>Respiratory, surface contact</td><td>None confirmed</td><td>RNA</td><td>Vaccines for Poliovirus and Hepatitis A</td></tr><tr><td>Pneumoviridae</td><td>Respiratory Syncytial Virus (RSV)</td><td>Respiratory</td><td>None confirmed</td><td>RNA</td><td>Vaccine and monoclonal antibodies against RSV</td></tr><tr><td>Poxviridae</td><td>Variola virus, Varicella virus, Mpox virus*</td><td>Respiratory, surface contact</td><td>Confirmed</td><td>DNA</td><td>Vaccines against Variola and varicella</td></tr></tbody></table>

Apart from viruses, considering bacterial pathogens, the emergence of antibiotic multi-resistant bacteria poses a significant threat to global public health, prompting consideration of such strains as potential candidates for Disease X, representing an unknown and potentially catastrophic infectious disease. Treating antibiotic multi-resistant bacteria as Disease X has advantages and disadvantages. While it may enhance global preparedness and collaboration, there are potential negative consequences, such as stigmatization and resource diversion (20-22).

Emergence of newly multi-drug-resistant bacterial strains is often regarded as an existing, endemic condition that requires a different approach to address AntiMicrobial Resistance (AMR) including surveillance of AMR and antimicrobial consumption/use and ensuring universal access to quality diagnosis and appropriate treatment of infections (23, 24). Whereas current research towards Disease X is exclusively focused on research and development (R&D) for medical

<footnote>*Diseases included in the 2023 updated WHO Blueprint priority diseases (1)
adapted from "Viral Families and Disease X: A Framework for U.S. Pandemic Preparedness Policy", 2023 (18)</footnote>
countermeasures and health systems optimization against viral threats (25, 26). Therefore, in this report, while acknowledging the importance of AMR as global public health threat of high importance, we consider only viral threats for Disease X.

# Description of the potential disease agents

In this report, we discuss the characteristics of the nine viral families mentioned above that the Pathogen X could belong to.

## Adenoviridae

Adenoviruses are large (~950 Å) and complex non-enveloped virions with an icosahedral capsid (70 to 100 nm) is made up of 252 capsomeres with 240 hexons forming the faces and 12 pentons at the vertices (27). Each penton bears a trimetric fibre that aids in attachment to the host cell via the receptor on its surface (28). The double-stranded linear DNA, between 26K and 48K base pairs (bp) on length, is associated with two major core proteins and carries a 55-kDa protein covalently attached to its 5' end (27).

## Arenaviridae

*Arenaviridae* are spherical or pleomorphic in shape, 40-200 nm in diameter, with dense lipid envelopes containing bi-segmented negative strand uncapped RNA segments (29), L (ca. 7.3 kbp) and S (ca. 3.5 kbp), that are encapsidated independently (30–32). The virion surface layer is covered with club-shaped projections which consist of trimeric spike structures of two virus-encoded membrane glycoprotein subunits (GP1 and GP2) and, in some, a third component (stable signal peptide (SSP)) (33). Isolated ribonucleoprotein (RNP) complexes are organized into “beads-on-a-string”-like structures (34).

## Coronaviridae

This family consists of large, enveloped particles decorated with 20nm long club- or petal-shaped surface projections (the “peplomers” or “spikes”), and single-stranded capped, polyadenylated, positive-strand RNA viruses with genomes ranging from 25 to 32 kbp and a roughly spherical virion of 118-136 nm in diameter (35,36). Within the envelope is a flexible (subfamily *Coronavirinae*) or a doughnut-shaped (subfamily *Torovirinae*) nucleocapsid.

## Filoviridae

Filoviruses are filamentous, enveloped particles with a nucleocapsid containing non-segmented, negative-sense, single-stranded RNA genome, approximately 19 kb long and four viral structural proteins (37, 38).

## Orthomyxoviridae

Influenza viruses are spherical or pleomorphic enveloped particles 80 to 120 nm in diameter (39, 40). The helically symmetric nucleocapsid consists of a nucleoprotein and a multipartite genome (10.0 to 14.6 kbp) of single-stranded negative sense RNA in seven or eight segments, 50-
150 nm in length. The envelope carries 500 distinct spike-like surface projections including the hemagglutinin attachment protein and a neuraminidase in a 10:1 ratio respectively (41).

### Paramyxoviridae

All paramyxoviruses are enveloped virions 150 to 300 nm in diameter with a tubelike, helically symmetrical nucleocapsid containing a monopartite, single-stranded, negative-sense RNA genome (14 to 20 kbp in size) and an RNA-directed RNA polymerase (42, 43). The genome is uncapped, and the genome 3'-end is not polyadenylated. The nucleocapsid associates with the matrix protein (M) at the base of a double-layered lipid envelope.

### Picornaviridae

Picornaviruses are small (22 to 30 nm) nonenveloped, single-stranded, uncapped positive sense (7 to 8.5 kbp) RNA viruses with cubic symmetry (44, 45). The virus capsid is composed of 60 identical subunits called protomers, each consisting of four polypeptides VP1-VP4 (46).

### Pneumoviridae

This family consists of large filamentous enveloped negative-strand RNA viruses of 70-190 nm in diameter and up to 2 µm in length (47). Virions consist of a lipid envelope surrounding a nucleocapsid. The RNA length genome varies between genera, 13 to 15 kbp.

### Poxviridae

Poxvirus virions are large and brick shaped, approximately 220-450 nm long, 140-260 nm wide and 140-260 nm thick with short surface tubules 10 nm wide in the lipoprotein surface membrane (48, 49). The genome consists of double-stranded DNA, from 128 to 375 kbp, and the core contains enzymes for virus entry and replication.

## Priority level for EU

The COVID-19 pandemic highlighted limitations of outbreak response strategies and systems adopted within the European Union and its Member States, to respond quickly and effectively to a pandemic of this unprecedented scale, caused by an unknown pathogen (50, 51).

The pandemic demonstrated the ease with which a respiratory pathogen could spread via air travel and as a result of global connectivity (52). Coupled with densely populated cities with an immunologically naïve population, such an introduction of a novel pathogen led to an unprecedented public health crisis.

To prevent and ensure improved response to future threats, Health Emergency preparedness and Response Authority (HERA) was launched as a new European Commission Directorate-General on 16 September 2021 (53). HERA focuses on research on Pathogen X including threat assessment, horizon scanning for the identification of potential medical countermeasures and innovative technologies, and the development of standardized research protocols.
# Drivers of the disease emergence and spread

Anthropogenic changes in the environment, such as human encroaching on forests and wild areas, biodiversity loss, and changes in host and vector population dynamics, have been associated with an increased risk of disease outbreaks and emerging diseases (54-58). Here we discuss the drivers of disease emergence and spread established for the high-risk viral families as summarised in Table 2.

<table><caption>Table 2. Drivers of Disease X emergence and spread for each high-risk viral family in an EU context</caption><thead><tr><td>Viral family</td><td>Common drivers</td><td>Threat to the EU</td><td>Potential emergence and spread in the EU</td><td>Previous outbreaks in the EU</td></tr></thead><tbody><tr><td>Adenoviridae</td><td>Land use changes and wildlife trade</td><td>Medium</td><td>Endemic with reports of antropozoonosis (57)</td><td>Endemic species in Europe</td></tr><tr><td>Arenaviridae</td><td>Land use changes, biodiversity loss, climate change</td><td>Low</td><td>Possibility of imported cases but currently no known reservoirs for arenaviruses in Europe</td><td>None</td></tr><tr><td>Coronaviridae</td><td>Land use changes, biodiversity loss, wildlife trade, monoculture. climate change and global travel</td><td>High</td><td>Although the risk of emergence is low in EU, coronaviruses being respiratory pathogens are easily transmitted</td><td>COVID-19 pandemic</td></tr><tr><td>Filoviridae</td><td>Land use changes, biodiversity loss, wildlife trade, global travel and conflict</td><td>Low</td><td>Possibility of imported cases but currently no known reservoirs for filoviruses in Europe</td><td>Laboratory outbreaks of Marburg virus in Germany in 1976 and EBOV in England in 1976. One imported case of Ebola in 2014</td></tr><tr><td>Orthomyxoviridae</td><td>Land use changes, monoculture, climate change, war/conflict, and global travel and trade</td><td>High</td><td>Avian and seasonal influenza are well established in Europe</td><td>Several outbreaks of different influenza strains have been recorded in Europe</td></tr><tr><td>Paramyxoviridae</td><td>Land use changes, biodiversity loss, wildlife trade, monoculture, and climate change</td><td>Low-medium</td><td>Rubeola and mumps are endemic vaccine preventable diseases in Europe, however the recent zoonotic diseases are currently geographically restricted to South and South-East Asia and Australia</td><td>Endemic species in Europe</td></tr><tr><td>Picornaviridae</td><td>No known environmental drivers.</td><td>Low-medium</td><td>Hepatitis A and Polio are vaccine preventable diseases</td><td>Hepatitis A endemic in Europe</td></tr><tr><td>Pneumoviridae</td><td>No known environmental drivers.</td><td>Low-medium</td><td>Human metapneumovirus and RSV are only known virus affecting humans in this family and they are endemic to Europe</td><td>Human outbreaks of known viruses endemic</td></tr><tr><td>Poxviridae</td><td>Biodiversity loss and wildlife trade</td><td>Medium-high</td><td>Imported cases from West Africa with sustained human to human</td><td>Mpox outbreak in May 2022 declared by WHO European region</td></tr></tbody></table>
## Land-use changes

The extension of agricultural cultivation into forests through deforestation and habitat fragmentation may perturb the existing zoonotic transmission cycles (59, 60).

The emergence of bat-associated viruses: bats play a critical role as reservoirs for new infectious diseases due to their unique immune systems, extended lifespans, and ability to host diverse viruses. Their natural behaviours, such as roosting in large colonies and migrating over vast distances, facilitate viral transmission and spread (61). Zoonotic spillover events, where viruses jump from bats to other animals and eventually to humans, have been implicated in outbreaks like Ebola, SARS, and COVID-19. Outbreaks from the *Paramyxoviridae* family, Nipah and Hendra viruses were associated with loss of bat habitat due to deforestation and agricultural expansion (59, 62, 63).

The emergence of rodent-associated viruses: Rodents serve as reservoirs for various infectious diseases, harbouring pathogens with zoonotic potential. Their proximity to human habitats increases the risk of disease transmission. The adaptability and widespread distribution of rodents contribute to the emergence and spread of infectious diseases. Like bats, they provide examples of disease emergence due to agricultural encroachment into forests, like the outbreak of Argentine Haemorrhagic Fever (AHF), caused by the Junín virus, an arenavirus. As the pampas of Argentina was cleared for maize cultivation, the rodent *Calomys musculinus*, a natural host of Junín virus, increased in population (64,65). The infected *Calomys* species shed virus in their urine, which was aerosolised by agricultural machinery infecting agricultural workers. Similar outbreaks due agricultural expansion has documented with other arenavirus diseases such as Bolivian HF and Lassa fever, caused by the Machupo virus and Lassa fever virus, respectively, via their natural respective host rodents *Calomys callosus* and *Mastomys natalensis* (66, 67).

## Biodiversity loss

Studies demonstrate that reforestation can increase biodiversity loss and disease transmission when land conversion of grasslands, savannas, and open-canopy woodlands in temperate regions are reforested (56, 68, 69). For example, the incidence of tick-borne encephalitis in humans in Italy was explained by the ratio of coppice to high stand forest in Italy with natural reforestation that favoured the abundance of the roe deers, reservoirs of tick-borne viruses (70). On the other hand, loss of biodiversity is found to play a major role with frequent emergence and transmission of zoonoses (71-73). Biodiversity loss in ecological communities is a consequence of loss of large-bodied species with slower life histories (74) and increasing abundance of smaller-bodied species with fast life histories (75), which are more likely to transmit zoonotic pathogens (76). Furthermore, land-use changes caused by humans has been found to increase in the abundance of a single species, often zoonotic host species and reduce the diversity of non-hosts due to habitat loss (60). Thus, biodiversity loss potentially leads to increase in of zoonotic reservoirs population and therefore, increased risk of spillover.

## Wildlife trade

Wildlife trade for consumption and recreation plays a major role in zoonoses emergence. Hunting, preparing, and selling bushmeat has been linked with spillover due to frequent contact with infectious materials from wildlife (77, 78). For example, EBOV spillover events and subsequent outbreaks in the Congo Basin have been traced back to hunters who were exposed to Ebola contaminated ape carcasses (79, 80). Another example of spillover from bushmeat
consumption, is the SARS epidemic (2003-2004) that emerged from the Pearl River delta region in Guangdong, China (81). The first cases of the SARS, a disease caused by a coronavirus, were likely wild animal handlers in markets and restaurants serving bushmeat (82, 83). Wildlife trade such as importation of experimental mammals have led outbreak events as seen with Marburg virus in Marburg, Germany and EBOV in Virginia, US in 1969 (84) and 1989 (85), respectively.

## Monoculture

Monocultures of livestock and poultry for the purpose of increasing productivity and improving management, promote susceptibility to infection (15) and facilitates disease transmission by increasing population size and density (86-88). Avian influenza which is of relatively low pathogenicity in wild water birds (89, 90) is highly pathogenic in domestic poultry (54) especially with some strains. Studies demonstrate that both extensive and intensive farming practices drive influenza virus spillover from wild birds to domestic birds and pigs and thereby, infecting humans (89, 91). Rice fields combined with free-grazing duck farming in wetlands bring wild water birds into proximity with domestic ducks (91) and can transmit the pathogen to domestic poultry (92). Low genetic diversity in the immunologically naïve domestic population encourages rapid dissemination and amplification of infection (71, 88). In regions where swine farming is in proximity to poultry farming, pigs serve as “mixing vessels” to generate reassortants which are potential candidates for new pandemic strains, are they are susceptible to both avian and human influenza viruses (93,94). Intensification of camel herding in the Arabian Peninsula was associated with Middle East Respiratory Syndrome (MERS) outbreak (95). MERS is caused by MERS-CoV, a member of Coronaviridae.

## Climate change

Climate change such as increasing temperatures and precipitation changes causes shifts in the geographical range of various pathogen hosts, vectors, and reservoirs (96, 97). Climate change also has an indirect impact on vegetation and ecosystems which affects the geographical range and migration of pathogen hosts and reservoirs (98,99). Extreme climatic events such as drought, heatwaves, wildfires, storms, and floods cause habitat destruction and are linked to Nipah (100) and Ebola (101) spillovers from wildlife moving to safer areas foraging for limited food resources. Global warming was related to melting ice and thawing of the permafrost exposing once-frozen pathogens (102). Studies demonstrated a genetic analysis of an anthrax outbreak in the Arctic circle suggesting that the bacterial strain emerged from a once infected reindeer corpse as the frozen ground thawed (103). Climate also influenced human social gatherings and the transmissibility of viruses such as influenza (104) and COVID-19 (105). Some studies suggested that heavy rainfall could induce social isolation, thereby reducing COVID-19 cases (105). However, in Indonesia and Pakistan, increased rainfall and temperatures were associated with increased cases of COVID-19 reflecting different behavioural responses to weather (106). Destruction and damage to infrastructure such as sewage systems and portable drinking water due to climatic disasters influence disease outbreaks such as hepatitis A (107).

## Global travel and conflict

Global travel enables asymptomatic viraemic travellers to transmit across borders, introducing a novel pathogen to a seeming naïve population. The effect of travel on global pandemics as in the case of the SARS outbreak 2003-2004 demonstrated that virus can be spread around the world
by international air travellers (108). Travelers from Singapore, Canada and Vietnam became infected following their visit to Hong Kong in March 2003, leading to SARS-CoV-1 infections in twenty countries in a month's time. A similar consequence of global connectivity was observed with SARS-CoV-2 transmission across 229 countries with a total of 698,446,176 confirmed cases of COVID-19 that originated from Wuhan, China (109). War and conflict lead to the displacement of large populations into temporary overcrowded settlements with inadequate safe water and sanitation, need for sustenance hunting of wildlife, and higher risk of exposure to disease vectors and reservoirs (110). Exacerbation and increased geographical spread of Lassa fever (110) and Ebola (111, 112) outbreaks, in West and Central Africa respectively, can be attributed to local unrest and conflict.

## Diagnostic procedures and notification systems used at local, national, and European scale

The Zoonoses Directive 2003/99/EC obliges EU Member States to collect relevant and, when applicable, comparable data on zoonoses, zoonotic agents, antimicrobial resistance, and foodborne outbreaks (113). In 2004, the European Commission entrusted European Food Safety Authority (EFSA) with the task of setting up an electronic reporting system and database for monitoring zoonoses (EFSA Mandate No 2004-0178, continued by M-2015-0231) (114). However, this monitoring system was exclusively meant for assessing zoonoses and pathogens emerging within the European region, and not for detecting outside threats. To detect imported infectious disease threats, TropNet (European Tropical and Travel Medicine Network) was founded in 1999 to create a "European Network on Imported Infectious Disease Surveillance" (http://tropnetdev.netsysco.net/about-us/).

Epidemic intelligence systems rapidly detect and assess outbreak events of any origin to ensure the EU's health security by collating information from a variety of sources, which is then validated and analysed. Epipulse (restricted access) and Epitweeter (public access via a R package) are tools developed by the European Centre for Disease Prevention and Control (ECDC) for public health threat detection (115). Early Warning and Response System (EWRS) is another tool with restrictive access that monitors public health threats in the EU (116).

Surveillance of animal health and zoonotic threats in the EU is assessed by various disease intelligence and surveillance systems. Platform for Automated extraction of Disease Information from the web (PADI web) is a multilingual event-based surveillance system dedicated to the monitoring of online news sources for the detection of animal health infectious events (117). Nationally, France monitors animal diseases are a potential threat to public health, the economy and the environment via the epidemiological surveillance in animal health, ESA platform coordinated by ANSES (the French Agency for Food, Environmental and Occupational Health & Safety) and the French Ministry of Agriculture (Directorate General for Food, DGAL) (118).

The other EU agencies involved in outbreak preparedness for future threats include the Directorate-General for Health and Food Safety (DG-SANTE), The Directorate-General for Research and Innovation (DG-RTD), European Medicines Agency (EMA), EU Civil Protection Mechanism, and more recently, HERA (50).

Monitoring Outbreak events for Disease surveillance project (MOOD: https://mood-h2020.eu/about-mood/), a part of the European program Horizon 2020, aims to improve epidemic intelligence tools and services for the early detection, assessment, and monitoring of current and future infectious disease threats such as Disease X across Europe in the context of continuous global, environmental, and climatic change.
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# INFLUENZA A

Claudia Cataldo (a), Maria Bellenghi (a), Francesca Dagostin (b), Marzia Facchini (c), Sara Piacentini (c), Simona Puzelli (c), Luca Busani (a)

(a) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(b) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(c) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

## Biological, ecological, and molecular features
of the causative agent

### Disease name

Influenza A (IA).

### Disease agent

#### Common, scientific and Latin name

Influenza A viruses (IAVs) are the causative agents of one of the most critical viral respiratory diseases in humans, swine, poultry, and other species. Influenza is a descriptive term for a respiratory epidemic disease presenting with several symptoms: fever, cough, sore throat, runny nose, headache and muscle pain (1, 2). Common name of the disease is “influenza” in humans and other animal species (e.g., swine influenza in pigs, equine influenza in horses, and avian influenza in wild and domestic birds). In avian species, “fowl plague” is also used when the influenza virus subtypes responsible for the infection can cause systemic and highly lethal disease. Seasonal influenza occurs almost every winter in humans.

### Taxonomy

IAVs belong to the family of *Orthomyxoviridae*. The multiple influenza A subtypes are defined by the two surface proteins, hemagglutinin (H) and neuraminidase (N). There are a total of 18 different hemagglutinin subtypes (H1 through H18) and 11 different neuraminidase subtypes (N1 through N11), including the bat-specific influenza A-like subtypes H17N10 and H18N11, unable to reassort with influenza A viruses. The remaining 16 HA and 9 NA could theoretically be found in any combination (3-5).

### Disease agent characteristics

IAVs are enveloped viruses with a negative sense, single-stranded, segmented RNA genome, organized in 8 segments encoding for the 11 viral genes: hemagglutinin (HA), Viral Attachment Protein (VAP), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), nucleoprotein (NP), non-structural protein 1 (NSP1), non-structural protein 2 (NS2; also known as nuclear export protein, NEP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase basic protein 1-F2 (PB1-F2). The lipid membrane of the virion harbours the HA and the NA, that project from the surface of the virion, and the M2 integral ion channel. The matrix M1 protein lies just beneath the envelope which, along with the viral proteins,
encloses and protects the virion core that contains the viral RiboNucleoProteins (vRNP) complexes and the Nuclear Export Protein (NEP). At one end of the vRNPs are the three polymerases (3P) proteins (PB1, PB2, and PA) that make up the viral RNA polymerase complex (3) (6). The influenza viruses are pleomorphic, mostly roughly spherical but filamentous particles have frequently been observed.

The influenza virus life cycle can be divided into the following stages: binding of the VAP at the N-acetylneuraminic (sialic) acid expressed on the host cell surface, internalization by receptor-mediated endocytosis; endosomal uptake and release; entry into the host cell; entry of viral ribonucleoproteins into the nucleus; transcription and replication of the viral genome; export of the viral ribonucleoproteins from the nucleus; assembly and budding of new virions at the host cell plasma membrane (2) (3).

IAVs are divided into subtypes based on the HA and NA proteins on the virus surface. All known subtypes of IAVs have been found among birds, except subtypes H17N10 and H18N11, which have only been found in bats, as mentioned before.

IAVs commonly circulating among other animal species, including humans, are fewer H and N subtypes than in birds.

The two most common IAVs in humans are H3N2 and H1N1(7). They are the causative agents of seasonal influenza characterized by a sudden rise in body temperature to > 38.5°C 1-3 days following infection. Other symptoms include headache, limb ache, tiredness, general faintness, and dry cough. The most severe outcomes are peracute death, primary influenza pneumonia, encephalitis, and myocarditis. In addition, severe and fatal consequences of primary viral and viral-bacterial pneumonia are known, particularly in older patients with underlying diseases (chronic heart or lung disease, metabolic disorders such as diabetes, immune disorders, etc.).

Avian Influenza Viruses (AIVs) found in wild aquatic birds worldwide present the most remarkable diversity of virus subtypes, from H1 to H16 and from N1 to N9. Avian IAVs can spill over from wild aquatic birds and can infect domestic poultry and other bird and animal species.

AIV of H5 and H7 subtypes exhibit two pathotypes in poultry: Low Pathogenic (LP) and Highly Pathogenic (HP).

LP strains result in mild or asymptomatic infections, whereas HP strains cause up to 100% morbidity and mortality. Therefore, any AIV that exhibits an Intravenous Pathogenicity Index (IVPI) in 6-week-old chickens greater than 1.2 or kills at least 75% of 4- to 8-week-old chickens during a 10-day-observation period is defined as a HP strain (8).

The LPAI of H5 and H7 subtypes can spontaneously mutate to the HP phenotype under natural conditions. The HA cleavage site mutation causes the HP phenotype, but additional mutations may play a role. Two different classes of proteases are responsible for cleavage-activation of the hemagglutinin of influenza viruses: the trypsin-like proteases that cleave LPAIV are present only in a limited number of cells or tissues, so that these viruses commonly cause localized infections in, for example, the respiratory tract of mammals or intestinal tract of birds. In contrast, furin and subtilisin-like proteases that activate HPAIV are ubiquitously expressed causing the systemic spread of the virus (9).

Three different subtypes of Influenza A Viruses of Swine (IAV-S) co-circulate worldwide: H1N1, H3N2, and H1N2. However, the origin, genetic background, and antigenic properties of those IAV-S vary considerably from region to region. For example, recently, the main IAV-S circulating in U.S. pigs have been swine triple reassortant (tr) H1N1, trH3N2 virus, and trH1N2 virus (10). Pigs could also be affected by avian influenza viruses and may play a role in the adaptation of avian IAVs to humans and other mammalian hosts, either as intermediate hosts in which avian influenza viruses may adapt to humans or as a "mixing vessel" in which influenza viruses from various origins may reassort, generating novel progeny viruses capable of replicating and spreading among humans.
For other species, Equine Influenza (EI) is mainly caused by two subtypes of IAVs, namely H7N7 (first isolated in the year 1956) and H3N8 (first isolated in the year 1963) (11), while H17-N1 and N10-N11 subtypes have been detected in bats only (4).

## Physiochemical properties

These properties were mainly studied in avian viruses, which were most stable at slightly basic pH (7.4-8.2), low temperatures (<17°C), and mild to brackish salinities (0-20,000 parts per million (ppm)). Under acidic conditions (pH <6.6), warmer temperatures (>32°C), and high salinities (>25,000 ppm), AI viruses have much shorter stability (12).

## Priority level for EU

IAVs cause one of the most important respiratory diseases in humans, avian species, and pigs. In addition, the IAVs constantly mutate to evade the host's immune systems, and new viruses could emerge and spread in naïve populations. In particular, influenza diversification occurs by two main mechanisms, known as “antigenic drift” and “antigenic shift”. The first mechanism, which drives annual influenza epidemics, describes gradual antigenic changes in the HA or NA as a result of the accumulation of point mutations in the antigenic epitopes. The second process may occur when two different influenza viruses coinfect the same cells within an individual, causing the mixing and matching of viral genome segments; a change in HA and NA antigenic characteristics can occur due to this reassortment (2).

Interspecies transmission in animals can result in genetic reassortment of viral RNA segments during co-infections with different influenza A viruses and this is central to the emergence of novel influenza A viruses, typically through zoonotic transmission.

Repeated outbreaks and the rapid spread of genetically and antigenically distinct IAVs represent a considerable challenge for animal and public health. Spillover of IAVs from birds to animals and/or to humans could occur, and epidemiological and environmental processes influence the occurrence. Moreover, bidirectional transmission of IAV between pigs and people has altered the evolutionary dynamics of IAV, and a “One Health” approach is required to ameliorate morbidity and mortality in both hosts and improve control strategies.

Although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine worldwide, considerable diversity can be found in their H, N, and the other six genes. Human and swine IAVs have demonstrated a particular propensity for interspecies transmission, leading to regular and sometimes sustained incursions from man to pig and vice versa. The diversity of IAVs in swine remains a critical challenge in diagnosing and controlling this important pathogen for swine health, contributing to a significant public health risk (13). From late 2016 to date, avian influenza continues to evolve in Europe and globally, with reports of new bird outbreaks and occasional infections in mammals. Sporadic human infections have been reported in countries outside the EU, while the risk to the public in the EU remains low. However, a surprising number of HPAI virus detections in sea birds were recently (2022-2023) observed, mainly in gull species and particularly in black-headed gulls (14).

The genetic analyses indicate that the virus persisted in Europe in residential wild birds during and after the summer months. Although the virus retained a preferential binding for avian-like receptors, several mutations associated with increased zoonotic potential were detected.
**Distribution of the pathogen**

IAVs have a broad host range and are among the most clinically and economically important pathogens for humans, food animals, companion animals, and wild birds. Influenza in humans occurs every year in the cold season, and the causative IAVs circulate in all parts of the world.

Avian Influenza occurs worldwide, but some subtypes can be restricted to specific geographical areas or periods. There is often a “wave” pattern of avian influenza viruses in both wild and domestic avian species, with periods of many outbreaks and others with few cases. In temperate climates, seasonal epidemics occur mainly during winter, while in tropical regions, influenza may occur throughout the year, causing outbreaks more irregularly.

In 2021/2022, the HPAI epidemic in the EU was the largest observed so far in terms of outbreaks, dead wild birds, and geographical spread. In Europe, between 2005 and 2022, all countries experienced at least one episode of HPAI (Figure 1). In recent years, the range of wild bird and mammal species affected by HPAI viruses has also expanded, with the detection of HPAI viruses showing genetic markers of adaptation to replication in mammals. In addition, animal-to-human transmission has occasionally occurred, while no transmissions between humans have been reported.

<figure><img src="image_25.png" /><figcaption>Figure 1. Cumulative cases of Avian influenza in wild and domestic birds in Europe and the Balkans from 2005 to 2022 (Data from WOAH-WAHIS)</figcaption></figure>

Swine influenza in pigs regularly causes outbreaks worldwide, characterized by high levels of illness in pig herds. Still, since vaccination is standard in pigs, the disease's severity is usually
limited. Swine influenza viruses can circulate among swine throughout the year, but most outbreaks occur during the late fall and winter months, like human outbreaks.

## Ecology and transmission routes

The ecology of IAVs is dynamic and complex, involving multiple host species (Table 1). Aquatic wild birds of the orders *Anseriformes* (ducks and geese) and *Charadriiformes* (shorebirds and gulls) are the primary reservoir of avian influenza viruses in nature, harbouring H1-H16 subtypes. In contrast, H17 and H18 subtypes were recently discovered in bats (5) (15). Almost all HA and NA subtypes have been detected in dabbling ducks (*Anas* spp.), suggesting that these species are the major reservoir of IAVs (16).

<table><caption>Table 1. List of influenza A animal species included in the selected studies in decreasing order according to the number of references</caption><thead><tr><th>Genera</th><th>Type</th><th>Species</th><th>n. of papers#(n=15)</th><th>% of impact*(n=29)</th></tr></thead><tbody><tr><td rowspan="10">Avian</td><td rowspan="6">Domestic</td><td>Poultry</td><td>4</td><td>13.8%</td></tr><tr><td>Domestic birds</td><td>3</td><td>10.3%</td></tr><tr><td>Turkeys</td><td>1</td><td>3.4%</td></tr><tr><td>Guinea fowl</td><td>1</td><td>3.4%</td></tr><tr><td>Quail</td><td>1</td><td>3.4%</td></tr><tr><td>Chicken</td><td>1</td><td>3.4%</td></tr><tr><td rowspan="2">Goose</td><td>1</td><td>3.4%</td></tr><tr><td>Pigeon</td><td>1</td><td>3.4%</td></tr><tr><td rowspan="4">Wild</td><td>Anatidae</td><td>5</td><td>17.2%</td></tr><tr><td>Wild bird</td><td>2</td><td>6.9%</td></tr><tr><td>Ducks</td><td>2</td><td>6.9%</td></tr><tr><td>Aquatic bird</td><td>1</td><td>3.4%</td></tr><tr><td>ND</td><td>Other avian</td><td>1</td><td>3.4%</td></tr><tr><td rowspan="2">Mammals</td><td rowspan="2">Domestic</td><td>Swine</td><td>2</td><td>6.9%</td></tr><tr><td>Equine</td><td>1</td><td>3.4%</td></tr><tr><td>ND</td><td>ND</td><td>Host</td><td>2</td><td>6.9%</td></tr></tbody></table>

ND=not defined, # number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document; *% calculated on the total number of references

From an ecological point of view, IAVs are natural components of wetland ecosystems in which they occupy trophic niches represented by susceptible hosts while interacting with other biotic and environmental elements. However, ecosystem interactions underlie possible bidirectional viral flows between natural and anthropogenic habitats. Domestic pigs and poultry are two other critical reservoirs of IAVs (17).

Natural avian reservoirs, mainly aquatic wild birds, maintain the environment's low-pathogenic avian influenza viruses (LPAIVs). In these animals, virus prevalence can be >20% in the autumn migration season. The IAV can occasionally move from aquatic bird reservoirs and jumps to poultry or various mammalian species, including humans, resulting in sporadic infections, disease epidemics, or pandemics. If IAVs are transmitted to poultry, they can occasionally evolve into highly pathogenic (HP) strains. In recent years, a new scenario was observed with the increasing potential involvement of wild birds in the long-distance spread by migratory populations of HP avian influenza viruses, particularly H5 subtypes.
From an evolutionary perspective, sequence data generated about influenza viruses have provided a general understanding of the extent and structure of virus genetic diversity, the evolutionary processes that gave rise to it, from where influenza viruses originate, and the mutations that underpin host adaptation, antigenic drift, and antiviral resistance (18).

Human seasonal influenza waves display a complex spatiotemporal pattern resulting from biological, sociodemographic, and environmental factors. Wave-like spread on a small scale (country or area) and long-range seeding events synchronizing distant populations are the two main observed patterns, mainly due to short-distance commuting and air travel flows that facilitate inter-human virus transmission (19).

Animal IAVs follow different transmission patterns. Commercial poultry farms, “wet markets” (where live birds and other animals are sold), backyard poultry farms, commercial and family poultry slaughtering facilities, swine farms, human dietary habits, and the global trade in exotic animals have all been implicated in the spread of IAVs (20).

Direct transmission of IAV from wild birds to humans is rare; most IAV spillover events have occurred through close contact with infected domestic poultry. An example of a persistent transmission from domestic poultry to humans of an avian influenza virus is the outbreak of H7N9 in China, which has become one of the more severe zoonotic infections from avian IAV, with about 1600 human cases from 2013 to 2022, with high morbidity and 40% of case fatality. Most H7N9 human infections have resulted from close exposure to live poultry markets, whereas human-to-human virus transmission is limited (15).

Live poultry markets act as a major source of reassortment of IAV, causing new HA and neuraminidase (NA) subtype combinations and gene constellations, with the H9N2 subtype acting as a significant donor of internal gene segments to other subtypes (15).

Transmission of IAV in wild bird populations depends on several factors, including virus shedding, virus stability in the environment, and the degree of close contact/mixing with other hosts. The maintenance of this diverse pool of viruses globally is in part due to the migratory nature of the bird species, a mechanism in which the viruses are shed in bird faeces and later acquired by other birds that share the same habitat along migratory flyways (15). It has also been suggested that switching transmission dynamics might be a critical strategy for pathogens, such as IAVs, associated with mobile hosts, such as wild water birds, and that both intraspecies and interspecies transmission is essential to maintaining gene flow across seasons (21). In backyard poly-culture farming, domestic poultry often comes into contact with wild birds. The movement and mixing of domestic poultry to live poultry markets enhance the spread and mixing of IAVs (15). AIVs continue to cause both morbidity and mortality in poultry worldwide. Increased mortality is strongly related to infection with highly pathogenic IAVs (HPAIVs) (21).

The IAV hemagglutinin (HA) binds to sialic acid (SIA) receptors, widely represented in mammals and birds. Most avian and human influenza viruses preferentially bind to specific receptor types having SAA2,3Gal (avian receptor)- or SAA2,6Gal (mammalian receptor)-terminated saccharides, respectively. Thus, pigs are widely recognized as a “mixing vessel” of IAV with the presence of both α-2,3-SA and α-2,6-SA residues distributed throughout their respiratory tracts, where avian, swine, and human IAV strains reassort following co-infection and give rise to new genetic variants, potentially leading to epidemics and/or epizootics (20).

The transmission of Equine influenza viruses (EIVs) occurs by inhalation through aerosol that can spread effectively through the air up to 1-2 km of distance. Droplet infection plays a significant role in the transmission as nasal discharge/fomites aid in animal-to-animal transfer (22). Horse-to-horse spread is relatively rapid and faster than other respiratory infections in the equine species (23). International trade and traffic also lead to the spread of disease to disease-free zones of the world. The virus can persist for three days in the environment leading to the spread in other animals through fomites. The incubation period is 1-3 days, and the infected horses
can shed the virus for up to 10 days via nasal discharge (24). EIV is a self-limiting sterile disease in horses since the virus does not persist in recovered animals (25).

# Drivers of the disease emergence and spread

## Ecological drivers

Patterns of influenza outbreaks are different in the tropics than in temperate regions. Although considerable experimental progress has been made in identifying climate-related drivers of influenza, the apparent latitudinal differences in outbreak patterns raise fundamental questions about how potential environmental variables combine and act across the globe.

Some studies clarify that absolute humidity drives influenza outbreaks across latitudes, find that the effect of absolute humidity on influenza is U-shaped, and show that this U-shaped pattern is mediated by temperature (26). A study on seasonal influenza in children showed socio-demographic drivers regarding the role of children in influenza transmission to their elderly contacts. It was demonstrated that Influenza-Like Illness (ILI) was 3.4 times lower in the elderly contacts of immunized children than in contacts of the control group (27).

Different environmental drivers of Highly Pathogenic Avian Influenza H5N1 outbreaks in poultry and wild birds were described. In wild birds, outbreaks were strongly associated with an increased Normalized Difference Vegetation Index (NDVI) and lower elevation, though they were similarly affected by climatic conditions as poultry outbreaks. Outbreaks in poultry mainly occurred in areas where the location of farms or trade areas overlapped with habitats for wild birds. In contrast, outbreaks in wild birds were primarily found in areas where food and shelters are available. The different environmental drivers suggest that other spread mechanisms might be involved: HPAI H5N1 spread to poultry via poultry and wild birds, whereas contact with wild birds alone seems to drive the outbreaks in wild birds (28, 29). Table 2 reports leading influenza A environmental drivers.

<table><caption>Table 2. List of influenza A environmental covariates included in the selected studies in decreasing order according to the number of references</caption><thead><tr><td>Environmental drivers</td><td>n. of papers# (n=26)</td><td>% of impact* (n=54)</td></tr></thead><tbody><tr><td>Temperature (average daily, monthly or seasonal temperature)</td><td>19</td><td>35.2%</td></tr><tr><td>Humidity (absolute and relative)</td><td>8</td><td>14.8%</td></tr><tr><td>Time (month/season of the year)</td><td>6</td><td>11.1%</td></tr><tr><td>Precipitation (monthly/total seasonal rainfall)</td><td>5</td><td>9.2%</td></tr><tr><td>Chemical Characteristic (salinity and pH of the water)</td><td>3</td><td>5.5%</td></tr><tr><td>Water (if stagnant or flowing)</td><td>3</td><td>5.5%</td></tr><tr><td>Wind (speed and direction)</td><td>3</td><td>5.5%</td></tr><tr><td>Distance (to the nearest wetland)</td><td>2</td><td>3.7%</td></tr><tr><td>Normalized Difference Vegetation Index (NDVI)</td><td>2</td><td>3.7%</td></tr><tr><td>Altitude</td><td>1</td><td>1.8%</td></tr><tr><td>Land use (CORINE)</td><td>1</td><td>1.8%</td></tr><tr><td>Light (daylight time length)</td><td>1</td><td>1.8%</td></tr></tbody></table>

<footnote># number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document; *% calculated on the total number of references</footnote>
# Natural history of disease in humans and animals, including symptoms, morbidity, and mortality

## Brief history of the pathogen and disease

Records show that the flu has existed for at least 1,500 years. The history of influenza begins with Hippocrates (5th century BC), who first reported that an influenza-like illness spread from northern Greece to the islands south and elsewhere. In the 1300s, a flu epidemic hit Florence, Italy, which they called “influenza da freddo” (“cold flu”). History records various large flu epidemics or pandemics, from one in 1580 that spread from Asia to Europe and Africa to others that came over the centuries both on the continent of Europe and to Britain. Influenza pandemic episodes have been described since the end of the 19th century, and virus characteristics and associated host responses vary from one pandemic to another.

Different “pandemic events” have been documented, such as the 1889 “Russian flu” (H3N8) followed by the 1918 “Spanish flu” (H1N1), the “Asian flu” in 1957 (H2N2), the “Hong Kong flu” in 1968 (H3N2), the re-emergence of H1N1 viruses in 1977 - “Russian flu” (H1N1) and most recently the 2009 “swine flu” (H1N1pdm09).

Interestingly, the most well-known influenza pandemic, the 1918 Spanish flu, and the most recent pandemic, the swine influenza from 2009, were caused by an H1N1 virus. Also called “the mother of all pandemics”, the 1918 virus infected around one-third of the world population and was responsible for the death of at least 50 million people within a year, appearing in three successive waves. Although the 1918 pandemic has been extensively studied, the virus characteristics responsible for its fast spread, associated with a high mortality rate, especially in the population aged 20 to 40, largely remain obscure. The 2009 “swine flu,” since then renamed as “novel influenza A (H1N1)” or “pandemic 2009 H1N1 flu”, resulted in 18,500 reported laboratory cases, and a modelling study estimates that, in total, the 2009 H1N1 virus caused more than 200,000 influenza-associated deaths due to respiratory and 80,000 deaths due to cardiovascular failure. Notably, more than 80% of these fatal cases affected a young population (<65 years) (30).

The first record of Avian Influenza (AI) dates back to 1878 in northern Italy when it was described as a contagious disease of poultry associated with high mortality called “fowl plague”. It was not until 1955 that the classical fowl plague virus was shown to be a type A influenza virus based on the presence of type A influenza virus type-specific ribonucleoprotein. The term fowl plague was substituted by the more appropriate term, Highly Pathogenic Avian Influenza (HPAI), at the First International Symposium on Avian Influenza in 1981 (30).

Swine influenza was first proposed to be a disease related to human flu during the H1N1 pandemic in 1918 when pigs showed similar symptoms simultaneously as humans. The first identification of an influenza virus as a cause of disease in pigs occurred about ten years later, in 1930. For the following 60 years, swine influenza strains were almost exclusively H1N1. Then, between 1997 and 2002; H3N2 strains followed by H1N2, a reassortant between H1N1 and H3N2, were observed first in North America, and in the following years, they circulated in the pig population worldwide. Unlike human influenza viruses, swine viruses have different epizootiological patterns according to the area of the world with enzootic and geographic dependence. Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2, and these include classical swine H1N1, avian-like H1N1, human-like H3N2, reassortant H3N2, and various genotype H1N2 viruses. In Europe, North America, and China, IAVs circulating in pigs are distinct in their genetic characteristics and genetic sources.
## Disease in humans

The IAVs currently responsible for seasonal influenza in humans are H1N1 pdm09 and H3N2. Clinically, human influenza is characterized by an acute onset of symptoms after 24 to 48 hours from infection (incubation time). These symptoms constitute influenza-like illness (ILI) and include headache, cough, myalgias, malaise, chills, and fever that can persist for 2 to 8 days. The severity of the disease varies from mild to severe, and the elderly and immunocompromised are at higher risk of complications and coinfections. Pandemic and, to a lesser extent, seasonal IAV have been described to cause gastrointestinal illness with vomiting or diarrhoea, especially in children. Frequently, coinfection with colonizing bacteria aggravates the course of the disease. Coinfections with *Streptococcus pneumoniae* and *Staphylococcus aureus* are the most frequent and have been observed at a high rate during a pandemic, leading to an increase in pneumonia-associated death (31). Coinfection with other respiratory viruses, such as respiratory syncytial virus, aggravates the severity of the disease (32), especially in immunocompromised patients (33). Avian influenza viruses infecting humans are many; the most important are H5N1 and H7N9, confirmed to induce severe disease in humans (34, 35). Avian, swine, and other zoonotic influenza virus infections in humans may cause diseases ranging from mild upper respiratory tract infection (fever and cough) to severe pneumonia, sepsis with shock, acute respiratory distress syndrome, and even death. Conjunctivitis, gastrointestinal symptoms, encephalitis, and encephalopathy have also been reported to varying degrees depending on subtype.

## Disease in animals

In domestic poultry, AIV can cause LPAI with asymptomatic or mild disease, with HA subtypes H1, H3, H5, H6, H7, and H9 most commonly isolated. In contrast, specific AIV lineages in subtypes H5 and H7 can cause severe disease and rapid mortality and are referred to as HPAI (15). Low pathogenicity avian influenza viruses typically produce respiratory signs such as sneezing, coughing, ocular and nasal discharge, and swollen infraorbital sinuses in poultry. Sinusitis is common in domestic ducks, quail, and turkeys.

Lesions in the respiratory tract typically include congestion and inflammation of the trachea and lungs. In layers and breeders, there may be decreased egg production or infertility, ova rupture or involution, or mucosal oedema and inflammatory exudates in the lumen of the oviduct. A few layer and breeder chickens may have acute renal failure and visceral urate deposition (visceral gout). The morbidity and mortality are usually low unless accompanied by secondary bacterial or viral infections or aggravated by environmental stressors. In wild water birds, LPAIV infection probably does not affect movements during the stopover, resulting in the potential for the virus to spread along the migration route (21). HPAI viruses cause severe, systemic disease with high mortality in chickens, turkeys, and other gallinaceous poultry; mortality can be as high as 100% in a few days. Clinical signs or gross lesions may be lacking in peracute cases before death. However, in acute cases, lesions may include cyanosis and oedema of the head, comb, wattle, and snood (turkey); ischemic necrosis of comb, wattles, or snood; oedema and red discoloration of the shanks and feet due to subcutaneous ecchymotic haemorrhages; petechial haemorrhages on visceral organs and in muscles; and blood-tinged oral and nasal discharges.

Since the mid-2000s, spillover of highly pathogenic H5Nx viruses has occurred on multiple occasions, from poultry to wild birds. It has resulted in subsequent inter and trans-continental spread of H5Nx viruses via wild bird movements across Eurasia, Africa, and North America.

Birds that survive the peracute infection may develop CNS involvement as torticollis, opisthotonos, incoordination, paralysis, and drooping wings. The location and severity of microscopic lesions are highly variable and may include oedema, haemorrhage, and necrosis in
parenchymal cells of multiple visceral organs, skin, and CNS. In recent years, evidence of HPAI infections in wild birds without signs of illness was provided by several studies (8, 36). However, when migrating, they can carry the disease to new areas, potentially exposing domestic poultry to the virus. On the other hand, in wild birds, HPAI viruses can cause mass mortality, frequently observed along migratory routes.

Swine influenza virus infection causes acute respiratory distress in pigs. The incubation period for the swine influenza virus ranges from 1 to 3 days. The virus is inhaled and deposited on the surface of the lower respiratory tract. It has been documented that these pigs can lose from 5 to 12 pounds of body weight over a 3 to 4-week period. Often the bronchial and mediastinal lymph nodes are enlarged. Severe cases may result in fibrinous pleuritis (37).

About EIV, the three most common signs of equine influenza are pyrexia (peak 42°C), a serous and subsequently mucopurulent nasal discharge, and a persistent, harsh, dry cough. Other clinical signs include depression, anorexia, myalgia, limb oedema and enlarged mandibular lymph nodes. Haematological changes are non-specific but may consist of anaemia, leukopenia and lymphopenia. Secondary bacterial infection may occur with persistent pyrexia, coughing, purulent nasal discharge, pneumonia, or pleuritis (25).

# Availability of preventive, therapeutic, and control measures, including licensed or pipeline vaccines

## Therapy in humans

Treatment for most patients with influenza is symptomatic; it includes rest, hydration, and antipyretics as needed. Complicating bacterial infections require appropriate antibiotics. Antiviral drugs given within 1 to 2 days from the symptom onset decrease the duration of fever, severity of symptoms, and time to return to normal activity. Treatment with antiviral drugs is recommended for high-risk patients (including all hospitalized patients) who develop influenza-like symptoms; this recommendation is based on data suggesting that early treatment may prevent complications in these patients.

The H7N9 and H5N1 viruses are resistant to the earlier antiviral drugs amantadine and rimantadine; resistance or reduced susceptibility to oseltamivir has also been reported. The antiviral agent, baloxavir, has also shown *in vitro* activity against various avian influenza viruses, but clinical data still need to be provided.

## Therapy in animals

There is no effective treatment, although antimicrobials may reduce secondary bacterial infections, and antipyretics may provide symptomatic relief. Expectorants also may help relieve signs in severely affected pig herds. Vaccination and strict import controls are the only specific preventive measures to control influenza in pigs and horses. Sow vaccination, pre-farrowing or the entire herd at once (mass vaccination), are the most common vaccination protocols. Sow vaccination attempts to maximize the transfer of maternal immunity to the progeny. Piglet vaccination is possible, but the reduced efficacy due to maternal antibodies is an issue. Good management practices, such as strict all-in/all-out procedures, limiting movement of pigs and sows within farrowing rooms and between pens, rooms, and barns, and stress reduction, mainly due to crowding and dust, help reduce virus transmission and losses.
## Licensed or pipelined vaccines

Specific vaccines against the most important virus subtypes are widely used in pigs and horses. In poultry, some vaccines are available, but due to the many immunologically distinct viral subtypes that cause influenza in poultry and the virus's ability to rapidly evolve new strains, the preparation of effective vaccines is complicated. While avian flu vaccines are currently available, they are not used on a large scale on poultry farms. This hinders the ability to conduct surveillance testing, which helps detect the virus in unvaccinated flocks and limits the spread of the disease. Thus, the most effective control of outbreaks in poultry remains rapid culling of infected farm populations and decontaminating farms and equipment. This measure also reduces the chances of human exposure to the virus. However, vaccines in poultry against some H5 and H7 avian influenza viruses have been used in several countries. In 2007 the U.S. Food and Drug Administration approved a vaccine to protect humans against one subtype of the H5N1 virus. It was the first vaccine approved for use against bird flu in humans. No vaccines are currently available for the other avian influenza viruses rarely associated with human disease (H7N7, H9N2, H7N3, and H7N9).

## Other prevention measures

In addition to vaccination, people may take several personal measures to reduce their risk of acquiring influenza. Influenza spreads from person to person principally when people cough or sneeze or by direct or indirect contact with respiratory secretions from infectious people on their hands or surfaces. Avoid close contact with sick people, washing hands frequently, and increase ventilation in all settings are general measures that, applied consistently, can help reduce the spread of the infection.

Prevention is indicated for all patients but is especially important for high-risk patients and health care practitioners.

At the farm level, appropriate biosecurity and management measures to prevent the direct or indirect contact of poultry with wild birds and preventive hygiene measures such as cleaning and disinfection are crucial. Biosecurity is a set of practices you can use to avoid exposing animals to the disease, and can be summarized in:

- restrict traffic onto and off of the farms;

- disinfect shoes, clothes, hands, egg trays or flats, crates, vehicles, and tires;

- avoid contact with other poultry farms or bird owners.

Disease awareness among farmers and cooperation by all persons in the poultry sector must ensure that the strictest bio-security measures are applied to prevent the introduction of the HPAI virus in the establishments and the (further) spread of the disease.

Similar measures are also implemented in pig farms.

## Disease specific recommendations

The competent authorities deliver specific recommendations for preventing and controlling human influenza to the population and are also in charge of Influenza pandemic preparedness. Pandemics require a multisectoral response over several months or even years, and operational plans at national and subnational levels support the national strategies for responding to a pandemic. A pandemic plan is a living document reviewed at intervals and revised if there is a change in global guidance or evidence-based; lessons learned from a pandemic, an exercise, or other relevant outbreak; or changes to national or international legislation related to
communicable disease prevention and control. The Directive 2005/94/EC on Community measures for the control of AI sets out a list of specific provisions concerning preventive biosecurity measures, surveillance, and mass poultry vaccination. In addition, it establishes the measures to be applied in case of outbreaks, like the epidemiological investigation, tracking back and forward, and the restriction to animal movement in the area around the outbreaks to prevent further spread. The restricted zone consists of a protection zone and a surveillance zone with a radius of at least 3 and 10 km around the outbreak.

## Epidemiological situation at different spatial scales: past and current trends

Seasonal influenza is a vaccine-preventable disease that each year infects approximately ten to thirty percent of Europe's population and causes hundreds of thousands of hospitalizations across Europe. Older people, younger children, and those with chronic conditions suffer the most. Still, everyone is at risk of developing severe complications, including pneumonia, myocarditis, and encephalitis, that may result in death. The European Centre for Disease Prevention and Control (ECDC) coordinates European influenza surveillance through the European Influenza Surveillance Network (EISN), which combines epidemiological and virological surveillance. Epidemiological and virological surveillance data are regularly sent by all the EU Member States, Iceland, and Norway to the European Surveillance System (TESSy) database hosted by ECDC (info at https://www.ecdc.europa.eu/en/about-us/partnerships-and-networks/disease-and-laboratory-networks/eisn). Analysing the data of the influenza seasons 2018-2021, there are differences among countries. France and Norway reported the highest positive specimens in Europe between 2019 and 2021 (Figure 2).

<figure><img src="image_26.png" /><figcaption>Figure 2. Number of positive specimens of influenza A reported in 29 countries of the European Union and European Economic Area, 2019-2021 (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>
The weekly number of positive specimens reported over the years (Figures 3-5) showed a remarkable decrease in 2021 due to the restrictive measures implemented during the COVID-19 pandemic (38). Trends were more apparent in individual countries: Sweden showed an increasing number of cases while decreasing case numbers were apparent in Portugal and Slovakia (Figure 4).

<figure><img src="image_27.png" /><figcaption>**Figure 3.** Number of weekly positive specimens by month and year of onset of influenza A in the European Union and European Economic Area countries, 2019-2021 (data from TESSy-ECDC)</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 4.** Number of positive specimens of influenza A in the European Union and European Economic Area countries, 2012-2021 (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>**Figure 5.** Number of weekly positive specimens by month and year of onset of influenza A in the European Union and European Economic Area countries, 2012-2021 (data from TESSy-ECDC)</figcaption></figure>

**Sociological and demographical dimensions affecting susceptibility and exposure, including gender**

At-risk groups include infants, older adults, farmers, veterinarians, live animal sellers, swine workers, and poultry workers. Serological screenings in rural populations in China found different proportions of positivity to IAVs of animal origin, including H1, H3, H5, H7, H9, and others. The risk of infection is also related to contact with wild birds, as serological evidence of H5N1 infection among Alaskan hunters who handled dead wild avian species demonstrated (15).

The severity of the consequences is related to additional risk factors or comorbidities that comprise age, chronic respiratory, renal, hematologic, neurologic or cardiovascular disease, diabetes, obesity, immunosuppression/-deficiency, and pregnancy/postpartum period, as many clinical studies on hospitalized 2009 influenza have demonstrated (39-41). The impact of sex differences on morbidity and mortality after multiple behavioural, environmental, and social factors influence IAV infection.

Studies comparing male and female susceptibilities and disease outcomes after IAV infection reported different results, with some suggesting that young males are more susceptible. In contrast, others indicate that females are at an increased risk for severe and fatal outcomes (42).

Table 3 shows the most important influenza A human covariates. Among them, the infection rate and the human density are the most critical.
<table><caption>Table 3. List of influenza A human covariates included in the selected studies in decreasing order according to the number of references</caption><thead><tr><td>Human drivers</td><td>n. of papers #(n=7)</td><td>% of impact*(n=31)</td></tr></thead><tbody><tr><td>Infections (rate of infection)</td><td>11</td><td>35.5%</td></tr><tr><td>Population (human density)</td><td>10</td><td>32.2%</td></tr><tr><td>Economic condition (high/low-income countries)</td><td>3</td><td>9.7%</td></tr><tr><td>Outcomes (ICl admission, chronic disease, mortality)</td><td>3</td><td>9.7%</td></tr><tr><td>Migration (average length of weekly movements)</td><td>2</td><td>6.45%</td></tr><tr><td>Vaccination</td><td>2</td><td>6.45%</td></tr></tbody></table>

# number of papers extracted, the number of references per covariates is higher because more covariates may have been extracted from a document; *% calculated on the total number of references

# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay applied to organic material taken using a nasal or throat swab is the routine method to confirm IA infection in humans and animals. Patients with lower respiratory tract illness can have samples taken from sputum, endotracheal aspirate, or bronchoalveolar lavage fluid. The culture of the organism should not be attempted because special precautions are required for these highly pathogenic viruses.

In pigs, IAV is primarily diagnosed by RT-PCR, virus isolation, and occasionally by detecting antibodies against IAV in non-vaccinated animals. Viruses can be isolated from nasal and oral secretions in the febrile phase, affected lung tissue in the early acute stage, or udder wipes collected from sows with infected suckling piglets. Sequencing and characterization of the influenza viral isolates may be needed to select epidemiologically relevant strains for evaluating custom vaccine production. A clinical diagnosis (presumptive diagnosis) can be made by observing the sudden onset of coughing, fever, and nasal secretions in many pigs. However, subclinical and chronic influenza infections are common; cough and nasal secretions may be sporadic in those cases. A retrospective diagnosis can be made by demonstrating a rise in virus-specific antibody titres in acute and convalescent serum samples using the hemagglutination inhibition test. Both H3 and H1 subtype antigens should be included. This test is also used for herd surveys, and an ELISA (Enzyme-Linked Immunosorbent Assay) against the nucleoprotein (not subtype specific) is also available.

All suspected cases of HPAI in poultry or captive birds must be investigated. Appropriate measures must be taken according to the Regulation (EU) 2016/429 ("Animal Health Law"), and the rules for the prevention and control of certain diseases laid down in Commission Delegated Regulation (EU) 2020/687 have to be taken in case of confirmation. Since 2003 EU Member States must carry out surveillance programs for avian influenza aimed at the early detection of highly pathogenic avian influenza viruses and at detecting infections with low pathogenic avian influenza viruses of the H5 and H7 subtypes in poultry which have the potential to mutate to the highly pathogenic form of the virus. The surveillance for avian influenza is compulsory and, by Implementing Regulation (EU) 2020/690, highly pathogenic avian influenza and infection with
low pathogenic avian influenza viruses are subject to European Union surveillance programs. Surveillance of avian influenza in poultry and wild birds must be implemented on the entire territory of all EU Member States and by the provisions laid down in Annex II to Delegated Regulation (EU) 2020/689. By Implementing Regulation (EU) 2020/2002, Member States shall submit to the Commission every year data on the results of the implementation of the Union surveillance programs. The data shall be submitted electronically via the Animal Disease Information System (ADIS).

## Infrastructure capacity to identify pathogens for each Member State

In Europe, the Istituto Zooprofilattico Sperimentale delle Venezie, a veterinary public health regional laboratory in Italy, is the designated European Reference Laboratory (EURL) for avian influenza and Newcastle disease, according to the Decision (EU) 2018/662. A European reference laboratory for IA in humans is still not available, but the activities are carried out at the national level, according to national infrastructures and regulations.

## Estimated influence of environmental change on the disease's future trends

It is thought that the most significant change in AI epidemiology resulting from climate change will be brought about by changes in the distribution, composition, and migration behaviour of wild bird populations that harbor the genetic pool of AI viruses and in which natural AI transmission cycles occur. In contrast, HPAI, primarily confined to domestic poultry, has spread worldwide successfully in various climatic conditions. Although the effect of the environment on HPAI transmission and persistence is poorly understood, these observations support the idea that climate change will have minimal impact on HPAI epidemiology. However, the indirect effects are mainly those occurring due to the influence of climate change on agroecosystems associated with duck and crop production and changes in the distribution of domestic-wild waterfowl contact points (43). In addition, including abiotic factors such as temperature, UV index, and other meteorological parameters in IV surveillance systems could further our understanding of virus stability and transmissibility and help develop accurate predictive models of influenza epidemics. Moreover, combining epidemiological, meteorological, and genetic studies could unravel the evolution of influenza viruses and improve early intervention and long-term control strategies for future influenza outbreaks (44).

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# LEPTOSPIROSIS

Claudia Cataldo (a), Maria Bellenghi (a), Alessandra Ciervo (b), Francesca Dagostin (c), Luca Busani (a)

(a) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(b) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

## Biological, ecological and molecular features of the causative agent

### Disease name

Leptospirosis.

### Disease agent

#### Common, scientific and Latin name

*Leptospira* species are the causative microbial agents of leptospirosis, an emerging zoonotic disease of worldwide and ubiquitous distribution.

#### Taxonomy

From 1989, the genus *Leptospira* was separated into *L. interrogans*, including pathogenic strains, and *L. biflexa*, including saprophytic strains (1). Due to the biological intricacy, leptospires were classified by their serological and genomic characteristics.

#### Serological classification

*Leptospira* species are separated in different serovars through the agglutination test with homologous antigens. Over 200 and 60 serovars are recorded for *L. interrogans* and *L. biflexa*, respectively (Table 1).

#### Genomic classification

The genetic heterogeneity of *Leptospira* does not match with the previous two species differentiation, and pathogenic and non-pathogenic serovars occur within the same species (Table 2).

The genotypic classification of *Leptospira* is taxonomically correct, but it must be taken into consideration that the molecular typing is problematic for clinical microbiologists for its incompatibility with the serogroup system identification.

From 1998, phylogenetic studies on 16S rRNA *Leptospira* strains identified three clades based on the pathogenicity status: pathogenic, saprophytic, and intermediate. The last group includes strains of uncertain pathogenicity in humans (2).
<table><caption>Table 1. *L. interrogans* serogroups and some serovars</caption><thead><tr><td>Serogroup</td><td>Serovar(s)</td></tr></thead><tbody><tr><td>Icterohaemorrhagiae</td><td>Icterohaemorrhagiae, copenhageni, lai, zimbabwe</td></tr><tr><td>Hebdomadis</td><td>hebdomadis, jules, kremastos</td></tr><tr><td>Autumnalis</td><td>autumnalis, fortbragg, bim, weerasinghe</td></tr><tr><td>Pyrogenes</td><td>pyrogenes</td></tr><tr><td>Bataviae</td><td>bataviae</td></tr><tr><td>Grippotyphosa</td><td>grippotyphosa, canalzonae, ratnapura</td></tr><tr><td>Canicola</td><td>canicola</td></tr><tr><td>Australis</td><td>australis, bratislava, lora</td></tr><tr><td>Pomona</td><td>pomona</td></tr><tr><td>Javanica</td><td>javanica</td></tr><tr><td>Sejroe</td><td>sejroe, saxkoebing, hardjo</td></tr><tr><td>Panama</td><td>panama, mangus</td></tr><tr><td>Cynopteri</td><td>cynopteri</td></tr><tr><td>Djasiman</td><td>djasiman</td></tr><tr><td>Sarmin</td><td>sarmin</td></tr><tr><td>Mini</td><td>mini, georgia</td></tr><tr><td>Tarassovi</td><td>tarassovi</td></tr><tr><td>Ballum</td><td>ballum, aroborea</td></tr><tr><td>Celledoni</td><td>celledoni</td></tr><tr><td>Louisiana</td><td>louisiana, lanka</td></tr><tr><td>Ranarum</td><td>ranarum</td></tr><tr><td>Manhao</td><td>manhao</td></tr><tr><td>Shermani</td><td>shermani</td></tr><tr><td>Hurstbridge</td><td>hurstbridge</td></tr></tbody></table>

<table><caption>Table 2. *Leptospira* serogroups associated with genospecies</caption><thead><tr><td>Serogroup</td><td>Genospecies</td></tr></thead><tbody><tr><td>Andamana</td><td>L. biflexa</td></tr><tr><td>Australis</td><td>L. interrogans, L. noguchii, L. borgpetersenii, L. kirschneri</td></tr><tr><td>Autumnalis</td><td>L. interrogans, L. noguchii, L. santarosai, L. borgpetersenii, L. kirschneri</td></tr><tr><td>Ballum</td><td>L. borgpetersenii</td></tr><tr><td>Bataviae</td><td>L. interrogans, L. noguchii, L. santarosai, L. borgpetersenii, L. kirschneri</td></tr><tr><td>Canicola</td><td>L. interrogans, L. inadai, L. kirschneri</td></tr><tr><td>Celledoni</td><td>L. weilii, L. borgpetersenii</td></tr><tr><td>Codice</td><td>L. wolbachii</td></tr><tr><td>Cynopteri</td><td>L. santarosai, L. kirschneri</td></tr><tr><td>Djasiman</td><td>L. interrogans, L. noguchii, L. kirschneri</td></tr><tr><td>Grippotyphosa</td><td>L. interrogans, L. santarosai, L. kirschneri</td></tr><tr><td>Hebdomadis</td><td>L. interrogans, L. weilii, L. santarosai, L. borgpetersenii, L. kirschneri, L. alexanderi</td></tr><tr><td>Hurstbridge</td><td>L. fainei</td></tr><tr><td>Icterohaemorrhagiae</td><td>L. interrogans, L. weilii, L. inadai, L. kirschneri</td></tr><tr><td>Javanica</td><td>L. weilii, L. santarosai, L. borgpetersenii, L. meyeri, L. inadai, L. alexanderi</td></tr><tr><td>Louisiana</td><td>L. interrogans, L. noguchii</td></tr><tr><td>Lyme</td><td>L. inadai</td></tr><tr><td>Manhao</td><td>L. weilii, L. inadai, L. alexanderi</td></tr><tr><td>Mini</td><td>L. interrogans, L. weilii, L. santarosai, L. borgpetersenii, L. meyeri, L. alexanderi</td></tr><tr><td>Panama</td><td>L. noguchii, L. inadai</td></tr><tr><td>Pomona</td><td>L. interrogans, L. noguchii, L. santarosai, L. kirschneri</td></tr><tr><td>Pyrogenes</td><td>L. interrogans, L. noguchii, L. weilii, L. santarosai, L. borgpetersenii</td></tr><tr><td>Ranarum</td><td>L. interrogans, L. meyeri</td></tr><tr><td>Sarmin</td><td>L. interrogans, L. weilii, L. santarosai</td></tr><tr><td>Sejroe</td><td>L. interrogans, L. weilii, L. santarosai, L. borgpetersenii, L. meyeri</td></tr><tr><td>Semaranga</td><td>L. meyeri, L. biflexa</td></tr><tr><td>Shermani</td><td>L. noguchii, L. santarosai, L. inadai</td></tr><tr><td>Tarassovi</td><td>L. noguchii, L. weilii, L. santarosai, L. borgpetersenii, L. inadai</td></tr></tbody></table>
The number of available *Leptospira* genome sequences increased rapidly in the genomic era and with the whole-genome sequencing (WGS) tools. It forced a revisiting of the taxonomy of the genus in two major clades and four subclades. The first clade includes pathogen species for humans and animals and is divided into two subclades, P1 pathogens group and P2 intermediate group. The second clade contains saprophyte species, which is also subdivided into two subclades, S1 and S2 (Table 3) (3).

<table><caption>Table 3. *Leptospira* species belonging to subclades: pathogenic (P1, P2) and saprophytic (S1, S2)</caption><thead><tr><th>Species</th><th>Subclade</th><th>Species</th><th>Subclade</th></tr></thead><tbody><tr><td>L. adleri</td><td>P1</td><td>L. saintgironsiae</td><td>P2</td></tr><tr><td>L. ainazelensis</td><td>P1</td><td>L. sarikeiensis</td><td>P2</td></tr><tr><td>L. ainlahdjerensis</td><td>P1</td><td>L. selangorensis</td><td>P2</td></tr><tr><td>L. alexanderi</td><td>P1</td><td>L. semungkikensis</td><td>P2</td></tr><tr><td>L. alstonii</td><td>P1</td><td>L. venezuelensis</td><td>P2</td></tr><tr><td>L. barantonii</td><td>P1</td><td>L. wolffii</td><td>P2</td></tr><tr><td>L. borgpetersenii</td><td>P1</td><td>L. abararensis</td><td>S1</td></tr><tr><td>L. ellisii</td><td>P1</td><td>L. bandrabouensis</td><td>S1</td></tr><tr><td>L. gomenensis</td><td>P1</td><td>L. biflexas.s.</td><td>S1</td></tr><tr><td>L. interrogans.s.</td><td>P1</td><td>L. bourretii</td><td>S1</td></tr><tr><td>L. kirschneri</td><td>P1</td><td>L. bouyouniensis</td><td>S1</td></tr><tr><td>L. kmetyi</td><td>P1</td><td>L. brenneri</td><td>S1</td></tr><tr><td>L. mayottensis</td><td>P1</td><td>L. chreensis</td><td>S1</td></tr><tr><td>L. noguchii</td><td>P1</td><td>L. congkakensis</td><td>S1</td></tr><tr><td>L. santarosai</td><td>P1</td><td>L. ellinghauseni</td><td>S1</td></tr><tr><td>L. stimsonii</td><td>P1</td><td>L. harrisiae</td><td>S1</td></tr><tr><td>L. tipperaryensis</td><td>P1</td><td>L. jelokensis</td><td>S1</td></tr><tr><td>L. weilii</td><td>P1</td><td>L. kanakyensis</td><td>S1</td></tr><tr><td>L. yasudae</td><td>P1</td><td>L. kemamanensis</td><td>S1</td></tr><tr><td>L. andrefontaineae</td><td>P2</td><td>L. levettii</td><td>S1</td></tr><tr><td>L. broomii</td><td>P2</td><td>L. meyeri</td><td>S1</td></tr><tr><td>L. dzoumogneensis</td><td>P2</td><td>L. montravelensis</td><td>S1</td></tr><tr><td>L. fainei</td><td>P2</td><td>L. mtsangambouensis</td><td>S1</td></tr><tr><td>L. fletcheri</td><td>P2</td><td>L. noumeaensis</td><td>S1</td></tr><tr><td>L. fluminis</td><td>P2</td><td>L. perdikensis</td><td>S1</td></tr><tr><td>L. haakeii</td><td>P2</td><td>L. terpstrae</td><td>S1</td></tr><tr><td>L. hartskeerlii</td><td>P2</td><td>L. vanthielii</td><td>S1</td></tr><tr><td>L. inadai</td><td>P2</td><td>L. wolbachii</td><td>S1</td></tr><tr><td>L. johnsonii</td><td>P2</td><td>L. yanagawae</td><td>S1</td></tr><tr><td>L. koniamboensis</td><td>P2</td><td>L. idonii</td><td>S2</td></tr><tr><td>L. langatensis</td><td>P2</td><td>L. ilyithenensis</td><td>S2</td></tr><tr><td>L. licerasiae</td><td>P2</td><td>L. kobayashii</td><td>S2</td></tr><tr><td>L. neocaledonica</td><td>P2</td><td>L. ognensis</td><td>S2</td></tr><tr><td>L. perolatii</td><td>P2</td><td>L. ryugenii</td><td>S2</td></tr></tbody></table>

## Disease agent characteristics

Leptospires are Gram-negative coiled spirochetes with typical hook-ends and thickness of about 0.1 to 0.15 µm and 6 to 20 µm in length. Commonly with other spirochetes, leptospires have a double membrane structure consisting of the cytoplasmic membrane and peptidoglycan cell wall. This structure is associated with an outer membrane, other functional proteins, and a periplasmic flagellum that allow the bacteria to be motile. All species are obligate aerobes with an optimum growth temperature of 28 to 30°C.
## Physiochemical properties

Leptospiral survival studies have thus far only been performed for environmental matrices such as water, soil and mud. Once they are excreted into the environment, many factors such as temperature, pH value, moisture and humidity, UV light, salt and mineral concentrations, and other microorganisms, affect survival. In soil, the reported survival times span from a few hours to 193 days. In tap water, distilled water, sea- and river water, different *Leptospira* species survived between a few hours and 20 months. Survival is temperature-dependent and increases with increasing incubation temperatures. Increasing the temperature survival time also increases, from 130 days at 4°C to 263 days at 20°C and to 316 days at 30°C in fresh water (4). In Table 4 *Leptospira* serovars are grouped based on their capability to cause disease (5).

<table><caption>**Table 4.** Relationship between *Leptospira* serovars and biological and physiochemical properties in relation to their capability to cause disease</caption><thead><tr><th>Leptospira serovars</th><th>Biological and physiochemical properties</th></tr></thead><tbody><tr><td>L. illina</td><td></td></tr><tr><td>L. biflexa</td><td></td></tr><tr><td>L. meyeri</td><td>Saprophytic</td></tr><tr><td>L. wolbachii</td><td>Do not cause disease</td></tr><tr><td>L. yanagawae</td><td>1°C&gt;T&lt;35°C</td></tr><tr><td>L. vanthielii</td><td></td></tr><tr><td>L. terpstrae</td><td></td></tr><tr><td>L. inadai</td><td></td></tr><tr><td>L. parva</td><td></td></tr><tr><td>L. broomi</td><td>Biochemical intermediate</td></tr><tr><td>L. inadai</td><td>Do live as saprophytic or pathogen</td></tr><tr><td>L. licerasiae</td><td>1°C&gt;T&lt;37°C</td></tr><tr><td>L. wolffii</td><td></td></tr><tr><td>L. fainei</td><td></td></tr><tr><td>L. kirschnerii</td><td></td></tr><tr><td>L. interrogans</td><td></td></tr><tr><td>L. weilii</td><td>Pathogenic</td></tr><tr><td>L. noguchii</td><td>Do cause disease in humans and rodents</td></tr><tr><td>L. borgpetersenii</td><td>20°C&gt;T&lt;37°C</td></tr><tr><td>L. santarosai</td><td></td></tr></tbody></table>

## Priority level for EU

Leptospirosis is among the communicable diseases that, according to the Commission Implementing Decision (EU) 2018/945 are covered by epidemiological surveillance. In Europe, the number of human cases of leptospirosis is about 800 per year. France is the country that reports the highest number of cases, followed by Germany. There are several European countries where leptospirosis has just been reported in recent years (Luxemburg, Cyprus, Iceland). In Europe, leptospirosis occurs mainly in the Mediterranean and East European regions. About 160 mammalian species have been identified as natural carriers of pathogenic leptospires. These include feral, semi-domestic and farm and pet animals as important infection sources. The
infectious period of natural hosts can be lifelong. Accidental hosts can act as intermediate infection sources and may shed leptospires for days or months.

## Distribution of the pathogen

Since warm and humid conditions facilitate the transmission of leptospirosis (6), outbreaks typically occur in tropical areas (7) and sometimes during summer or fall in temperate regions. Table 5 lists the maintenance and incidental host linked with different *Leptospira* serovars (8).

<table><caption>Table 5. Maintenance and Incidental Hosts for the most important Serovars of *Leptospira interrogans*</caption><thead><tr><td>Serovar</td><td>Maintenance host</td><td>Incidental host</td></tr></thead><tbody><tr><td>L. bratislava</td><td>Pig</td><td>Horse, Dog</td></tr><tr><td>L. canicola</td><td>Dog</td><td>Pig, Cattle</td></tr><tr><td>L. grippotyphosa</td><td>Rodent</td><td>Cattle, Pig, Horse, Dog</td></tr><tr><td>L. hardio</td><td>Cattle</td><td>Human</td></tr><tr><td>L. Icterohaemorrhagiae</td><td>Brown rat</td><td>Domestic animals and Human</td></tr><tr><td>L. pomona</td><td>Pig, Cattle</td><td>Sheep, Horse, Dog</td></tr></tbody></table>

## Ecology and transmission routes

The main modes of transmission of pathogenic *Leptospira* spp. to humans are through direct contact or by contact with contaminated water and soil, as well as infected animals (9). Contact with contaminated water due to floods or recreational activities (such as swimming, fishing, kayaking, surfing, canoeing, rafting, and triathlons) in lakes, rivers, and ponds was associated with an increased risk of leptospirosis (10). The risk is also increased for occupations, especially in developing countries, that may have direct or indirect contact with rodent urine, such as sewage workers, garbage collectors, and agricultural workers (10). Manual labourers tend to be more prone to skin abrasions, which may further increase the risk of infection (11). Increased shedding of the pathogen into the environment results in a heightened risk of occupational and nonoccupational exposure to *Leptospira* spp. in both rural and urban settings (12). *L. borgpetersenii* (mainly host-to-host transmission) and *L. interrogans* (mainly via contaminated water) are the two most common pathogenic species causing leptospirosis in animals (13). Generally, all animal pathogenic strains can be transmitted and be pathogenic to humans. Rats and other rodents are known to be maintenance hosts. Studies have shown that these animals are chronic carriers of *Leptospira* spp., commonly not manifesting any signs of infection when examined (14). Small mammals, particularly rats, are the main reservoir hosts for *L. interrogans*, with large herbivores as significant additional sources of infection. The leptospiral life cycle involves shedding in the urine by an infected animal, persistence in the environment, acquisition of a new host, and hematogenous dissemination to the kidneys. Once leptospires gain access to the renal tubular lumen of the kidney, they colonize the brush border of the proximal renal tubular epithelium, from which urinary shedding can persist for a long time without significant pathogenic effects on the reservoir host (12). Leptospirosis is primarily a zoonosis, with humans
serving as accidental hosts. However, it is worth noting that transient leptospiral shedding occurs during human infection and human-to-human infection, although extremely rare, through sexual intercourse and during lactation (12). Portals of entry include cuts and abrasions on the skin or mucous membranes such as the conjunctival, oral, or genital surfaces. Exposure may occur through either direct contact with an infected animal or through indirect contact via soil or water contaminated with urine from infected animals. Individuals with occupations at risk for direct contact with potentially infected animals include veterinarians, abattoir workers, farm workers (particularly in dairy milking situations), hunters and trappers, animal shelter workers, personnel in laboratories or during fieldwork.

# Drivers of the disease emergence and spread

## Ecological drivers

According to the Leptospirosis Burden Epidemiology Reference Group (LERG), leptospirosis increases due to rainfall, flooding, open sewers, crowding, animal contacts, and poor hygiene (15). Recent studies have demonstrated that the trend of leptospirosis is spreading worldwide as several countries are prone to become seriously affected after the effects of global warming and severe floods. Besides that, the prevalence of outbreaks is highly associated with various outdoor activities, such as recreational wildlife programs, adventure travels, and army expeditions or training (16,17). In Table 6 are reported the environmental drivers impacting the most on *Leptospira* spreading, while in Table 7 are reported the animal drivers.

<table><caption>**Table 6.** List of *Leptospira* environmental covariates included in the selected studies in decreasing order of importance according to the number of references</caption><thead><tr><td>Environmental drivers</td><td>n. of papers# (n. 54)</td><td>% of impact* (n. 63)</td></tr></thead><tbody><tr><td>Time (seasonal rate of infection or seasonal incidence in a period)</td><td>15</td><td>23.8%</td></tr><tr><td>Temperature (average daily/monthly)</td><td>10</td><td>15.8%</td></tr><tr><td>Location (area where cases or outbreaks occurred)</td><td>9</td><td>14.3%</td></tr><tr><td>Precipitation (total, monthly or seasonal rainfall)</td><td>9</td><td>14.2%</td></tr><tr><td>Water (eventually contaminated water)</td><td>8</td><td>12.69%</td></tr><tr><td>Altitude</td><td>3</td><td>4.7%</td></tr><tr><td>Land Use (Corine)</td><td>3</td><td>4.7%</td></tr><tr><td>Distance (Rho/spatial correlation or mean distance between infected colonies)</td><td>2</td><td>3.17%</td></tr><tr><td>Soil</td><td>2</td><td>3.17%</td></tr><tr><td>Chemical Characteristics (soil pH)</td><td>1</td><td>1.58%</td></tr><tr><td>Food (contact with animal excreta-contaminated water, soil or food)</td><td>1</td><td>1.58%</td></tr></tbody></table>

<footnote># number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document; *% calculated on the total number of references</footnote>
<table><caption>Table 7. List of *Leptospira* animal covariates identified from the selected studies in decreasing order according to the number of references</caption><thead><tr><td>Animal drivers</td><td>n. of papers# (n. 54)</td><td>% of impact* (n. 115)</td></tr></thead><tbody><tr><td>Rodent</td><td>28</td><td>24.3%</td></tr><tr><td>Dog</td><td>15</td><td>13.0%</td></tr><tr><td>Wild boar</td><td>8</td><td>6.9%</td></tr><tr><td>Cattle</td><td>7</td><td>6.1%</td></tr><tr><td>Fox</td><td>7</td><td>6.1%</td></tr><tr><td>Horse</td><td>7</td><td>6.1%</td></tr><tr><td>Shrew</td><td>5</td><td>4.3%</td></tr><tr><td>Wolf</td><td>3</td><td>2.6%</td></tr><tr><td>Bears</td><td>3</td><td>2.6%</td></tr><tr><td>Goat</td><td>3</td><td>2.6%</td></tr><tr><td>Lynx</td><td>3</td><td>2.6%</td></tr><tr><td>Pig</td><td>3</td><td>2.6%</td></tr><tr><td>Hare</td><td>2</td><td>1.7%</td></tr><tr><td>Jackal</td><td>2</td><td>1.7%</td></tr><tr><td>Mustelidae</td><td>2</td><td>1.7%</td></tr><tr><td>Porcupine</td><td>2</td><td>1.7%</td></tr><tr><td>Sheep</td><td>2</td><td>1.7%</td></tr><tr><td>Badger</td><td>1</td><td>0.9%</td></tr><tr><td>Bat</td><td>1</td><td>0.9%</td></tr><tr><td>Cat</td><td>1</td><td>0.9%</td></tr><tr><td>Cow</td><td>1</td><td>0.9%</td></tr><tr><td>Deer</td><td>1</td><td>0.9%</td></tr><tr><td>Erinaceomorphae</td><td>1</td><td>0.9%</td></tr><tr><td>Foina</td><td>1</td><td>0.9%</td></tr><tr><td>Herpestedae</td><td>1</td><td>0.9%</td></tr><tr><td>Lagomorph</td><td>1</td><td>0.9%</td></tr><tr><td>Otter</td><td>1</td><td>0.9%</td></tr><tr><td>Swine</td><td>1</td><td>0.9%</td></tr><tr><td>Wild bird</td><td>1</td><td>0.9%</td></tr><tr><td>Wild cat</td><td>1</td><td>0.9%</td></tr></tbody></table>

## Natural history of disease in humans and animals, including symptoms, morbidity and mortality

### Brief history of the pathogen and disease

The modern history of leptospirosis began in 1886 when Adolph Weil described a particular type of jaundice accompanied by splenomegaly, renal dysfunction, conjunctivitis, and skin rashes. It was subsequently named Weil's disease. Although the aetiology of the disease is unknown, it appeared to be infectious in nature and often associated with outdoor occupations in which people came into contact with water (sewer workers, rice-field workers, and coal miners). The first demonstration of leptospires was made by Stimson (18), who used the recently described Levaditi

<footnote># number of papers extracted, the number of reference per species is higher because from one paper more species could have been extracted) *% calculated on the total number of references</footnote>
silver deposition staining technique to observe spirochetes in kidney tissue sections of a patient described as having died of yellow fever. Stimson called the organism *Spirocheta interrogans*. The first isolation of *Leptospira* followed just a few years later. In Japan, where Weil's disease was common in coal miners (19). The organism was named *Spirochaeta icterohaemorrhagiae*. One of the first isolates survives to these days, and Ictero No. 1 was accepted by the Subcommittee on the Taxonomy of *Leptospira* in 1990 as the Type Strain of *L. interrogans* (20). The Japanese group Campo reported the key finding that rats were renal carriers of *Leptospira* (21). Ido and colleagues observed and cultured spirochetes from the kidneys and urine of a range of house and wild rat species and identified them as *S. icterohaemorrhagiae* based on specific reactivity with immune sera. They also observed that leptospires were restricted to the kidneys and that the rats appeared healthy, the first observation of the asymptomatic carrier state. The following decades saw significant advances in the understanding of leptospirosis: one of the more important was the recognition of leptospirosis as an infectious disease of almost all mammalian species, especially in an increasing range of rodent species, and the importance of domestic animals as a source of human infection. The genus name *Leptospira* was first proposed by Noguchi (22) to differentiate the Weil's disease spirochete from others known at the time, especially *Treponema pallidum*, *Spirochaeta* and *Spironema* (later *Borrelia*) *recurrentis*; the differentiation was based almost entirely on morphological characteristics. As new serovars were isolated, they were given species status, e.g. *L. pomona*, *L. canicola*, *L. hardjo*, *L. copenhageni*, and so on. Species (serovars) with related antigens were grouped together in serogroups. Even with the limited taxonomic tools available for *Leptospira* at the time, it was apparent that there were not >200 species, and so in 1982 the Subcommittee on the Taxonomy of *Leptospira* adopted the notion of two species of *Leptospira*, with *L. interrogans* containing the pathogenic serovars and *L. biflexa* containing the saprophytic serovars (23).

## Disease in humans

Clinical symptoms of leptospirosis are highly variable and nonspecific. Most cases remain subclinical or asymptomatic, while symptoms typically manifest 2-30 days after the initial exposure (7). The infection is responsible for various clinical features ranging from subclinical symptoms to fatal pulmonary haemorrhage and Weil's syndrome (a combination of jaundice, renal failure, and haemorrhage). Most symptomatic cases (up to 90%) follow a biphasic pattern, consisting of an initial symptomatic leptosiremic phase lasting 5 to 7 days and an immune phase during which symptoms can gradually improve as the host mounts an antibody response (7). Muscle pain is often focused in the calves and lower back, gastrointestinal symptoms (anorexia, nausea, vomiting, and diarrhoea) are common, and nonproductive cough occurs in approximately half of the cases (8). Septic meningitis is also relatively frequent (up to 80% of cases) and usually manifests approximately seven days from the onset of the illness, as the immune phase begins. In a minority of cases, leptospirosis can progress to severe, fulminant disease with a mortality rate of 5-40% (7). Kidney involvement is expected because of the organism's predilection for renal tubules in their natural hosts, and renal failure occurs in 16-40% of cases (9).

Renal dysfunction in leptospirosis is typically non-oliguric and associated with hypokalaemia. Pulmonary manifestations of severe leptospirosis include alveolar haemorrhage (termed Severe Pulmonary Haemorrhagic Syndrome or SPHS) and pulmonary oedema, both of which can result in Acute Respiratory Distress Syndrome (ARDS) (24). Pulmonary involvement is associated with significantly higher mortality from leptospirosis, with case fatality rates estimated at 50-70% (25). *Leptospira* infection can also involve the heart, most commonly causing nonspecific echocardiogram abnormalities (even in mild disease). Myocarditis, pericarditis, heart block, and arrhythmias may occur, and repolarization abnormalities are a poor prognostic sign (26). Even
after recovery, patients may have continued late sequelae, including neuropsychiatric and ocular
symptoms.

Disease in animals

Infection most frequently occurs through the mucous membranes of the eye, mouth, nose, or genital tract. The oral infection has also been shown in predators. Vertical transmission can also occur. A period of bacteraemia, which may last for a week, begins 1 or 2 days after infection. During this period, leptospires can be isolated from blood, most body organs, and cerebrospinal fluid. This primary bacteremic phase ends with the appearance of circulating antibodies, which are usually detectable after 10–14 days. A secondary bacteremic period (after 15–26 days) has rarely been reported (27). Acute clinical disease coincides with the bacteremic phase of the disease. It is seen mainly in young animals. It is usually associated with incidental infections, particularly hemolysin-producing strains such as *pomona* or *icterohaemorrhagiae* serogroup strains, which cause haemolytic disease, haemoglobinuria, jaundice, and death. Renal damage can be essential, particularly in *canicola* infection in dogs. Antileptospiral agglutinins appear detectable in the blood at approximately 10-14 days after infection and reach maximum levels at around 3-6 weeks. Peak titres vary considerably (1,000 to 100,000 in the Microscopic Agglutination Test, MAT). Depending on the species, these may be maintained for up to 6 weeks, after which a subsequent gradual decline occurs.

Low titres may be detectable for several years in many animals. The duration and intensity of
urinary shedding vary from species to species, animal to animal, and with the infecting serovar.
In the case of *pomona* infection in pigs, the intensity of excretion is highest during the first month
of shedding (28). Leptospires may also localize in the uterus of pregnant females; abortion,
abortion, stillbirth, and neonatal disease may result from intrauterine infections in late gestation.
An additional feature seen in host-maintained infection is the persistence of leptospires in the
oviduct and uterus of non-pregnant females and in the genital tracts of males (29).

Availability of preventive, therapeutic, and control
measures, including licensed or pipelines vaccines

Therapy in humans

Most cases of leptospirosis are mild and self-limiting, and patients often do not present for
care, while for milder cases, oral doxycycline, azithromycin, ampicillin or amoxicillin are
indicated (30). Azithromycin or doxycycline are the drugs of choice as per standardized
guidelines in geographical locations where also rickettsial diseases are endemic (31). Among
pregnant and young children, doxycycline is contraindicated (32). In severe cases, intravenous
penicillin G has proven to be equally effective as cefotaxime and ceftriaxone (32). Administration
of fluids is recommended to correct hypovolemia, hypotension, or bleeding; transfusion with
saline/blood is mainly suggested. In patients suffering from complications like acute kidney
injury, treatment with fluids or diuretics is initiated in mild cases, and dialysis can be performed
in severe stages of the disease. Ventilator support is often needed for patients who encounter
complications like ARDS and pneumonia.
## Therapy in animals

The treatment of acute leptospirosis in individual animals or herds depends on antibiotics plus supportive symptomatic treatment. Antibiotics used may vary according to their safety in a particular species, their availability for a specific country, the cost, and the route of administration. A combination of penicillin and streptomycin has been the antibiotic therapy of choice for the treatment of acute leptospirosis, but ampicillin, amoxycillin, tetracyclines, tulathromycin and third generation cephalosporins have also been used (33).

## Licensed or pipelined vaccines

Vaccines composed of inactivated whole cells (bacterins) are the only vaccines currently licensed for the control of leptospirosis (34). They are mainly for animal use, but in countries such as France, Cuba, China and Japan, bacterins are approved for use in at-risk human populations (35). Although there are some negative aspects to the use of bacterins, e.g., short-term protection and the lack of cross-protection, they have significantly reduced the incidence of the disease and remain one of the most viable strategies to control the infection in humans (36).

The most extensively studied vaccine candidates to date are the Leptospiral immunoglobulin-like (Lig) proteins (37), which are highly conserved and only found in the pathogenic *Leptospira* spp. Vaccines using the LigA or LigB recombinant proteins have demonstrated a wide range of protection, even if LigA is not present in all pathogenic species, potentially limiting its role in a universal vaccine against leptospirosis. Also, several Outer Membrane Proteins (OMP: Lp11, Lp21, Lp22, Lp25, Lsa30, and Lp35), identified using a bioinformatics approach, induced a partially protective immune response when pooled in a single vaccine preparation (38).

## Other prevention measures

Prevention of leptospirosis in humans starts by reducing exposure risk by avoiding contact with water contaminated with animal urine, either by not wading or swimming in contaminated water or wearing protective clothing for those with an occupational risk of exposure to contaminated water.

Measures to decrease the risk of infection should include: a) surveillance for both human and animal populations; b) control of rodents to reduce the risk of infection; c) covering skin lesions with impermeable dressings to prevent contact with contaminated water; d) use of protective clothing, such as gloves and galoshes, for those working in contact with animals, sewage or during heavy rains; e) post exposure prophylactic treatment with antibiotics may be advised in situations of accidental exposure to rodent, or contaminated water or soil (39).

## Disease specific recommendations

Contaminated urine is highly infectious for people and for susceptible animal species; therefore, contact with urine on mucous membranes or skin abrasions should be avoided. Handling infected animals or working in contaminated areas should be done with protective gloves, eye protection, and face masks (8).
# Epidemiological situation at different spatial scales: past and current trends

Leptospirosis is the most widespread zoonotic disease. The disease is (re-) emerging globally and numerous outbreaks have occurred worldwide during the past decade (Factsheets about leptospirosis by the European Centre for Disease Prevention and Control, ECDC). France has consistent annual numbers of infections, and France and Germany reported the highest number of cases notified in Europe between 2018 and 2020 (Figure 1).

<figure><img src="image_30.png" /><figcaption>**Figure 1.** Number of reported cases of leptospirosis in 29 countries of the European Union and European Economic Area, 2018–2020 (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>

On the other hand, in Europe, countries like Luxemburg, Finland, Cyprus, and Iceland did not notify cases in the last years. Other European countries that consistently notified human cases were Netherlands, Portugal, Romania, Slovenia, and Spain, almost all with an increasing trend, probably due in part to better surveillance and increased public awareness. Based on the reporting of human cases the significant hotspots for leptospirosis are in central and Mediterranean areas and East Europe. The data reported over the 2012-2020 period (Figure 2) has been relatively consistent over the years, and considering all EU/EEA (European Union/European Economic Area) countries, a slight upward trend can be observed. Trends were more apparent in individual countries: Czech Republic and Ireland showed an increasing number of cases, while decreasing case numbers was apparent in Italy, Greece and Austria.
<figure><img src="image_31.png" /><figcaption>Figure 2. Number of reported cases of *Leptospira* in 29 countries of the European Union and European Economic Area, 2012-2020 (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>

**Sociological and demographical dimensions affecting susceptibility and exposure, including gender**

Occupation, migratory behaviour, gender and age are all significant risk factors of leptospirosis. In the past, leptospirosis was first considered an occupational disease, whereby coal miners were the first occupational risk groups to be documented (40). In addition, various mammals including feral, farm, and pet animals can harbour leptospires. This extends the occupational risk to other professions, including farmers, miners, slaughterhouse labourers, pet traders, veterinarians, rodent catchers, sewer workers, garbage collectors and livestock ranchers (41). A report submitted from the second leptospirosis Burden Epidemiology Reference Group (LERG) meeting showed that the median case-fatality percentage was higher in women than men. However, that does not mean that women are more likely to be infected with the disease. The same report shows men are more likely to be infected with leptospirosis as they are more prone to occupational exposure in outdoor settings. The median incidence of the disease was the highest in men older than 59 years, followed by those between 20 to 29 years. For women, approximately 37% of leptospirosis cases were reported in the 40 to 49 age category (42). Table 8 reported the most important human covariates linked with leptospirosis spreading, while Figure 3 reported leptospirosis distribution by sex and age.