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

## Brief history of the pathogen and disease

WNV was isolated for the first time in Africa in 1937 in the West Nile district of the northern province of Uganda, following a campaign aimed at monitoring the circulation of Yellow Fever virus. A new virus was isolated in a febrile 37-years-old woman and named WNV in relation to the district where it was found.

After these first investigations, cases were reported in Israel, Egypt, France, and South Africa throughout the 1950s to 1970s, followed by large outbreaks in Romania and Russia in the 1990s. WNV infections were reported for the first time in Algeria and Morocco in 1994 and 1996, respectively. In 1999, the first WNV cases in the Western Hemisphere were identified in New York City. WNV is currently considered as the most widespread flavivirus on the globe (15, 17, 112, 125-127).

## Disease in humans

In humans, the WNV incubation period varies between 2 and 15 days after infection. WNV infection is often asymptomatic in humans (80%), however some patients (20%) may present febrile illness WNF, characterized by fevers and maculopapular rash (50% of cases), headache, myalgia, arthralgia, malaise, anorexia, nausea, vomiting, diarrhea, abdominal pain, retro-orbital pain, pharyngitis, and lymphadenopathy (128-132). Acute form of WNV may persist for up to 60 days and can be characterized by a biphasic condition (2, 132).

In less than 1% of cases, mainly in elderly or immunocompromised people, it can lead to WNND characterized by encephalitis, meningitis and/or acute flaccid paralysis and sometimes to fatal outcomes. Pancreatitis, orchitis, myocarditis, haemorrhagic fever, nephritis, hepatitis, and rhabdomyolysis may be rare non-neurological presentations associated with WNV infection. A biphasic fever, usually helpful for WNV diagnosis, is an additional characteristic feature. After recovery, patients may present long-term sequelae, such as persistent movement disorders, functional disabilities, weakness, cognitive impairments, depression and, in some cases, early death (133).

In Europe, the case fatality ratio among patients with WNV disease reported by the ECDC from 2017 to 2022 was 9% in 2017 (26 deaths out of 288 confirmed cases in EU and neighbouring countries), 8.6% in 2018 (180/2083), 10.8% in 2019 (50/463), 11.3% in 2020 (38/336), 8.2% in 2021 (13/159) and 8% in 2022 (92/1113) (Figure 6).

Age and gender are the main intrinsic predisposing factors for the disease and its relative severity. In general, older individuals are more susceptible. In fact, the risk of acquiring the disease increases by 1.5 folds for every 10 years of age. It has also been demonstrated that patients older than 75 years might succumb to the infection, and this could be explained by aging related innate immunosenescence. Thus, aging affects several antiviral pathways, including cellular pathways (macrophage related defence) and cytokines such as type I Interferon (IFN) and Toll-like receptor 3 (TLR 3)-mediated antiviral pathways, leading to increased susceptibility to viral infection including WNV (134). In addition, males have been shown to be more at risk than females. Pre-existing conditions and diseases such as cancers, cardiovascular, renal and cerebrovascular diseases, and diabetes are also other major risk factors. Immunosuppressed individuals have 40 times higher risk of contracting the disease and dying from WNV infection (reviewed in 2, 132, 135-137).
<figure><img src="image_74.png" /><figcaption>**Figure 6.** Number of reported WNV human cases, deaths and fatality rate, European Union and European Economic Area countries, 2017-2022 (data extracted from ECDC Epidemiological update reports)</figcaption></figure>

In Europe, based on the analyses of TESSy data on infection for the period 2012-2022, males resulted more affected than females (Figure 7). WNV was more common in males (61.6%) than in females (38.4%) with a male-to-female ratio of 1.6:1. Most infections occurred in people over the age of 50 (77.6% of the total, of which 48.6% occurred in males and 29% in females over 50 years old). Notification rates increased with age in both sexes, peaking at 0.13 cases per 100,000 population in males aged 70-79 years.

<figure><img src="image_75.png" /><figcaption>**Figure 7.** Incidence of WNV infections per 100,000 population, by sex and age group and male-to-female ratio by age group, European Union and European Economic Area countries, 2012-2022 (data from TESSy-ECDC)</figcaption></figure>
## Common human exposure routes

WNV is mostly transmitted to humans through mosquito bites. The transmission occurs especially when mosquitoes are active (i.e. between spring and autumn) and most infections in humans and equids are observed between July and September in temperate and subtropical areas. Human behaviour, occupation and leisure activities can influence the likelihood of contracting WNV disease. In fact, the exposure to mosquito bites, when coupled with the presence of infected mosquitoes and birds, can result in an increased probability of getting infected (132).

Other transmission routes include the possibility of infection through SoHO as blood transfusion, organ, tissues or cells transplantation from an infected donor, intrauterine transmission and through breastfeeding. Technician and laboratory workers may also be infected during manipulations of infected material through percutaneous injections or inhalation of droplets in the air (132).

## Disease in animals

From a veterinary standpoint, WNV is the causative agent of WNF, which might develop into asymptomatic forms, benign forms (flu-like syndrome) and neuro-invasive forms. In Europe, most of the clinical signs are reported in equids and birds.

### Horses

Among many flaviviruses causing disease in mammals, WNV is probably the one with a major impact on equid health. Horses are sporadically infected by the virus and, although in most cases they remain asymptomatic, around 20% can develop clinical signs that use to be more severe than in humans and have important health and economic consequences (138, 139).

Overall, the combination, severity, and duration of clinical signs can be highly variable. Low grade fever, obtunded mentation, inappetence, colic, or lameness can be among the first recognized signs in diseased animals. In different outbreaks the most frequent clinical signs observed are ataxia (57%-100%), weakness (30%-100%), and muscle fasciculations (42%-100%). Ataxia can be symmetrical or asymmetrical, such as weakness affecting either fore/hindlimbs or all. Fewer horses present with hyper-responsiveness and cranial nerve deficits such as facial paralysis, vestibular ataxia, drooping lip and/or inability to swallow, photophobia and central blindness. Abnormal behaviour such as obtunded mentation, somnolence, disorientation, hyperexcitability and aggressive behaviour as well as changes in personality have also been associated with the infection.

### Birds

Birds are the natural hosts of WNV and play a key role on the virus epidemiology, with many species susceptible to the infection (40). The disease shows up due to the virus invasion of different organs, liver, spleen, kidney, heart, and mainly the central nervous system, and can lead to death within 24-48 h later (138). Clinical signs in susceptible infected avian species include ruffled feathers, lethargy, ataxia, unusual posture, inability to fly or to hold head upright, head tremors, seizures, leg paralysis, nystagmus, and weight loss (40). Corvids and raptors appear particularly susceptible to WNV infection, with the latter often exhibiting neurological signs, in some cases leading to the death (40, 81, 82).

Although abundant information exists regarding experimental infections of birds with WNV, especially with the WNV/NY99 strain (89-91), studies about the pathogenicity of Euro-Mediterranean strains in different avian species are still scarce (82, 83, 92, 103, 104, 140, 141).
In fact, the pathogenicity of new strains is worth to be studied in order to obtain a better understanding of WNV eco-epidemiology (82).

## Common animal exposure routes

Vector borne transmission is the main transmission route for WNV in animals (91). However, birds can also get infected through direct contact, since they are able to shed high-virus titres through oral and cloacal secretions. Particularly, direct transmission in contact birds might occur through faecal-oral or oral-oral routes, as demonstrated in a few species, mainly in *Corvidae* and *Laridae* families, or by skin or feather picking. Moreover, in laboratory experiments, transmission through direct contact has also been described in common goose, chicken and only in one occasion in red-legged partridge (91). Direct contact transmission could play a role in WNV epidemiology in those situations in which wild birds aggregate in high densities, as in breeding colonies, roosting and feeding areas, or stopovers during migration (142). Another means of WNV transmission is through the ingestion of infected mosquitoes, infected prey or infected tissues and organs (91). Persistent infection, defined as the detection of the virus in host tissues after viremia has subsided, might increase the probability of the viral transmission through the oral route. In fact, predation of infected birds characterized by a persistent infection associated to high viral load in their organs might likely result in the WNV transmission, months after the end of the mosquito season (23, 143).

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

To date, no WNV-specific therapy is available and vaccines are only licensed for use in horses but not in humans. While several methodologies for the vaccine development have been successfully applied and have contributed to significantly reducing the WNV incidence in horses in the US, none have progressed to phase III clinical trials in humans (139).

### Therapy in humans

Despite the great efforts invested in recent years in the development of prophylactic measures against this pathogen, there is currently no specific drug or therapy licensed for its treatment (139). Some therapies that have shown variable effectiveness against WNV include IVIG, interferon, and ribavirin (128). Patients with severe meningeal symptoms often require pain control for headaches and antiemetic therapy other than rehydration for associated nausea and vomiting. Patients with encephalitis require close monitoring for the development of high intracranial pressure and seizures. Furthermore, they should be monitored for the inability to protect their airway, also in the case of poliomyelitis. In fact, acute neuromuscular respiratory failure may develop rapidly, and prolonged ventilatory support may be required. Various drugs have been evaluated or empirically used for WNV disease; however, none have shown specific benefit to date.

### Therapy in animals

In horses, survival rate for WNV encephalitis is high compared with other infectious encephalitis (55-70%). Treatment of WNND is mainly supportive (138).
# Licensed or pipelined vaccines

## Humans

There is no vaccine authorized for humans, nor candidates close to being licensed. Although several veterinary vaccines have been licensed, WNV vaccines for humans have not progressed beyond phase 1 or 2 clinical trials (139, 144).

## Animals

### Horses

Four of the six licensed vaccines are currently on the market. The WN-Innovator, with a classic inactivated whole virion-based approach, was the first to be developed and was licensed by the United States Department of Agriculture (USDA) in 2003. Live attenuated recombinant viruses have also been used (either based on canary poxvirus or yellow fever virus), as well as a plasmid DNA vaccine, which was the first licensed by the USDA, although it was subsequently withdrawn from the market by the manufacturers. However, despite their proven efficacy, these vaccines still exhibit some limitations, such as the need for repeated administrations to get a solid initial immunization, and the relatively short duration of the induced immunity, which makes necessary annual boosters (139).

### Birds

Several commercial and experimental vaccine candidates have been assayed in wild and domestic birds, although no one has yet been authorized. Overall, they induced humoral and, although less analysed, cellular responses, and reduced disease, injury, viremia, viral shedding, and mortality associated with WNV. Furthermore, if they induce herd immunity, they could help prevent outbreaks and the spread of the virus. For example, a prospective vaccination of the entire population of California condors (*Gymnogyps californianus*), an endangered species, performed before the WNV arrival would have helped preventing its infection and possible extinction. Likewise, vaccination also greatly reduced virus incidence in domestic geese in Israel. However, the implementation of bird vaccines faces several drawbacks, such as the feasibility of access to the target host, mainly for wild species, and the administration route. In any case, its availability could benefit domestic populations (farm birds, including those for hunting and restocking activities), as well as wild ones (as those housed in rehabilitation centers and wildlife reserves, and in recreational facilities, like zoos (139).

## Other prevention measures

In the absence of a vaccine, one prevention measure to reduce the risk of infection in humans consists in raising awareness of personal and community behaviours. In particular, it would be important to increase protection against mosquito bites by avoiding human exposure, through i) the reduction of outdoor activities at peak biting times and sleeping outside, ii) the wearing of light coloured, long-sleeved shirts and trousers, iii) the use of mosquito nets and repellents (131). In addition, it is necessary to implement mosquitoes surveillance and control measures such as removing or treating mosquitoes breeding sites located near cities and villages, reduce the risk of animal-to-human transmission wearing protective equipment when handling animals and during slaughtering and culling operations and, prevent infectious disease transmission associated with organ and tissue transplantation and blood transfusion, implementing donors epidemiological screening and laboratory testing tools, specifically during WNV outbreak (2).
In countries with endemic circulation of WNV along with other arbovirosis such as Italy and Spain, national integrated One Health surveillance and control plan are established (32, 35) For example, in Italy, a specific surveillance plan (National Plan for Arbovirosis), coordinated by the Istituto Superiore di Sanità (ISS) and by the Istituto Zooprofilattico Sperimentale di Abruzzo e Molise (IZSAM), in collaboration with the Ministry of Health, is in place to ensure the early detection of potential cases of WNV and minimize any spread of the disease. The plan also includes guidelines and recommendations for mosquito surveillance and control. 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

### Regulation on substances of human origin

In July 2022, the European Commission adopted the proposal for a Regulation on standards of quality and safety for Substances Of Human Origin (SoHO) intended for human application. By repealing the Blood Directive (2002/98/EC) and the Tissues and cells Directive (2004/23/EC) both evaluated in 2019), the proposed Regulation concludes the revision of the legal framework for blood, tissues and cells, which was included in the REFIT Annex (#37 p.15) of the Commission's Work Programme for 2021.

Currently, as recommended by ECDC (https://www.ecdc.europa.eu/en/west-nile-fever/facts) to prevent transfusion-transmitted WNV infections, European Union/European Economic Area (EU/EEA) countries should implement 28-day blood donor deferral or individual donation nucleic acid testing (ID-NAT) of prospective donors who have visited or live in an affected area.

In affected areas, blood establishments* should follow recommendations provided in the EU preparedness plan for blood safety. Donors of organs, tissues and cells living in or returning from an affected area should be tested for WNV infection.

Systematic collection of epidemiological information on WNV infection among donors and recipients of SoHO is an important tool for national authorities to better assess the risk of transmission and impact of preventive measures on the availability of SoHO.

According to the preparedness plan for WNV blood safety in the EU, blood establishments in affected areas should:

- temporarily interrupt blood collection or implement NAT screening for blood donations from WNV affected areas

- quarantine, retest and discard positive blood components in storage at the time of implementation of measures and retrieve and quarantine blood components derived from whole blood donated 120 days prior the date of collection of the ID-NAT-positive donation

- enhance donor post-donation information, especially about fever, influenza-like illness or other acute symptoms within 15 days after donation

- strengthen post-transfusion haemovigilance and perform look-back analysis in any case of transfusion-transmitted WNV infection for a period dating 120 days prior to the donation of implicated blood components; and

- consider the use of pathogen inactivation procedures.

<footnote>* According to Directive 2002/98/EC, 'blood establishments' are structures or bodies that are responsible for any aspect of the collection and testing of human blood or blood components, whatever their intended purpose, and their processing, storage, and distribution when intended for transfusion</footnote>
# Epidemiological situation at different spatial scales: past and current trends in Europe

## Past trends

WNV circulation in Europe has been reported since 1958 (20). Before 2010, major WNV outbreaks occurred throughout Europe, notably in Romania and Russia in the 1990s, Hungary in 2008, Greece in 2010 (17). Since 2010, WNV has been reported in an increasing number of EU countries including Austria, Bulgaria, Croatia, Cyprus, France, Greece, Hungary, Italy, the Netherlands, Portugal, Romania, Slovenia and Spain, other than northerly regions that had not previously reported cases, like Germany and the Czech Republic. Moreover, it has been described in five EU neighbouring countries including Albania, Montenegro, Serbia, Turkey, and Kosovo. The epidemiological situation of WNF in Europe is heterogeneous: some European countries report outbreaks in humans and animals every year while others have never reported any autochthonous cases (8).

Outbreaks have occurred annually in multiple regions, with a peak in 2018 that affected more regions than had been recorded in previous years (39).

In Europe, there were, on average, 18 newly affected areas annually between 2011 and 2017, and 45 additional areas reported in 2018, with most infections occurring from early summer to early autumn and peaking in August (145).

Until 2017, the countries with the highest number of cases in the EU were Greece, Italy, Romania, and Hungary and one of the neighbouring countries, Serbia. ECDC reported a 7.2-fold increase in cases from 2017 to 2018, especially in Bulgaria (15-fold), France (13.5-fold), and Italy (10.9-fold), which is partially attributed to the unusually hot spring and summers. The same was true for Serbia with notification numbers 8.5 times higher in 2018 compared to 2017. In 2020, a major outbreak was reported in Spain with 77 confirmed cases, of which 72 developed neuroinvasive disease and seven died, a strong impact that could be, among others, partly attributed to reduced vector control activities during that season (145, 35, 17).

## Current trends

For the period 2008-2022 a total number of 5358 (4142 confirmed) human cases have been reported to the ECDC (TESSy data), with the highest numbers observed in 2018 and 2022 (Figure 8).

Over the past years, there has been an increase in the number of countries reporting surveillance data in animals (146). In 2021, the number of human cases in the EU was 158, being the lowest figure of the period 2017-2021. 54 infections were reported on October: 7 in Greece, 52 in Italy, 7 in Hungary, 7 in Romania, 6 in Spain, 3 in Austria, 3 in Germany, and 17 in Serbia, with cases reported for the first time in Spree-Neiße in Germany and La Spezia in Italy.

While the number of tested equids appears to correlate with WNV circulation, increasing in years when a greater number of cases are reported, there has been an overall increase in the number of tested birds over time, reflecting an intensification of the surveillance system in place.

Since the beginning of the 2022 transmission season and as of 23 November 2022, EU/EEA countries have reported 965 human cases of WNV with higher infection observed in Italy (586), Greece (284), Romania (46), Hungary (14), Germany (11), Croatia (8), Austria (6), Spain (5), France (4) and Slovakia (1).
EU/EEA countries have reported 92 deaths in Italy (37), Greece (31) and Romania (5). EU-neighbouring countries have reported 246 human cases of WNV infection in Serbia (226) and 12 deaths in Serbia (25, 147).

<figure><img src="image_76.png" /><figcaption>Figure 8. Number of reported WNV human infections by week of onset, European Union and European Economic Area countries, 2012-2022 (data from TESSy-ECDC)</figcaption></figure>

During the 2022 transmission season, within the reporting countries, human cases of WNV infection were reported from 107 different NUTS 3 or GAUL 1 regions, of which the following regions reported human cases of WNV infection for the first time ever: Bouches-du-Rhône in France, Harz, Vogtlandkreis and Salzlandkreis in Germany, Pistoia, Lucca, Monza e della Brianza, Biella, Cagliari and Catania in Italy, Brasov in Romania, Moravicki in Serbia and Tarragona and Córdoba in Spain.

Moreover, 93 outbreaks among equids and 314 outbreaks among birds have been reported by EU/EEA countries.

Outbreaks among equids have been reported by Italy (44), Germany (15), Greece (9), Croatia (8), Spain (6), France (5), Hungary (3), Portugal (2) and Austria (1).

Outbreaks among birds have been reported by Italy (249), Germany (51), Spain (9), Austria (2), Croatia (2) and Hungary (1).
# Current trend in some European countries

## Italy

WNV human infections in Italy are mostly located along the Po valley (northern part of the Country), with a mean of 167 cases reported annually and an average notification rate of 0.28 per 100 000 inhabitants over the period 2012-2022. The total number of 1841 cases were reported between 2012 and 2022 (Figures 9 and 10).

<figure><img src="image_77.png" /><figcaption>Figure 9. ITALY - WNV: cases and incidence per 100,000 inhabitants by year and incidence by gender and age group (n = 1841) (data from TESSy-ECDC)</figcaption></figure>
<figure><img src="image_78.png" /><figcaption>Figure 10. ITALY - WNV: average annual incidence rate per 100,000 inhabitants by NUTS3 (n=1703, period 2012-2022) (data TESSy-ECDC)</figcaption></figure>

## France

Few human infections of WNV are reported annually in France, with a mean of about 5 cases and an average notification rate of 0.009 per 100 000 inhabitants over the period 2012-2022. Particularly, between 2012 and 2022, 56 cases were reported (Figure 11).

Although the enzootic WNV circulation is often observed in the South of France (34), most of the human infections from France seems to be acquired abroad, as only seven locally acquired cases were reported from the Var (n=5) and Bouches-du-Rhône (n=2) departments (Figure 12).

## Spain

Spain is characterized by an average of 9 cases with a mean notification rate of 0.04 per 100,000 inhabitants over the period 2012-2022.

The total number of cases reported between 2012 and 2022 is 93, with most of the infections happening in 2020 (77) (Figure 13).

Geographically, WNV infections are located in the South-Western part of the Country, mainly in the Andalusia region (n=83) (Figure 14).
<figure><img src="image_79.png" /><figcaption>Figure 11. FRANCE - WNV: cases and incidence per 100,000 inhabitants by year and incidence by gender and age group (n = 56) (data from TESSy-ECDC).</figcaption></figure>
<figure><img src="image_80.png" /><figcaption>Figure 12. FRANCE - WNV average annual incidence rate per 100,000 inhabitants by NUTS3 (n=7, period 2012-2022) (data from TESSy-ECDC)</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>Figure 13. SPAIN - WNV: cases and incidence per 100,000 inhabitants by year and incidence by gender and age group (n = 93) (data from TESSy-ECDC)</figcaption></figure>
<figure><img src="image_82.png" /><figcaption>Figure 14. SPAIN - WNV: average annual incidence rate per 100 000 inhabitants by NUTS3 (n=87, period 2012-2022) (data from TESSy-ECDC).</figcaption></figure>

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

Age and gender are among the most critical factors in determining the WNV infection risk. For example, the number of WNV reported cases is estimated to be greater in areas with a higher number of elderly (age > 65 years) people, consistently with the fact that age is one of the main risk factors for developing severe symptoms upon infection (120) (see also the paragraph “Availability of preventive, therapeutic and control measures, including licensed or pipelined vaccines”).

However, several other factors such as economic conditions and sociodemographic characteristics are considered to be important in the WNV epidemiology. As reported in a number of studies, a range of local-level socio-economic factors such as income, sanitation, wastewater management, and population density tend to influence the distribution and intensity of mosquito-borne diseases, including WNV, both pre-infection and post-infection (39). Poorer communities are less likely to have air-conditioned homes, tap water and adequate drainage, and therefore may be more exposed to biting mosquitoes. However, factors associated with higher economic status can also bring humans into closer contact with mosquitoes, for example, homeowners with gardens and potted plants, swimming pools and ponds or having good access to recreational space where mosquitoes can breed (39). As for the land-use variables, regions with a larger proportion of arable land and wetlands are associated with higher WNV incidence. Humans are particularly at risk in areas close to rice paddies, irrigated agriculture and wetlands, since these areas tend to attract susceptible mosquitoes and birds (39). The percentage of discontinuous urban fabric
populated areas of low to medium density often characterized by peri-urban forest, gardens, parks, and ponds, such as residential suburbs and villages, are also often cited as a driver of WNV infections in humans (39, 44). For example, WNV infection outbreaks have been associated with urban settings in some countries such as Romania, where people living in the basements of tall buildings were more at risk than the rest of the population. This has been linked to a peri-domestic ecological behaviour of some mosquito vector species such as *Cx. pipiens*, which are urban mosquitoes commonly found indoors (2). Other risk factors include outdoor activities and travelling. In fact, it has been demonstrated that spending more time outdoors or travelling to endemic regions constitute a risk of contracting WNV since individuals might get more exposed to mosquito bites. The main outdoor occupations at risk appear to be farm workers, loggers, landscapers and groundskeepers, construction workers, painters, summer camp workers, pavers, soldiers and security guards, among others. Healthcare, laboratory workers, veterinarians, animal handlers, animal slaughterers, and butchers appear also to be at risk of contracting WNV due to the possibility of getting exposed to the virus via needlesticks, accidental cuts, or contamination of open wound coming in contact with WNV infectious materials (2).

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

## Diagnosis

Cases of WNV infection should be notified following the EU case definition outlined to the ECDC. List of case definitions for reporting communicable diseases to the Community network follow the Commission Implementing Decision (EU) 2018/945 of 22 June 2018 on the communicable diseases and related special health issues to be covered by epidemiological surveillance as well as relevant case definitions:

- **Clinical criteria**

At least one of the following:

- any person with fever

- encephalitis; and/or

- meningitis

- **Laboratory criteria**

- Laboratory test for case confirmation at least one of the following:

- isolation of WNV from blood or cerebrospinal fluid (CSF).

- detection of WNV nucleic acid in blood or CSF.

- WNV-specific antibody response (immunoglobulin M; IgM) in CSF; and/or

- WNV IgM high titre, detection of WNV IgG and confirmation by neutralisation

- Laboratory test for probable case:

- WNV-specific antibody response in serum.

- **Epidemiological criteria**

At least one of the following epidemiological links:

- Animal to human transmission (residing, having visited or having been exposed to mosquito bites in an area where WNV is endemic in horses or birds).

- Human to human transmission (vertical transmission, blood transfusion, transplants).
- Case classification:

A. Possible case
Not applicable

B. Probable case
Any person meeting the clinical criteria and with at least:

- an epidemiological link; and

- a laboratory test for a probable case.

C. Confirmed case
Any person meeting laboratory criteria for case confirmation.

Note: Serological results should be interpreted according to previous exposure to other flavivirus infections and vaccination status. Confirmed cases in such situations should be validated by serum neutralisation or other equivalent assays.

Notification system

WNF is a notifiable disease in humans and in equids at the EU level. Notifications of human WNF cases in Europe are collected through the European Surveillance System (TESSy) of the ECDC. Between June and November, the period of high vector activity, the ECDC publishes weekly updated maps of human cases and complementary information on animal and vector WNV infections based on data provided by the World Organisation for Animal Health (WOAH) and European countries. The yearly analyses of TESSy data are published in the ECDC annual epidemiological report and jointly with the European Food Safety Authority (EFSA). EU countries report outbreaks of WNV encephalomyelitis in horses to the European Commission (EC) via the Animal Disease Notification System (ADNS) and regular summaries are posted online. The data from WNV monitoring in animals is reported annually by EU countries under Directive 2003/99/EC and presented in the annual EFSA/ECDC EU Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks. Animal WNF outbreak data reported to the WOAH are publicly available on the World Animal Health Information Database (WAHIS) interface (8).

In 2021, 25 Member States reported information on WNV infections in humans (146). Surveillance is mandatory in 23 countries; in France it is voluntary, while Germany did not specify its surveillance system. There is no surveillance system in place in Denmark and the disease is not notifiable or reported at EU level. The EU case definition was used by 23 Member States, France reported another case definition, while Germany did not specify the case definition used. All countries conducting surveillance had a comprehensive surveillance system with full national coverage, except Germany, which did not specify the surveillance system but had full national coverage. All countries reported case-based data.

WNV surveillance in animals involves mostly passive surveillance, including surveillance based on the diagnosis of neuro-invasive cases in equids, but some countries implement active surveillance of equids and/or captive birds and/or wild birds. Alongside EU Member States, Switzerland submits reports to EFSA on animal surveillance and monitoring activities in animals. Two sources of information are used to complete this report. Firstly, data are submitted to EFSA by EU Member States and Switzerland from annual surveillance and monitoring activities in accordance with Directive 2003/99/EC. WNV is listed in Annex I, Part B (viruses transmitted by arthropods) as a virus to be monitored, if warranted by the epidemiological situation in an MS, in compliance with Article 4.1 of the same Directive. Secondly, it is mandatory for Member States
to notify outbreaks of equine and avian WNV to ADIS,41 in accordance with CIR (EU) 2020/2002.

## Infrastructure capacity to identify pathogens for each Member State

Based on a survey conducted by the ECDC to collect information on the current WNV surveillance and control capacities across the EU/EEA countries, European Neighbourhood Policy (ENP) partner countries and EU candidate/potential candidate countries, eighty-three percent of the EU/EEA countries, ENP partner countries and EU candidate/potential candidate countries have implemented at least one method of WNV surveillance. The most common passive WNV surveillance method for all countries is the detection of human cases, followed by surveillance on dead animals. The most common method of active WNV surveillance is mosquito screening, followed by sentinel bird screening and sentinel equid screening. The majority of the countries conduct routine vector surveillance (abundance monitoring), but less than 15% perform pesticide resistance testing, and even where this is done, this is not implemented systematically.

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

After the two major peaks observed in 2018 and 2022, which coincide with two of the hottest years recorded in Europe, changes in the epidemiological pattern of WNV circulation in Europe are expected to occur in the coming years (148).

As explained in the “drivers of disease emergence” section, global warming characterized by increased temperatures and extreme weather events is expected to create a suitable environment for diverse populations of mosquito species, as *Cx. pipiens*, and to increase the WNV transmission in Europe. This highlights the importance of European-wide integrated surveillance to cover human animals and vectors, detecting WNV host and vector distribution (118, 148).

Italy and other EU countries experienced an important peak of WNV circulation in 2022. Particularly, the Italian 2022 vector season was marked by an early onset of viral circulation in mosquitoes and birds while human infections started at the beginning of July, with a rapid increase in the number of cases (13). Furthermore, most human cases of WNND reported in Europe occurred in Italy in 2022.

Projections show up to 5-fold increase in WNV risk for 2040-60 in Europe, depending on geographical region and climate scenario, compared to 2000-20 (14). The proportion of disease-reported European land areas could increase from 15% to 23-30%, putting 161 to 244 million people at risk. Across scenarios, Western Europe appears to be facing the largest increase in the outbreak risk of WNV. The increase in the risk is not linear but undergoes periods of sharp changes governed by climatic thresholds associated with ideal conditions for WNV vectors. This will require a targeted public health response to manage the expansion of WNV with climate change in Europe (14).
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# ZIKA

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

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

(b) 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

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

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

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

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

### Disease name

Zika, Zika fever, Zika virus infection.

### Disease agent

Zika virus (ZIKV).

### Common, scientific and Latin name

The ZIKV is the causal agent responsible for Zika virus infection or Zika fever. Zika 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*. ZIKV was first identified in Uganda in 1947 in a Rhesus macaque monkey that was caged in the canopy of Zika Forest, near Lake Victoria (1). The second isolation was made from *Ae. africanus* mosquitoes caught in the same forest in January 1948 (2). Thus, ZIKV received its name from the geographical area where the initial isolations were made.

### Taxonomy

Zika virus is a small enveloped single-positive-stranded RNA virus belonging to the *Flavivirus* genus, of the *Flaviviridae* family. Its genome contains approximately 10.7 kb coding for three structural proteins forming the virion particle: the capsid (C), the pre-membrane/membrane (PrM/M), and the envelope (E), and seven non-structural proteins responsible for viral genome replication, modification of host cellular functions and immune responses: NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5 (3,4). Beyond ZIKV, the genus *Flavivirus* comprises 52 other viral species, including the West Nile virus, yellow fever virus, dengue virus (DENV), chikungunya virus (CHIKV), tick-borne encephalitis, St. Louis encephalitis, Langat virus, the Modoc virus, Rio Bravo virus, the Powassan virus, and the Japanese encephalitis virus. ZIKV is genetically and antigenically related to all of them and more specifically to the Spondweni virus (5). Both viruses form a unique clade (clade X or Spondweni serocomplex) within the mosquito-
borne flavivirus cluster. Based on phylogenetic analyses ZIKV strains were grouped into three major lineages: East African, West African, and Asian/American (6, 7).

## Physiochemical properties

Similar to other Flaviviruses, ZIKV is 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 also survive almost indefinitely at room temperature (8). More recent experiments showed that routinely used disinfectants and alcohols are sufficient to inactivate ZIKV in the laboratory. Complete loss of infectivity was observed after virus exposure to 1% hypochlorite, 2% paraformaldehyde, and 2% glutaraldehyde (9). Exposure for 10 minutes entirely inactivated ZIKV in the presence of 2.5% FCS serum; increasing concentrations of serum reduced the antiviral effects of UV light. Gloves routinely used in BSL-2 laboratories protect against ZIKV (9).

Regarding the environmental stability of ZIKV, experiments showed that commonly used disinfectants and UV radiation can inactivate dried ZIKV. Additional experiments demonstrated that dried ZIKV remained infectious for >3 days suggesting that dried droplets can be infectious, and confirming that proper surface disinfection is essential. The virus was stable at temperatures up to 50°C but lost all infectivity at temperatures of >60°C. Thus, virus-contaminated materials such as surgical instruments can be decontaminated by heat. Experiments also found that ZIKV infectivity was highest after adjusting the stock to a pH of ≈9. In contrast, adjusting ZIKV to pH 12 or to <pH 4 abrogated infectivity (9).

## Priority level for EU

ZIK is not endemic in Europe, but like other *Aedes*-borne viruses, it has the potential to emerge following the importation of the virus in the European regions and areas colonized by competent vectors: *Ae. aegypti* (currently established in Madeira) and to a lesser extend *Ae. albopictus* (currently established in 18 countries including Switzerland and UK, and 337 regions of mainland Europe) (10,11). Up to date, European Zika cases reported to the European Centre for Disease Control (ECDC) are sporadic and mostly travel-associated (travellers returning from endemic areas). To our knowledge, sporadic autochthonous sexually transmitted ZIKV infections were reported in France, Germany, Italy and Norway from 2016 to 2019 (12-15), three autochthonous vector-borne transmissions were identified in France in 2019 (16) and few unidentified transmission events occurred in Germany (17).

Considering the frequency of travellers between high-incidence areas in the world and the European Union (EU) and the past experiences in France (16), the risk of more ZIKV outbreaks in continental Europe is not unlikely. The large outbreak in South America in 2016 led to an increased concern about the virus getting introduced in the EU/European Economic Area (EEA) and potential local transmission. In March 2016, a surveillance of Zika started with the main objectives of detecting the locally acquired/autochthonous cases in the European Union/European Economic Area (EU/EEA) early, and timely reporting of travel-associated cases, particularly those residing in areas where *Ae. albopictus* is established, to trigger appropriate control measures (18).

The risk of larger-scale outbreaks in the European region, as the ones seen in South America in 2016, is currently considered moderate to low (as of 2022) mainly because *Ae. aegypti*, the main vector of ZIKV is not widely present in Europe, although it is established in limited areas,
such as Madeira Island and the north-eastern Black Sea coast. *Ae. albopictus*, a potential vector for ZIKV transmission is present in 18 European countries (as of February 2023), primarily in the Mediterranean Basin. However, the likelihood of ZIKV spread in countries where *Aedes* mosquitoes are present should be seen as high or moderate, since in several European countries there is a suitable climate for the establishment of competent mosquitoes, including some countries with a history of transmission of DENV or CHIKV, higher ship and flight connections as well as high population density and urbanization patterns favourable to anthropophilic populations of *Aedes*.

Distribution of the pathogen

ZIKV was first detected in 1947 in rhesus monkeys in the Zika jungle of Uganda. In humans, the first report was in 1952 from Uganda and the United Republic of Tanzania. Infections have since been reported in Africa, the Americas, Asia and equatorial Pacific Island countries. In 2015 and 2016, high temperatures and severe drought conditions provoked a major outbreak in South America. Although, the number of reported outbreaks decreased from 2017, in 2023, 92 countries and regions had already experienced current or past transmission events (19). As with every emerging arthropod-borne virus (arbovirus), ZIKV distribution and spread depend on the distribution and spread of its competent vectors. *Ae. aegypti* and *Ae. albopictus* are known to be the main vectors of ZIKV (20).

*Ae. aegypti* originated from Africa and has a worldwide distribution, mainly in tropical regions. This mosquito species was probably imported to America and the Mediterranean by ship from Africa (21). The ship carrying tanks of fresh water on board could retain colonies of *Ae. aegypti* favouring its dispersion from one continent to another. It was observed sporadically in Europe from the Atlantic coast (Britain, France, and Portugal) to the Black Sea, in the first half of the 20th century, which shows a wide dispersion compared to today (22). This species was common before the Second World War in the Mediterranean region (present in Spain up to 1953, Portugal up to 1956 and Madeira up until 1977-1979), and it disappeared from Southern Europe and North Africa after this period. This may be attributed to the use of DDT (dichloro-diphenyl-trichloroethane), a synthetic insecticide (now prohibited in many places due to its high impact on the environment and public health) used in malaria eradication programs (23). Sporadic occurrences were observed in Britain, France, Malta, Italy, Croatia, Ukraine, Russia and Turkey (24). It was recently re-established in the island of Madeira (Portugal) in 2004 and 2005 (24), observed for the first time in the Netherlands in 2010, in relation to imported used tires (25).

*Ae. albopictus* originally came from the tropical and subtropical areas of Southeast Asia; however, it spread to other countries through cold- and dry-resistant eggs in used tires and the plant Lucky Bamboo during the past three decades (26). It was first found in Europe in Albania in 1979 by a shipment of goods from China (27). Then, it was introduced to Italy through the port town of Genoa in used tires from Georgia (USA) in 1990. This species dispersed across mainland Italy, as well as parts of Sicily and Sardinia in 1990-1991. *Ae. albopictus* was then established in France in 1999, especially in the south. In 2002, it was also found in the tourist city on the island of Corsica. After that, the species spread in Belgium in 2000 and 2013, in Montenegro in 2001, in Canton Ticino in Southern Switzerland, Greece in 2003, 2004 Spain and Croatia in 2004, the Netherlands and Slovenia in 2005, and in Bosnia and Herzegovina in 2006 (28). The map of the distribution of *Ae. albopictus* in Europe in 2024 is presented in Figure 1.
<figure><img src="image_83.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>

## Ecology and transmission routes

Zika virus (ZIKV) is a neurotropic flavivirus. In humans, the incubation period of ZIKV may vary from 3 to 14 days and the infectious period from 2 to 7 days. Most of the infections are asymptomatic. Symptomatic infections may cause from mild symptoms such as fever, rash, conjunctivitis, muscle and joint pain, malaise and headache to severe neurological manifestations in adults and the developing foetus. Neurological manifestations include Guillain-Barré like syndrome, neuropathy and myelitis in adults and children (29,30). In February 2016, the causal relationship between ZIKV infections and microcephaly in developing foetus was confirmed and considered until November 2016 as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO). Mild symptoms are common to other arboviral and non-arboviral diseases; thus, the diagnosis of ZIKV infection requires laboratory confirmation. Transmission of ZIKV can be mosquito-borne, sexual, transfusion-based, or vertical (maternofoetal).

**Mosquito-borne:** The primary mode of transmission of ZIKV is mosquito-borne transmission with *Ae. aegypti* which is the major vector for ZIKV (31). *Ae. aegypti* is present in all tropical regions of the world, placing those at the highest risk of new outbreaks. The widely distributed *Ae. albopictus* can also transmit ZIKV but seems to play a minor role in the recent outbreaks (it was reported as the potential vector only in Gabon in 2007 and recently in sporadic cases in France) (32,33) (34). Other *Aedes* species with limited geographic distributions can also transmit Zika, for instance, *Ae. hensilli* on Yap Island or *Ae. polynesiensis* in French Polynesia (35, 36).

Conflicting studies suggest that mosquitoes belonging to other genera, mainly *Culex* mosquitoes, which transmit West Nile and Japanese encephalitis viruses, might be involved in ZIKV transmission (37-41).
Sexual: ZIKV sexual transmission has been suspected since 2008 when a traveller returning from an endemic area infected his partner who was in a region where vectors were absent (42). Sexual transmission is possible from both asymptomatic and symptomatic infections through genital, oral, and anal intercourse (43).

In men, ZIKV RNA shedding was demonstrated in two-thirds of semen samples tested within 30 days of illness onset, which decreased substantially within a month (44). ZIKV RNA can persist for several months in semen, but infective particles seem to be limited to the first weeks of illness, making the presence of ZIKV RNA an unreliable indicator of the presence of infectious ZIKV (44). The presence of ZIKV RNA in vaginal secretions is rare (around 2%) and its persistence is thus difficult to assess (45). The effect of Zika on male fertility is a matter of concern. The testis is an immune-privileged organ, protected by the blood-testis barrier to protect spermatogenesis. Despite this mechanism, pathogens such as ZIKV can persist in the male genital tract. The consequences of this on male fertility need to be determined. Acute Zika may alter the quality of sperm with evidence of a decreased sperm count between day 7 and day 60 after infection. Total motile sperm count decreased by 50% at day 60. Inhibin values increased until sperm count recovered (after 120 days) (46).

Transmission through Blood Transfusion: Given the presence of ZIKV in blood donors, and the report of four possible cases of transfusion-associated transmission of the virus, ZIKV should be classified as a potential transfusion-transmitted disease (47). This emerging disease is a challenge for blood banks, and strategies should be implemented to prevent transmission by blood transfusion.

Maternofoetal: Perinatal transmission of the ZIKV was first reported during the French Polynesian outbreak in 2013 (48). Viral RNA was detected in the amniotic fluid of pregnant women suffering from symptoms compatible with ZIKV infection, and later in foetal brains and products of miscarriages, supporting maternofoetal transmission of the virus (49,50). Vertical transmission will not occur in all pregnant women infected with the ZIKV and symptomatic congenital infection will not be observed in all exposed foetuses, similar to cytomegalovirus and toxoplasmosis (51). The exact rate of vertical transmission and congenital infection remains to be identified. Infectious viral particles have been detected in breast milk, but neonatal transmission by breastfeeding has so far not been described (52).

# Drivers of the disease emergence and spread

## Ecological drivers

ZIKV spreads primarily by vector-borne transmission. Therefore, its emergence and spread are conditioned, among other factors, by the distribution of the competent vectors. Mosquito population dynamics is itself affected by multiple components which partly depend on ecological drivers, such as temperature, precipitation or relative humidity (53, 54). Environmental conditions including land cover and urbanization also play a critical role in *Aedes* spp. establishment, favouring vector settlement and proliferation after its introduction (55). In urban environments, some facilities are favourable to the production of breeding sites, such as gardens, terraces and green spaces. Those facilities are conducive to the coexistence of high vector and human population densities (56). Although *Ae. albopictus* is known as less efficient to transmit the ZIKV than *Ae. aegypti*, its high level of eco-physiological plasticity allowed it to adapt to temperate environments and largely dispersed all over the world (57).

Moreover, the temperature seems to affect the vector/virus couple relationship including vector competence accelerating the ZIKV replication in the vector, the vector survival rate and
the hatching rate at high temperatures (58). Favoured by high temperatures, ZIKV vector transmission events appear to be stricter than those of other Aedes-transmitted viruses, such as dengue. One study showed that ZIKV transmission required a minimal temperature of 5°C warmer than DENV (59).

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

## Brief history of the pathogen and disease

ZIKV was first discovered in 1947 and is named after the Zika Forest in Uganda (1). In 1952, the first human cases were detected and since then, outbreaks of Zika have been reported in tropical Africa, Southeast Asia, and the Pacific Islands. Zika outbreaks have probably occurred in many locations. From the 1960s to the 1980s, sporadic human infections were detected across Africa and Asia. Since 2007, outbreaks of Zika have been recorded in Africa, the Americas, Asia, and the Pacific. Because the symptoms of Zika are similar to those of many other diseases, many cases may not have been recognized. The first European autochthonous Zika cases were reported in 2016 for sexual transmission and in 2019 for vector-borne transmission (12, 15, 16).

## Disease in humans

Most ZIKV infections are asymptomatic or with mild clinical symptoms, however, infection during pregnancy can cause congenital microcephaly and other brain defects, Guillain-Barre syndrome, stillbirth and miscarriages (60). Serological evidence showed that ZIKV infection is associated with neurological disorders, such as increased intraocular pressure, resulting in damage to optic nerves which triggers a causal relationship with glaucoma (61). About 20-25% of infected patients usually develops, with an incubation period of about one week, the following symptoms: skin rashes, headache, fever, joint pains and conjunctivitis. Besides, some patients also vomit, have diarrhoea, redness of eyes, weakness, oedema, abdominal pain, loss of appetite, and experience of hematospermia (40). Detection of viral RNA in the amniotic fluid, placenta, and brain tissue of foetuses and infants with microcephaly, and the high rates of microcephaly among children born to mothers with proven acute ZIKV infection during pregnancy, provided strong evidence linking central nervous system anomalies to maternal infection (49,62).

Unlike some congenital pathogens, the ZIKV seemed to harbour a specific neurotropism (63). Major anomalies observed in foetuses and neonates include microcephaly, ventriculomegaly, diffuse calcifications, cerebral atrophy, signs of abnormal gyration, cortical development, and ocular anomalies that might lead to severe mental retardation and substantial motor disabilities, and (27) visual and auditory impairments (63). Microcephaly might not always be observed, as a normal head circumference was observed in 20% of ZIKV congenital syndromes (64); screening for microcephaly at birth is thus not sufficient to detect congenital syndromes (65). Head growth deceleration has been reported in infants born with a normal head circumference, leading to the development of microcephaly after birth (66). The clinical presentation of ZIKV infection is similar in pregnant and non-pregnant women, and congenital infection is possible even in asymptomatic women (64).
# Availability of preventive, therapeutic and control measures, including licensed or pipeline vaccines

## Therapy in humans

There is no antiviral-specific treatment.

## Licensed or pipeline vaccines

As of 2022, no effective vaccine to prevent Zika infection is licensed and available (67). A wide variety of drug formulations are being studied; and among those being tested are live virus vaccines, inactivated vaccines, whole-virus vaccines, subunit vaccines, and messenger RNA (mRNA), DNA, protein, and vector-based formulations (67).

## Other prevention measures

In the absence of therapeutic strategies and licensed vaccines, efficient vector control plays a crucial role in ZIKV prevention (68). Integrated anti-virus control is required and should include: a) epidemiological surveillance; b) environmental management focusing on educative actions to eliminate potential mosquito breeding sites and reduce standing water sites; c) chemical control using repellents (mainly for travellers and pregnant women) and insecticides, respecting the vectors resistance; and d) biological control against eggs, larvae and mosquitoes (69-72). In certain countries such as Germany, Italy, and Spain, tools or apps have been provided to citizens for reporting mosquito presence and biting incidents. Current guidelines also recommend 6 months with protected intercourse for travellers returning from endemic areas (73).

## Disease-specific recommendations

WHO recommends that pregnant women avoid travelling to areas with ZIKV transmission, particularly during outbreaks, based on the increased risk of microcephaly and other severe congenital malformations. All residents of, and travellers to areas with ongoing or historical ZIKV transmission should be mindful of preventing mosquito bites and be able to make informed decisions on whether to practise protected intercourse, abstain from sex, or avoid/delay pregnancy. Pregnant women and their partners, and anyone planning pregnancy should be provided with comprehensive information about the risks associated with ZIKV infection, especially before travelling (17, 74).

Recommendations of the United States (US) Centres for Disease Control and Prevention (CDC) regarding preconception counselling are that men should use condoms for 3 months after returning from endemic areas (or after the last possible ZIKV exposure) and that women wait for two months before trying to conceive. These recommendations seem reasonable as the longest interval between infection and sexual male-to-female transmission ever reported is 44 days with a peak infectivity at 2 weeks. The risk of intrauterine transmission among ZIKV-infected women trying to conceive near the end of the recommended 8-week period is thought to be small as 95% have no detectable ZIKV RNA in serum after 6 weeks from disease onset. According to the US CDC, all pregnant women returning with possible ZIKV exposure should be tested, regardless of symptom status. Women living in areas of active virus circulation should avoid pregnancy, those
not living in endemic areas should be advised to avoid travelling in endemic countries, and all
pregnant women should avoid sexual contact with partners with the same exposure risks.

Travellers who visit areas endemic for *Aedes*-borne diseases (e.g. CHIKV, DENV, ZIKV) and reside in areas of mainland EU/EEA where *Ae. albopictus* and/or *Ae. aegypti* mosquitos are established and should continue to apply personal protective measures after their return for a period of about two weeks (17).

Medical practitioners in travel clinics should be aware of the current ZIKV epidemiology during their pre-travel individual risk assessments. Health professionals providing antenatal care, obstetricians and paediatricians should also be aware of current ZIKV epidemiology to identify and investigate pregnant women exposed to ZIKV during their pregnancy and monitor the neurological development of their children. Clinicians should also remain alert to the risk of ZIKV infection in travellers returning from areas with ongoing or past transmission and the risk to their sexual contacts in the EU/EEA.

Epidemiological situation at different spatial scales:
past and current trends

ZIKV circulated sporadically in Africa and Asia between 1964 and 2007 without major public health impact. The first outbreaks that significantly affected public health occurred in 2007, in Micronesia, and in 2013 in the Pacific islands (75, 76). In 2015, a major outbreak stroked South and Central America (77). To our knowledge, to date, ZIKV circulates in approximately 87 countries and territories of both tropical and subtropical regions (78). The increasing number of cases since 1964 might be associated with the expansion of urban populations, global travel and commerce, climate change, and a paucity of mosquito control programs. Following the same drivers, the low number of cases since 2020 might be associated with both underreporting and a reduction of global travel during the covid pandemic.

In link with the global epidemic of Zika and in particular the epidemic in South America, the highest number of cases in the EU/EEA countries were reported in 2016 (n=1,925; 20 countries). The majority of cases were travel-related (imported cases). In Europe, there was no autochthonous vector-borne transmission reported; however, there were 21 locally acquired cases via a sexual transmission (n=20) and vertical transmission (n=1). Over half of the cases were reported by France and most were linked with travel to the French overseas territories in the Caribbean (79). The number of cases reported by EU/EEA countries has been declining steadily since 2016 when the highest number of notifications was observed. In 2021, three EU/EEA countries reported seven human cases, the lowest number ever reported since the start of the EU/EEA surveillance in 2016 (17). Surveillance was mostly passive in reporting countries. The cases were reported by Spain (n=4), Germany (n=2) and Luxembourg (n=1). Among the cases reported in 2021, two were reported in January, two between May and June, and three from September to November. For 2021, all the cases were imported from outside the EU/EEA. The location of infection of the index case was known for six cases: two occurred in Cameroon, one in Benin, one in Sierra Leone, one in Cuba, and one in Thailand (17). The only autochthonous vector-borne transmission cases in the EU/EEA occurred in France, in 2019 (16).
## Sociological and demographical dimension affecting susceptibility and exposure, including gender

As the ZIKV has circulated for several decades with sporadic or silent transmission to human beings and no reported epidemics, it was surprising when it suddenly emerged as a major public health problem. Although the epidemic potential of the virus has changed, allowing epidemic transmission, it is unclear whether the virulence has changed. Because of the close genetic and epidemiological relationship between DENV and ZIKV, the same demographic, societal, and technological factors that drove the emergence and spread of the pandemic dengue virus probably also were factors in the emergence and spread of ZIKV (80). Economic growth in tropical developing countries was a major driver of unprecedented and unplanned urban growth, which provided the ideal ecological conditions for increased *Aedes* mosquito populations living in intimate contact with crowded human populations (80).

This, combined with ineffective mosquito control and modern transportation, provided the ideal mechanism to transport both mosquitoes and viruses around the world. As with DENV, the resulting increased transmission expanded the probability of genetic change in ZIKV and thus led to the emergence of viruses with greater epidemic potential and virulence. Therefore, globalisation facilitated the geographical spread of these new viral strains.

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

### Diagnosis

The ZIKV can be detected only in plasma or serum during acute illness. Compared with serum, urine was reported to increase the detection rate of viral RNA within the first week after symptom onset and expand the window of detection, as viral RNA was detectable up to 39 days after exposure (81). For Diagnosis of ZIKV infection, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and serum IgM antibodies detection are the common practices. In pregnant women infected with the virus, both RT-PCR, on serum and whole blood, and serology should be considered at any time (82); however, laboratory confirmation of foetal infection during pregnancy is challenging. Detection of ZIKV RNA in blood, urine, and amniotic fluid can be negative or transient, despite the proven presence in the foetus (83).

Conversely, the virus can be detected in pregnant mothers or amniotic fluid without foetal abnormalities. Sensitivity, specificity, and negative and positive predictive values of detection of ZIKV RNA in amniotic fluids are unknown, challenging maternal counselling. For infants with possible congenital ZIKV infection, RT-PCR should be performed within the first 2 days of birth on both serum and urine, and IgM ELISA (Enzyme-Linked Immunosorbent Assay) should be performed on serum. Molecular and serology diagnosis tests are commercially available.
# Infrastructure capacity to identify pathogens for each Member State

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

1. Probable case

a. Detection of ZIKV-specific IgM antibodies in a serum sample.

2. Confirmed case at least one of the following:

a. Detection of ZIKV nucleic acid in a clinical specimen;

b. Detection of ZIKV antigen in a clinical specimen;

c. Isolation of ZIKV from a clinical specimen;

d. Detection of ZIKV specific IgM antibodies in serum sample(s) and confirmation by neutralization test;

e. Seroconversion or a four-fold increase in the titre of Zika-specific antibodies in paired serum samples.

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

As for other *Aedes*-borne diseases, Zika distribution will be affected by climate change (52). The effect of climate change on El Niño was suggested has impacting ZIKV transmission (51). An assessment of the relationship between climate change, in particular the future temperature change and ZIKV transmission, showed that in the worst-case scenario, over 1.3 billion new people could face suitable transmission temperatures for ZIKV by 2050. Substantially increased ZIKV transmission temperature suitability was predicted in North America and Europe, where naïve populations might be particularly vulnerable. Mitigating climate change even to moderate emissions scenarios could significantly reduce the global expansion of climates suitable for ZIKV transmission (84).

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Roma, settembre 2024