# Astrovirus Diagnostics.

Philippe Pérot, Marc Lecuit, Marc Eloit

► To cite this version:

Philippe Pérot, Marc Lecuit, Marc Eloit. Astrovirus Diagnostics.. Viruses, 2017, 9 (1), 10.3390/v9010010. pasteur-01441063

HAL Id: pasteur-01441063

https://pasteur.hal.science/pasteur-01441063

Submitted on 19 Jan 2017

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# Astrovirus Diagnostics

Philippe Pérot<sup>1,2</sup>, Marc Lecuit<sup>1,3,4</sup> and Marc Eloit<sup>1,5,*</sup>

Academic Editor: Stacey Schultz-Cherry

Received: 22 November 2016; Accepted: 30 December 2016; Published: 12 January 2017

**Abstract:** Various methods exist to detect an astrovirus infection. Current methods include electron microscopy (EM), cell culture, immunoassays, polymerase chain reaction (PCR) and various other molecular approaches that can be applied in the context of diagnostic or in surveillance studies. With the advent of metagenomics, novel human astrovirus (HAstV) strains have been found in immunocompromised individuals in association with central nervous system (CNS) infections. This work reviews the past and current methods for astrovirus detection and their uses in both research laboratories and for medical diagnostic purposes.

**Keywords:** astrovirus; virology; detection methods; molecular diagnostics

## 1. Introduction

Astroviruses were first discovered in 2008 by electron microscopy (EM) examination of stool samples from children with diarrhea [1]. The virus name was given due to the star-shaped morphology of the virus, which is observed on the surface of some of the particles. Before the development of molecular techniques, EM was the only tool for laboratory diagnostics, as no cell line permissive for a broad range of strains was identified, precluding routine virus isolation. EM, and later polymerase chain reaction (PCR), increasingly demonstrated the role of astroviruses in diarrheal human disease in babies and infants (as well as in numerous animal species such as birds and mammals), and most of the population has demonstrated exposure to the virus, as is evidenced by antibody detection [2–7]. Recently, unbiased high throughput sequencing (HTS) has identified the unexpected role of astroviruses from a specific clade in human [8–12] and bovine [13] encephalitis. This paper summarizes the current tools available for the identification of astroviruses in diagnostic or research applications.

## 2. Virus Characteristics

Astroviruses are non-enveloped, positive sense, single-stranded RNA viruses, with solid capsid shell ~35 nm in diameter (~44 nm with spikes) [14], classified into two genera: mammalian viruses (*Mamastroviruses* (*MAstVs*), 19 species recognized by the International Committee for Taxonomy of Viruses (ICTV)) and avian viruses (*Avastroviruses* (*AAstVs*), three species recognized by ICTV). The taxonomy does not take into account the species of origin anymore. The genome is 6.8 kb to 7.9 kb in length and harbors a 5' untranslated region (UTR), followed by three open reading frames (ORFs), namely ORF1a, ORF1b, and ORF2, a 3' UTR and a polyA tail [15].

<footnote n="1">¹ Institut Pasteur, Biology of Infection Unit, Inserm U1117, Laboratory of Pathogen Discovery, 75015 Paris, France; philippe.perot@pasteur.fr (P.P.); marc.lecuit@pasteur.fr (M.L.)</footnote>

<footnote n="2">² Institut Pasteur, Centre d'innovation et de Recherche Technologique (Citech), 75015 Paris, France</footnote>

<footnote n="3">³ Paris Descartes University, Sorbonne Paris Cité, 75005, Paris, France</footnote>

<footnote n="4">⁴ Necker-Enfants Malades University Hospital, Division of Infectious Diseases and Tropical Medicine, 75015 Paris, France</footnote>

<footnote n="5">⁵ Ecole Nationale Vétérinaire d'Alfort, 94700 Maisons-Alfort, France</footnote>

<footnote>* Correspondence: marc.eloit@pasteur.fr; Tel.: +33-144-38-9216</footnote>
Human astroviruses (HAstVs) are found in four *MAstV* species (*MAstV* 1, 6, 8, 9), as summarized in Table 1. *MAstV* 1 includes the eight serotypes of classic HAstVs (HAstV 1–8), a common cause of viral gastroenteritis in children, targeted by usual diagnostic PCRs. *MAstV* 1 and *MAstV* 6 form a monophyletic group, together with astroviruses from cats, pigs, dogs, rabbits, California sea lions, and dolphins. The two other genotypes, *MAstV* 8 and *MAstV* 9, are closely related to astroviruses from mink, sheep, California sea lions, bats, cattle, pigs, and mice.

<table><caption>**Table 1.** Human astroviruses (HAstVs).</caption><thead><tr><th>Genus</th><th colspan="4">Mamastrovirus (MAstV)</th></tr><tr><th>Species</th><th>MAstV 1</th><th>MAstV 6</th><th>MAstV 8</th><th>MAstV 9</th></tr></thead><tbody><tr><th rowspan="8">Serotypes and strains</th><td>HAstV 1*</td><td>MLB 1</td><td>VA2/HMO-A</td><td>VA1/HMO-C</td></tr><tr><td>HAstV 2*</td><td>MLB 2</td><td>VA4</td><td>VA3/HMO-B</td></tr><tr><td>HAstV 3*</td><td>MLB 3</td><td>VA5</td><td></td></tr><tr><td>HAstV 4*</td><td></td><td>BF34</td><td></td></tr><tr><td>HAstV 5*</td><td></td><td></td><td></td></tr><tr><td>HAstV 6*</td><td></td><td></td><td></td></tr><tr><td>HAstV 7*</td><td></td><td></td><td></td></tr><tr><td>HAstV 8*</td><td></td><td></td><td></td></tr></tbody></table>

Within genotypes, strains can be grouped by serotypes, based on their antigenicity, although variability is still high, even within the same serotype. For this reason, subtypes are defined, which are well-documented within the eight serotypes of *MAstV* 1, and most likely also exist for the other species as well. Two species defined by prototypal MLB1 (*MAstV* 6) and VA1 (*MAstV* 9) strains have been described [16,17] and more recently associated to severe cases of encephalitis in immunocompromised patients [8–11,18]. High genetic variability (even within each genotype) and concerns regarding capabilities for cross-species transmission [19] are challenges for the definition of adequate diagnostic tools capable of identifying distant strains. Moreover, association between astrovirus infection and disease is still being investigated, as well as the interaction of astroviruses with other enteric viruses in diarrhea physiopathology, all of which suggests that diagnostic tools will need to continue to evolve in the near future.

### 3. Clinical Aspects of Astrovirus Infection

HAstVs are a classic cause of viral diarrhea in children, along with rotavirus, norovirus, sapovirus and adenovirus. Seroprevalence studies indicate that most children in Europe encounter astrovirus before the age of two [2]. Astrovirus-associated diarrhea is not reported in immunocompetent adults, as infection in childhood is considered to confer protective immunity. Additionally, humoral immunity is considered to play a major protective role, along with cellular adaptive immunity [20]. Therefore, immunosuppressed patients and the elderly can also develop astrovirus-associated diarrhea.

In non-immunocompromised individuals, after an incubation period of 4–5 days, an astrovirus infection will induce a mild disease, characterized by mild and short watery diarrhea for two to three days, followed by nausea, vomiting, and abdominal pain, which usually resolves spontaneously. These symptoms are most often milder than a rotavirus infection [21]. Recent seroprevalence studies have indicated that some infections can be asymptomatic as well [22]. As reported for rotavirus and norovirus, astrovirus has also been associated with intussusception in infants [23]. Although virological diagnosis of astrovirus-associated diarrhea is not routinely used in medical practice, it is sometimes used in epidemiological studies in the context of diarrheal outbreaks [24] and surveillance of diarrheal diseases [25,26].

An astrovirus infection in immunocompromised individuals may induce gastroenteritis, but it can also lead to severe and sometimes fatal systemic and central nervous system (CNS) infections, as seen in multiple cases of astrovirus-associated encephalitis and meningitis [8–11,18]. These

<footnote>*, classic human astroviruses.</footnote>
reports are associated with newly identified HAstVs that belong to novel species (MAstV 6 and 9). Studies are under way to assess the actual disease burden associated with these novel neurotropic astroviruses in humans. These novel astroviruses are enteric viruses, associated with diarrhea and fecal carriage, but their pathogenicity in the non-immunosuppressed host has not yet been precisely determined, although a case of meningitis in an apparently healthy adult has recently been reported [27]. Therefore, these newly-discovered viruses seem to share some clinical characteristics with enteroviruses, due to their association with diarrhea, but may also induce meningitis and encephalitis in the immunosuppressed patients. For this reason, their detection should now be part of the laboratory diagnostic work-up in patients, in particular those who are immunosuppressed and are diagnosed with meningitis or encephalitis of unknown cause.

In mammals, astroviruses have been reported in piglets, minks and dogs with preweaning diarrhea, but accurate diagnosis is complicated due to the prevalence of fecal shedding in healthy animals, which complicates the interpretation of the results [28]. Therefore, etiological diagnostic is not a routine practice. In mink presenting with the so called "shaking mink syndrome", and cattle with encephalitis, astroviruses can be tested in necropsy brain samples [29,30]. Additionally, astroviruses have been associated with severe avian diseases (i.e., chicken diarrhea, duck hepatitis, turkey enteritis, and avian nephritis), and diagnosis can be made in severely affected flocks by reverse transcription polymerase chain reaction (RT-PCR), using necropsy samples in specialized laboratories [15].

# 4. Methods for Virus Identification

## 4.1. Electron Microscopy (EM)

In 1975, the first observation by EM of 28–30 nm particles was reported in the stool of babies with gastroenteritis [31]. The star-shaped surface configuration of the viruses rapidly led the author to propose the name of "astrovirus" (derived from the Greek "astron" which means "star") [32] and, since then, this morphological characteristic has been widely used for the detection of astrovirus infection in both humans and animals [33–35]. Direct EM is complicated by the fact that only a minority of virions exhibit a complete star-shaped structure, and careful searching may be necessary to distinguish between, for example, astrovirus and calicivirus [36], which are similar in size. Sensitivity of EM is also dependent on elevated concentrations of particles, usually around 10⁷ per gram of stool [37]. The use of immune electron microscopy (IEM) techniques using specific antibodies or convalescent sera can improve the sensitivity of the detection [38] and help with the typing [39] or the detection of new viral agents [40]. Due to the limitations described above, the use of EM for the diagnosis of viral infections has been superseded by molecular methods, and therefore, it is rarely available or used in clinical laboratories anymore.

## 4.2. Virus Isolation

Astroviruses, like other enteric viruses, can be difficult to propagate in conventional cell cultures. The first propagation of HAstVs was made possible by Lee and Kurtz in human embryo kidney (HEK) cells through the use of serum medium supplemented in trypsin [41]. After six passages in HEK, the adapted virus has been established in the continuous rhesus monkey kidney cells (LLC-MK2) and in primary baboon kidney cells, but in the absence of cytopathic effect (CPE). A 15 amino acid (aa) deletion in the non-structural polyprotein 1A may be responsible for this adaptation [42]. Other cell lines, such as African green monkey kidney Vero cells (e.g., MA-104), were not permissive for the virus even after initial passages in HEK cells [41]. However, similar experiments based on virus adaptation in embryonic kidney cells in the presence of trypsin have enabled the propagation of bovine [43] and porcine [44] astroviruses.

A major advance was made with the ability to grow MAstV 1 in the colonic carcinoma cells (CaCo-2) directly from feces, without prior adaptation to cell culture [45]. In these conditions, a cytopathic effect is generally observed after 2–3 days of infection. A study assessing the ability
of laboratory strains of HAstVs 1–7 to replicate in various human and simian cells showed that propagation can be successful in many different cells lines, including CaCo-2 and MA-104 [46]. Additionally, although adenocarcinoma cell lines appear to be the most commonly used cells to grow wild-type HAstV today, propagation directly from stool specimens is also possible in human hepatoma cell line PLC/PRF/5 [47]. While virus isolation can serve as a useful tool to investigate astrovirus biology, it is still not an ideal diagnostic tool for detection of astrovirus in diagnostic laboratories, due to slow turnaround times and difficulty of isolation.

### 4.3. Immunodetection and Antigenic Typing

The ability to grow astroviruses has simplified the production of antisera in experimental animals, allowing the characterization of serotypes [48] and the development of a radioimmune assay for detection of anti-*MAstV* 1 (serotypes HAstV 1–8) antibodies [49]. Further measurement of neutralizing antibodies [50] and the production of astrovirus-specific monoclonal antibodies [51–53] have followed. In 1990, the first evaluation of an indirect enzyme immunoassay (EIA) that used both a monoclonal antibody directed toward the capsid of *MAstV* 1 for the capture, and a polyclonal antibody for the detection, was achieved in a cohort of patients with gastroenteritis, showing a sensitivity of 91% and a specificity of 96% when compared to IEM [54]. Initially based on a peroxidase-labeled goat antibody, the detection system has been adapted with biotin-avidin, demonstrating similar performances, and was proved to be useful for large epidemiological studies and routine screening of fecal samples [55]. Of note, Moe et al. also developed an RNA-probe hybridization and tested it in parallel to their rapid biotin-avidin EIA, but although the sensitivity of the probe assay was high, it did not detect more astrovirus-positive fecal specimens than EIA [55].

Many examples of the use of EIA for clinical purposes have been observed. For instance, studies on serotype identification and prevalence in the United Kingdom [39,56] and South America [57], and efforts in typing specimen collected from several continents [58], have contributed to a better understanding of astrovirus epidemiology. More recently, rapid immunochromatography tests detecting astroviruses and claiming good sensitivity and specificity have been commercialized by several companies, but studies evaluating their performances are currently limited [59]. Although EIA tests are much easier to implement than EM and proved to be as efficient [34], their use and development have been hampered by the advent of molecular diagnostic techniques.

### 4.4. Molecular Diagnostics

#### 4.4.1. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Reverse Transcription PCR (RT-qPCR)

Molecular approaches based on the amplification of viral genome or transcripts have dramatically improved the sensitivity of detection in comparison to EM, immunoassays or virus isolation, making substantial gains. With thresholds of detection as low as 10 to 100 genome copies per gram of stool [37,60], and the ability to develop type-specific detection systems, RT-PCR has now become a very common tool for the diagnosis of astrovirus infection in clinical laboratories. However, the design of amplification systems, in particular the intrinsic properties of the primers, are key, especially with regard to the amplification efficiencies and the ability to detect variant strains. For example, among the RT-PCR systems that have been developed for the detection of *MAstV* 1 (serotypes HAstV 1–8), some are targeting non-coding regions of the virus in a very sensitive and specific manner, while others are designed into conserved motives of the capsid, thereby allowing subsequent typing but with a risk of sub-optimal amplification efficiencies (for a complete review including a table of the most commonly used RT-PCR systems, see [61]).

Alternative to RT-PCR, nucleic acid sequence-based amplification (NASBA) has also shown a good concordance with RT-PCR-based methods for the detection of *MAstV* 1 (serotypes HAstV 1–8) [62]. After the discovery of distant HAstV strains, MLB [16,63] and VA1/HMO-C [17], additional
primers have been developed and used to describe new populations of viruses [64–67]. Beyond human astroviruses, many RT-PCR systems were also developed to detect astroviruses in wildlife [68,69], livestock [70,71] or pets [72,73]. Although consensus primers can detect a large number of astroviruses among both animal and human strains, there is not yet a universal pan-astrovirus RT-PCR system.

In parallel to the development of PCR primers, the application of real-time PCR (qPCR) in a diagnostic setting has improved the diagnosis of astrovirus infections by reducing the risk of false positives, allowing quantitation of viral loads and shortening the time to results (a positive or negative result is usually available within 24 h of specimen collection) [74–77]. qPCR can be done using a nucleic acid stain (typically SYBR green) followed by melting curve analysis, or by the use of a specifically designed hydrolysis probe coupled with a fluorophore (typically Taqman). One-step RT-qPCR methods have also been developed [78,79]. Further refinements have been proposed with an integrated cell culture/RT-qPCR assay that is able to detect low levels of astrovirus after an incubation of seven days or less [80], but this approach has remained essentially of interest for research purposes only. Of note, although such advances have brought the ability to detect precise quantification of viral loads, the interpretation of very low amounts of virus in relation to clinical symptoms, especially in asymptomatic individuals, is still not always easy [81].

### 4.4.2. Multiplex RT-PCR for Enteric Pathogens Panels

To meet the need for a rapid, efficient and cost-effective diagnosis, multiplex RT-PCR panels, including astroviruses and other gastrointestinal pathogens, have been developed over time. Early attempts using either end-point [82,83] or qPCR [84] proved to be as efficient as singleplex PCR for the detection in stool samples of HAstVs, noroviruses, adenoviruses, sapoviruses and enteroviruses. In the latter, the analysis of melting curves allowed the determination of dual-infection by the formation of dual peaks, while being at least 10× more sensitive than end-point PCR [84]. Since then, several multiplex assays relying on different formats of detection have been developed [85–93] and used for the diagnosis of astrovirus infection in humans [94–102] or animals [103–107]. Remarkably, among commercially-available solutions, the FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City, UT, USA) allows for the simultaneous detection of 22 different enteric pathogens directly from stool specimens, with a reported sensitivity and specificity of 100% and 99.9%, respectively, for the detection of *MAstV* 1 (but not *MAstV* 6 and 9), and a turnaround time of around one hour [108].

Although no systematic evaluation of the clinical benefit of the many existing panels and methods has yet been published, several studies have pointed out the advantages to streamline the diagnosis of presumptive infectious diarrhea with the use of a comprehensive multiplex PCR panel for the detection of known pathogens, and also for the detection of pathogens not requested or unable to be tested by conventional tests [109,110]. For example, the FilmArray Gastrointestinal Panel has been used to detect astrovirus infections among diarrheic patients who were initially tested negative for *Clostridium difficile* and/or rotavirus [111]. In another study, the Seeplex Diarrhoea ACE Detection (Seegene, Seoul, Korea) multiplex PCR assay was used in parallel to routine assays, to detect 11 cases of astrovirus infection among 245 stool samples from pediatric patients [112]. Other broad multiplex PCR tests, including the Luminex technology (Austin, TX, USA), have been reported to offer an unprecedented ability to diagnose gastrointestinal infections in immunocompromised patients, with assay performances comparable to the tests examined [113].

### 4.4.3. Medium to High Density Detection Systems: Nanofluidic PCR and Microarrays

With progress made in miniaturization and the development of nanofluidic systems, it has now become possible to run qPCR in parallel to nanoliter-volume chambers, thereby reducing the cost per assay. For example, a microfluidic qPCR system based on multiple singleplex TaqMan qPCR assays could quantitatively detect 13 viruses, including human astroviruses, with a sensitivity as low as two copies per microliters [114]. In another more recent study, a nanofluidic qPCR assay was used for the detection and the quantification of 19 enteric viruses and demonstrated a sensitivity lower than
the limit of detection of conventional RT-qPCR and digital RT-PCR by 1.6 log and 2.7 log, respectively, for the detection of human astroviruses [115]. In both studies, a preamplification step is needed to increase the amount of target molecules present in nanovolumes. Another technique is based on parallelization of specific probe/target nucleotide hybridization on microarrays and represents an alternative for the detection of several pathogens in a single assay.

The applicability of the microarray technology for the detection of enteric pathogens had been initially evaluated for the detection of astroviruses and noroviruses in a panel of archival stool samples, allowing in some cases the characterization of known genotypes, although a considerable number of the astroviruses remained untypable [116]. Two years later, a DNA oligonucleotide microarray that used specific short capture probes of 17-20 nucleotides was proposed for the detection of all eight serotypes of HAstVs [117]. Another research initiative, referred to as the Combimatrix custom microarray, broadened the scope to the detection of human astroviruses, noroviruses, adenoviruses and rotaviruses, with the use of both conserved and variable probe sequences, showing, in particular, the absence of cross-reactivity among the four viruses [118].

Finally, a more recent format of microarray was designed to detect about 100 viral species associated with gastrointestinal infections in vertebrates in order to expand the understanding of the etiology of the disease in humans and animals [119]. It is also interesting to note that, although microarrays are based on sequence homology with predefined known pathogens, the use of a Virochip allowed the identification of an astrovirus in a domestic rabbit with gastroenteritis after traditional diagnostic approaches, including virus isolation, failed to identify the pathogen [120]. Unfortunately, the somewhat complex and costly procedures of microarray experiments may limit their use in clinic.

#### 4.4.4. High Throughput Sequencing (HTS)

The advent of HTS has opened the way to metagenomics, which is the parallel sequencing and subsequent description of all nucleic acid molecules present in a sample. Specifically, it represents a group of disruptive technologies over PCR or other hypothesis-driven detection methods. With a combination of random amplification of microbial genomes or transcripts and appropriate downstream data mining, deep sequencing has the ability to provide more detailed taxonomic information than diagnostic PCRs, and may also be used for the discovery of new pathogens without any prior hypothesis [121]. As a consequence, over the last few years, the use of HTS in research laboratories has allowed a leap forward in the identification and characterization of astroviruses in several species including humans [28,120,122–135].

Although the primary purpose of many of these studies was not the diagnosis of astrovirus infection in symptomatic situations, the identification of partial or complete astrovirus genomes among complex polymicrobial flora provided valuable insight into viral diversity, pathogenesis, and emergence of astrovirus strains. Additionally, metagenomics applied to the diagnosis of diseases of unknown origin have also resulted in the discovery of neurotropic astroviruses in humans [8–12,18,27], cattle [136–141] and mink [29]. However, the use of HTS for routine microbiological diagnosis remains challenging due to the high associated workload and costs. While it is true that the overall cost of HTS experiments is lower compared to other methods, a significant budget per sample is still required, in particular when only a few samples have to be analyzed independently of cohorts. Overall, the ability to deliver results in a timely manner provides significant optimizations in laboratory organization and bioinformatic workflow. For these reasons, and although HTS is now standard use for pathogen discovery, its translation into medical and actionable diagnosis still remains in infancy, and is now initially used to target the most severe and life-threatening illnesses.

### 5. Conclusions

Our understanding of the implication of astrovirus infection has greatly benefited from the evolution of technologies, from initial morphological identification to the most recent advanced high throughput molecular techniques. While several methods for the detection of astroviruses are now
available, the predominant method for diagnosis in a clinical setting still remains RT-qPCR. This is due to its highly sensitive and specific nature, fast turn-around times, relatively low cost compared to more advanced molecular methods, and the ability to multiplex with other targets of interest. The main limitation of RT-qPCR, as evidenced by recent metagenomics studies, is that infection with rare or novel astroviruses will not be detected.

**Acknowledgments:** This review paper was not funded by specific grants.

**Author Contributions:** P.P. wrote most of the manuscript. M.L. contributed more specifically to Section 3 and M.E. to Sections 1 and 2. All authors contributed to the improvement and editing of the initial draft.

**Conflicts of Interest:** The authors declare no conflict of interest.

## References

1. Appleton, H.; Buckley, M.; Robertson, M.H.; Thom, B.T. A search for faecal viruses in new-born and other infants. *J. Hyg. (Lond.)* **1978**, *81*, 279-283. [CrossRef] [PubMed]

2. Kriston, S.; Willcocks, M.M.; Carter, M.J.; Cubitt, W.D. Seroprevalence of astrovirus types 1 and 6 in London, determined using recombinant virus antigen. *Epidemiol. Infect.* **1996**, *117*, 159-164. [CrossRef] [PubMed]

3. Koopmans, M.P.; Bijen, M.H.; Monroe, S.S.; Vinjé, J. Age-stratified seroprevalence of neutralizing antibodies to astrovirus types 1 to 7 in humans in The Netherlands. *Clin. Diagn. Lab. Immunol.* **1998**, *5*, 33-37. [PubMed]

4. Mitchell, D.K.; Matson, D.O.; Cubitt, W.D.; Jackson, L.J.; Willcocks, M.M.; Pickering, L.K.; Carter, M.J. Prevalence of antibodies to astrovirus types 1 and 3 in children and adolescents in Norfolk, Virginia. *Pediatr. Infect. Dis. J.* **1999**, *18*, 249-254. [CrossRef] [PubMed]

5. Kobayashi, S.; Kobayashi, M.; Araki, K.; Shinozaki, T.; Yanagawa, Y. [Antibody prevalence against astrovirus according to age groups]. *Kansenshogaku Zasshi* **1999**, *73*, 578-583. [CrossRef] [PubMed]

6. Holtz, L.R.; Bauer, I.K.; Jiang, H.; Belshe, R.; Freiden, P.; Schultz-Cherry, S.L.; Wang, D. Seroepidemiology of astrovirus MLB1. *Clin. Vaccine Immunol. CVI* **2014**, *21*, 908-911. [CrossRef] [PubMed]

7. Burbelo, P.D.; Ching, K.H.; Esper, F.; Iadarola, M.J.; Delwart, E.; Lipkin, W.I.; Kapoor, A. Serological studies confirm the novel astrovirus HMOAstV-C as a highly prevalent human infectious agent. *PLoS ONE* **2011**, *6*, e22576. [CrossRef] [PubMed]

8. Frémond, M.-L.; Pérot, P.; Muth, E.; Cros, G.; Dumarest, M.; Mahlaoui, N.; Seilhean, D.; Desguerre, I.; Hébert, C.; Corre-Catelin, N.; et al. Next-Generation Sequencing for Diagnosis and Tailored Therapy: A Case Report of Astrovirus-Associated Progressive Encephalitis. *J. Pediatr. Infect. Dis. Soc.* **2015**, *4*, e53-e57. [CrossRef] [PubMed]

9. Naccache, S.N.; Peggs, K.S.; Mattes, F.M.; Phadke, R.; Garson, J.A.; Grant, P.; Samayoa, E.; Federman, S.; Miller, S.; Lunn, M.P.; et al. Diagnosis of Neuroinvasive Astrovirus Infection in an Immunocompromised Adult With Encephalitis by Unbiased Next-Generation Sequencing. *Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am.* **2015**, *60*, 919-923. [CrossRef] [PubMed]

10. Quan, P.-L.; Wagner, T.A.; Briese, T.; Torgerson, T.R.; Hornig, M.; Tashmukhamedova, A.; Firth, C.; Palacios, G.; Baisre-De-Leon, A.; Paddock, C.D.; et al. Astrovirus Encephalitis in Boy with X-linked Agammaglobulinemia. *Emerg. Infect. Dis.* **2010**, *16*, 918-925. [CrossRef] [PubMed]

11. Brown, J.R.; Morfopoulou, S.;ubb, J.; Emmett, W.A.; Ip, W.; Shah, D.; Brooks, T.; Paine, S.M.L.; Anderson, G.; Virasami, A.; et al. Astrovirus VA1/HMO-C: An Increasingly Recognized Neurotropic Pathogen in Immunocompromised Patients. *Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am.* **2015**, *60*, 881-888. [CrossRef] [PubMed]

12. Lum, S.H.; Turner, A.; Guiver, M.; Bonney, D.; Martland, T.; Davies, E.; Newbould, M.; Brown, J.; Morfopoulou, S.; Breuer, J.; et al. An emerging opportunistic infection: Fatal astrovirus (VA1/HMO-C) encephalitis in a pediatric stem cell transplant recipient. *Transpl. Infect. Dis. Off. J. Transplant. Soc.* **2016**, *18*, 960-964. [CrossRef] [PubMed]

13. Selimovic-Hamza, S.; Bouzalas, I.G.; Vandevelde, M.; Oevermann, A.; Seuberlich, T. Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958-1976. *Front. Vet. Sci.* **2016**, *3*, 91. [CrossRef] [PubMed]
14. Dryden, K.A.; Tihova, M.; Nowotny, N.; Matsui, S.M.; Mendez, E.; Yeager, M. Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis E virus. *J. Mol. Biol.* **2012**, *422*, 650–658. [CrossRef] [PubMed]

15. De Benedictis, P.; Schultz-Cherry, S.; Burnham, A.; Cattoli, G. Astrovirus infections in humans and animals—Molecular biology, genetic diversity, and interspecies transmissions. *Infect. Genet. Evol.* **2011**, *11*, 1529–1544. [CrossRef] [PubMed]

16. Finkbeiner, S.R.; Kirkwood, C.D.; Wang, D. Complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea. *Virol. J.* **2008**, *5*, 117. [CrossRef] [PubMed]

17. Finkbeiner, S.R.; Li, Y.; Ruone, S.; Conrardy, C.; Gregoricus, N.; Toney, D.; Virgin, H.W.; Anderson, L.J.; Vinjé, J.; Wang, D.; et al. Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak of acute gastroenteritis. *J. Virol.* **2009**, *83*, 10836–10839. [CrossRef] [PubMed]

18. Sato, M.; Kuroda, M.; Kasai, M.; Matsui, H.; Fukuyama, T.; Katano, H.; Tanaka-Taya, K. Acute encephalopathy in an immunocompromised boy with astrovirus-MLB1 infection detected by next generation sequencing. *J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol.* **2016**, *78*, 66–70. [CrossRef] [PubMed]

19. Lukashov, V.V.; Goudsmit, J. Evolutionary relationships among Astroviridae. *J. Gen. Virol.* **2002**, *83*, 1397–1405. [CrossRef] [PubMed]

20. Koci, M.D. Immunity and resistance to astrovirus infection. *Viral Immunol.* **2005**, *18*, 11–16. [CrossRef] [PubMed]

21. Dennehy, P.H.; Nelson, S.M.; Spangenberger, S.; Noel, J.S.; Monroe, S.S.; Glass, R.I. A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children. *J. Infect. Dis.* **2001**, *184*, 10–15. [CrossRef] [PubMed]

22. Kurtz, J.B.; Lee, T.W.; Craig, J.W.; Reed, S.E. Astrovirus infection in volunteers. *J. Med. Virol.* **1979**, *3*, 221–230. [CrossRef] [PubMed]

23. Lee, Y.W.; Yang, S.I.; Kim, J.M.; Kim, J.Y. Clinical features and role of viral isolates from stool samples of intussusception in children. *Pediatr. Gastroenterol. Hepatol. Nutr.* **2013**, *16*, 162–170. [CrossRef]

24. Afrad, M.H.; Karmakar, P.C.; Das, S.K.; Matthijnssens, J.; Ahmed, F.; Nahar, S.; Faruque, A.S.G.; Rahman, M.Z.; Rahman, M.; Azim, T. Epidemiology and genetic diversity of human astrovirus infection among hospitalized patients with acute diarrhea in Bangladesh from 2010 to 2012. *J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol.* **2013**, *58*, 612–618. [CrossRef] [PubMed]

25. Guan, H.; Zhang, J.; Xiao, Y.; Sha, D.; Ling, X.; Kan, B. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance. *Front. Microbiol.* **2016**, *7*, 386. [CrossRef] [PubMed]

26. Zhao, J.Y.; Shen, X.J.; Zhang, B.F.; Wang, Z.Q.; Xia, S.L.; Huang, X.Y.; Xu, B.L. [Surveillance for viral diarrhea in sentinel hospitals in Henan province, 2013–2015]. *Zhonghua Liu Xing Bing Xue Za Zhi Zhonghua Liuxingbingxue Zazhi* **2016**, *37*, 1392–1396. [PubMed]

27. Cordey, S.; Vu, D.-L.; Schibler, M.; L'Huillier, A.G.; Brito, F.; Docquier, M.; Posfay-Barbe, K.M.; Petty, T.J.; Turin, L.; Zdobnov, E.M.; et al. Astrovirus MLB2, a New Gastroenteric Virus Associated with Meningitis and Disseminated Infection. *Emerg. Infect. Dis.* **2016**, *22*, 846–853. [CrossRef] [PubMed]

28. Shan, T.; Li, L.; Simmonds, P.; Wang, C.; Moeser, A.; Delwart, E. The fecal virome of pigs on a high-density farm. *J. Virol.* **2011**, *85*, 11697–11708. [CrossRef] [PubMed]

29. Blomström, A.-L.; Widén, F.; Hammer, A.-S.; Belák, S.; Berg, M. Detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics. *J. Clin. Microbiol.* **2010**, *48*, 4392–4396. [CrossRef] [PubMed]

30. Boujon, C.L.; Selimovic-Hamza, S.; Bouzalas, I.; Seuberlich, T. Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus. *J. Virol. Methods* **2017**, *239*, 26–33. [CrossRef] [PubMed]

31. Appleton, H.; Higgins, P.G. Viruses and gastroenteritis in infants. *Lancet* **1975**, *305*, 1297. [CrossRef]

32. Madeley, C.R.; Cosgrove, B.P. 28 nm Particles in faeces in infantile gastroenteritis. *Lancet* **1975**, *306*, 451–452. [CrossRef]

33. Ashley, C.R.; Caul, E.O.; Paver, W.K. Astrovirus-associated gastroenteritis in children. *J. Clin. Pathol.* **1978**, *31*, 939–943. [CrossRef] [PubMed]
34. Cubitt, W.D.; Mitchell, D.K.; Carter, M.J.; Willcocks, M.M.; Holzel, H. Application of electronmicroscopy, enzyme immunoassay, and RT-PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit. *J. Med. Virol.* **1999**, *57*, 313–321. [CrossRef]

35. McNulty, M.S.; Curran, W.L.; McFerran, J.B. Detection of astroviruses in turkey faeces by direct electron microscopy. *Vet. Rec.* **1980**, *106*, 561. [CrossRef] [PubMed]

36. Madeley, C.R. Comparison of the features of astroviruses and caliciviruses seen in samples of feces by electron microscopy. *J. Infect. Dis.* **1979**, *139*, 519–523. [CrossRef] [PubMed]

37. Glass, R.I.; Noel, J.; Mitchell, D.; Herrmann, J.E.; Blacklow, N.R.; Pickering, L.K.; Dennehy, P.; Ruiz-Palacios, G.; de Guerrero, M.L.; Monroe, S.S. The changing epidemiology of astrovirus-associated gastroenteritis: a review. *Arch. Virol. Suppl.* **1996**, *12*, 287–300. [PubMed]

38. Berthiaume, L.; Alain, R.; McLaughlin, B.; Payment, P.; Trépanier, P. Rapid detection of human viruses in faeces by a simple and routine immune electron microscopy technique. *J. Gen. Virol.* **1981**, *55*, 223–227. [CrossRef] [PubMed]

39. Noel, J.; Cubitt, D. Identification of astrovirus serotypes from children treated at the Hospitals for Sick Children, London 1981–93. *Epidemiol. Infect.* **1994**, *113*, 153–159. [CrossRef] [PubMed]

40. Lavazza, A.; Tittarelli, C.; Cerioli, M. The use of convalescent sera in immune-electron microscopy to detect non-suspected/new viral agents. *Viruses* **2015**, *7*, 2683–2703. [CrossRef] [PubMed]

41. Lee, T.W.; Kurtz, J.B. Serial propagation of astrovirus in tissue culture with the aid of trypsin. *J. Gen. Virol.* **1981**, *57*, 421–424. [CrossRef] [PubMed]

42. Willcocks, M.M.; Ashton, N.; Kurtz, J.B.; Cubitt, W.D.; Carter, M.J. Cell culture adaptation of astrovirus involves a deletion. *J. Virol.* **1994**, *68*, 6057–6058. [PubMed]

43. Aroonprasert, D.; Fagerland, J.A.; Kelso, N.E.; Zheng, S.; Woode, G.N. Cultivation and partial characterization of bovine astrovirus. *Vet. Microbiol.* **1989**, *19*, 113–125. [CrossRef]

44. Shimizu, M.; Shirai, J.; Narita, M.; Yamane, T. Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney. *J. Clin. Microbiol.* **1990**, *28*, 201–206. [PubMed]

45. Willcocks, M.M.; Carter, M.J.; Laidler, F.R.; Madeley, C.R. Growth and characterisation of human faecal astrovirus in a continuous cell line. *Arch. Virol.* **1990**, *113*, 73–81. [CrossRef] [PubMed]

46. Brinker, J.P.; Blacklow, N.R.; Herrmann, J.E. Human astrovirus isolation and propagation in multiple cell lines. *Arch. Virol.* **2000**, *145*, 1847–1856. [CrossRef] [PubMed]

47. Taylor, M.B.; Grabow, W.O.; Cubitt, W.D. Propagation of human astrovirus in the PLC/PRF/5 hepatoma cell line. *J. Virol. Methods* **1997**, *67*, 13–18. [CrossRef]

48. Lee, T.W.; Kurtz, J.B. Human astrovirus serotypes. *J. Hyg. (Lond.)* **1982**, *89*, 539–540. [CrossRef] [PubMed]

49. Wilson, S.A.; Cubitt, W.D. The development and evaluation of radioimmune assays for the detection of immune globulins M and G against astrovirus. *J. Virol. Methods* **1988**, *19*, 151–159. [CrossRef]

50. Hudson, R.W.; Herrmann, J.E.; Blacklow, N.R. Plaque quantitation and virus neutralization assays for human astroviruses. *Arch. Virol.* **1989**, *108*, 33–38. [CrossRef] [PubMed]

51. Herrmann, J.E.; Hudson, R.W.; Perron-Henry, D.M.; Kurtz, J.B.; Blacklow, N.R. Antigenic characterization of cell-cultivated astrovirus serotypes and development of astrovirus-specific monoclonal antibodies. *J. Infect. Dis.* **1988**, *158*, 182–185. [CrossRef] [PubMed]

52. Sanchez-Fauquier, A.; Carrascosa, A.L.; Carrascosa, J.L.; Otero, A.; Glass, R.I.; Lopez, J.A.; San Martin, C.; Melero, J.A. Characterization of a human astrovirus serotype 2 structural protein (VP26) that contains an epitope involved in virus neutralization. *Virology* **1994**, *201*, 312–320. [CrossRef] [PubMed]

53. Bass, D.M.; Upadhyayula, U. Characterization of human serotype 1 astrovirus-neutralizing epitopes. *J. Virol.* **1997**, *71*, 8666–8671. [PubMed]

54. Herrmann, J.E.; Nowak, N.A.; Perron-Henry, D.M.; Hudson, R.W.; Cubitt, W.D.; Blacklow, N.R. Diagnosis of Astrovirus Gastroenteritis by Antigen Detection with Monoclonal Antibodies. *J. Infect. Dis.* **1990**, *161*, 226–229. [CrossRef] [PubMed]

55. Moe, C.L.; Allen, J.R.; Monroe, S.S.; Gary, H.E.; Humphrey, C.D.; Herrmann, J.E.; Blacklow, N.R.; Carcamo, C.; Koch, M.; Kim, K.H. Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe. *J. Clin. Microbiol.* **1991**, *29*, 2390–2395.

56. Lee, T.W.; Kurtz, J.B. Prevalence of human astrovirus serotypes in the Oxford region 1976–92, with evidence for two new serotypes. *Epidemiol. Infect.* **1994**, *112*, 187–193. [CrossRef] [PubMed]
57. Medina, S.M.; Gutierrez, M.F.; Liprandi, F.; Ludert, J.E. Identification and Type Distribution of Astroviruses among Children with Gastroenteritis in Colombia and Venezuela. *J. Clin. Microbiol.* **2000**, *38*, 3481–3483.
[PubMed]

58. Noel, J.S.; Lee, T.W.; Kurtz, J.B.; Glass, R.I.; Monroe, S.S. Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing. *J. Clin. Microbiol.* **1995**, *33*, 797–801. [PubMed]

59. Khamrin, P.; Dey, S.K.; Chan-it, W.; Thongprachum, A.; Satou, K.; Okitsu, S.; Maneekarn, N.; Ushijima, H. Evaluation of a Rapid Immunochromatography Strip Test for Detection of Astrovirus in Stool Specimens. *J. Trop. Pediatr.* **2010**, *56*, 129–131. [CrossRef] [PubMed]

60. Bosch, A.; Pintó, R.M.; Guix, S. Human astroviruses. *Clin. Microbiol. Rev.* **2014**, *27*, 1048–1074. [CrossRef] [PubMed]

61. Guix, S.; Bosch, A.; Pintó, R.M. Human astrovirus diagnosis and typing: Current and future prospects. *Lett. Appl. Microbiol.* **2005**, *41*, 103–105. [CrossRef] [PubMed]

62. Tai, J.H.; Ewert, M.S.; Belliot, G.; Glass, R.I.; Monroe, S.S. Development of a rapid method using nucleic acid sequence-based amplification for the detection of astrovirus. *J. Virol. Methods* **2003**, *110*, 119–127. [CrossRef]

63. Jiang, H.; Holtz, L.R.; Bauer, I.; Franz, C.J.; Zhao, G.; Bodhidatta, L.; Shrestha, S.K.; Kang, G.; Wang, D. Comparison of novel MLB-clade, VA-clade and classic human astroviruses highlights constrained evolution of the classic human astrovirus nonstructural genes. *Virology* **2013**, *436*, 8–14. [CrossRef] [PubMed]

64. Ahmed, S.F.; Sebeny, P.J.; Klena, J.D.; Pimentel, G.; Mansour, A.; Naguib, A.M.; Bruton, J.; Young, S.Y.N.; Holtz, L.R.; Wang, D. Novel astroviruses in children, Egypt. *Emerg. Infect. Dis.* **2011**, *17*, 2391–2393.
[CrossRef] [PubMed]

65. Finkbeiner, S.R.; Le, B.M.; Holtz, L.R.; Storch, G.A.; Wang, D. Detection of newly described astrovirus MLB1 in stool samples from children. *Emerg. Infect. Dis.* **2009**, *15*, 441–444. [CrossRef] [PubMed]

66. Kapoor, A.; Li, L.; Victoria, J.; Oderinde, B.; Mason, C.; Pandey, P.; Zaidi, S.Z.; Delwart, E. Multiple novel astrovirus species in human stool. *J. Gen. Virol.* **2009**, *90*, 2965–2972. [CrossRef] [PubMed]

67. Smits, S.L.; van Leeuwen, M.; van der Eijk, A.A.; Fraaij, P.L.A.; Escher, J.C.; Simon, J.H.; Osterhaus, A.D.M.E. Human astrovirus infection in a patient with new-onset celiac disease. *J. Clin. Microbiol.* **2010**, *48*, 3416–3418.
[CrossRef] [PubMed]

68. Chu, D.K.W.; Poon, L.L.M.; Guan, Y.; Peiris, J.S.M. Novel astroviruses in insectivorous bats. *J. Virol.* **2008**, *82*, 9107–9114. [CrossRef] [PubMed]

69. Rivera, R.; Nollens, H.H.; Venn-Watson, S.; Gulland, F.M.D.; Wellehan, J.F.X. Characterization of phylogenetically diverse astroviruses of marine mammals. *J. Gen. Virol.* **2010**, *91*, 166–173. [CrossRef] [PubMed]

70. Sharp, C.P.; Gregory, W.F.; Mason, C.; de Bronsvoort, B.M.C.; Beard, P.M. High prevalence and diversity of bovine astroviruses in the faeces of healthy and diarrhoeic calves in South West Scotland. *Vet. Microbiol.* **2015**, *178*, 70–76. [CrossRef] [PubMed]

71. Xiao, C.-T.; Giménez-Lirola, L.G.; Gerber, P.F.; Jiang, Y.-H.; Halbur, P.G.; Opriessnig, T. Identification and characterization of novel porcine astroviruses (PAstVs) with high prevalence and frequent co-infection of individual pigs with multiple PAstV types. *J. Gen. Virol.* **2013**, *94*, 570–582. [CrossRef] [PubMed]

72. Castro, T.X.; Cubel Garcia, R.C.N.; Costa, E.M.; Leal, R.M.; da Xavier, M.P.T.; Leite, J.P.G. Molecular characterisation of calicivirus and astrovirus in puppies with enteritis. *Vet. Rec.* **2013**, *172*, 557. [CrossRef] [PubMed]

73. Moschidou, P.; Martella, V.; Lorusso, E.; Desario, C.; Pinto, P.; Losurdo, M.; Catella, C.; Parisi, A.; Bányai, K.; Buonavoglia, C. Mixed infection by Feline astrovirus and Feline panleukopenia virus in a domestic cat with gastroenteritis and panleukopenia. *J. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc.* **2011**, *23*, 581–584. [CrossRef] [PubMed]

74. Dai, Y.; Xu, Q.; Wu, X.; Hu, G.; Tang, Y.; Li, J.; Chen, Q.; Nie, J. Development of real-time and nested RT-PCR to detect astrovirus and one-year survey of astrovirus in Jiangmen City, China. *Arch. Virol.* **2010**, *155*, 977–982. [CrossRef] [PubMed]

75. Le Cann, P.; Ranarijaona, S.; Monpoeho, S.; Le Guyader, F.; Ferré, V. Quantification of human astroviruses in sewage using real-time RT-PCR. *Res. Microbiol.* **2004**, *155*, 11–15. [CrossRef] [PubMed]

76. Logan, C.; O'Leary, J.J.; O'Sullivan, N. Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis. *J. Virol. Methods* **2007**, *146*, 36–44. [CrossRef] [PubMed]
77. Zhang, Z.; Mitchell, D.K.; Afflerbach, C.; Jakab, F.; Walter, J.; Zhang, Y.-J.; Staat, M.A.; Azimi, P.; Matson, D.O. Quantitation of human astrovirus by real-time reverse-transcription-polymerase chain reaction to examine correlation with clinical illness. *J. Virol. Methods* **2006**, *134*, 190–196. [CrossRef] [PubMed]

78. Royuela, E.; Negredo, A.; Sánchez-Fauquier, A. Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus. *J. Virol. Methods* **2006**, *133*, 14–19. [CrossRef] [PubMed]

79. Wei, H.; Zeng, J.; Deng, C.; Zheng, C.; Zhang, X.; Ma, D.; Yi, Y. A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus. *J. Virol. Methods* **2013**, *188*, 126–131. [CrossRef] [PubMed]

80. Grimm, A.C.; Cashdollar, J.L.; Williams, F.P.; Fout, G.S. Development of an astrovirus RT-PCR detection assay for use with conventional, real-time, and integrated cell culture/RT-PCR. *Can. J. Microbiol.* **2004**, *50*, 269–278. [CrossRef] [PubMed]

81. Corcoran, M.S.; van Well, G.T.J.; van Loo, I.H.M. Diagnosis of viral gastroenteritis in children: interpretation of real-time PCR results and relation to clinical symptoms. *Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol.* **2014**, *33*, 1663–1673. [CrossRef] [PubMed]

82. Rohayem, J.; Berger, S.; Juretzek, T.; Herchenröder, O.; Mogel, M.; Poppe, M.; Henker, J.; Rethwilm, A. A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples. *J. Virol. Methods* **2004**, *118*, 49–59. [CrossRef] [PubMed]

83. Yan, H.; Nguyen, T.A.; Phan, T.G.; Okitsu, S.; Li, Y.; Ushijima, H. Development of RT-multiplex PCR assay for detection of adenovirus and group A and C rotaviruses in diarrheal fecal specimens from children in China. *Kansenshōgaku Zasshi J. Jpn. Assoc. Infect. Dis.* **2004**, *78*, 699–709. [CrossRef]

84. Beuret, C. Simultaneous detection of enteric viruses by multiplex real-time RT-PCR. *J. Virol. Methods* **2004**, *115*, 1–8. [CrossRef] [PubMed]

85. Feeney, S.A.; Armstrong, V.J.; Mitchell, S.J.; Crawford, L.; McCaughey, C.; Coyle, P.V. Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis. *J. Med. Virol.* **2011**, *83*, 1650–1656. [CrossRef] [PubMed]

86. Khamrin, P.; Okame, M.; Thongprachum, A.; Nantachit, N.; Nishimura, S.; Okitsu, S.; Maneekarn, N.; Ushijima, H. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. *J. Virol. Methods* **2011**, *173*, 390–393. [CrossRef] [PubMed]

87. Liu, J.; Gratz, J.; Amour, C.; Kibiki, G.; Becker, S.; Janaki, L.; Verweij, J.J.; Taniuchi, M.; Sobuz, S.U.; Haque, R.; et al. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. *J. Clin. Microbiol.* **2013**, *51*, 472–480. [CrossRef] [PubMed]

88. Liu, Y.; Xu, Z.; Zhang, Q.; Jin, M.; Yu, J.; Li, J.; Liu, N.; Cui, S.; Kong, X.; Wang, H.; et al. Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex-based assay. *J. Clin. Microbiol.* **2012**, *50*, 2384–2389. [CrossRef] [PubMed]

89. Mo, Q.-H.; Wang, H.-B.; Dai, H.-R.; Lin, J.-C.; Tan, H.; Wang, Q.; Yang, Z. Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification. *Arch. Virol.* **2015**, *160*, 719–725. [CrossRef] [PubMed]

90. Mori, K.; Hayashi, Y.; Akiba, T.; Nagano, M.; Tanaka, T.; Hosaka, M.; Nakama, A.; Kai, A.; Saito, K.; Shirasawa, H. Multiplex real-time PCR assays for the detection of group C rotavirus, astrovirus, and Subgenus F adenovirus in stool specimens. *J. Virol. Methods* **2013**, *191*, 141–147. [CrossRef] [PubMed]

91. Ohrmalm, C.; Eriksson, R.; Jobs, M.; Simonson, M.; Strømme, M.; Bondeson, K.; Herrmann, B.; Melhus, A.; Blomberg, J. Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes. *J. Clin. Microbiol.* **2012**, *50*, 3208–3215. [CrossRef] [PubMed]

92. Wang, J.; Xu, Z.; Niu, P.; Zhang, C.; Zhang, J.; Guan, L.; Kan, B.; Duan, Z.; Ma, X. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea. *BioMed Res. Int.* **2014**, *2014*, 648520. [CrossRef] [PubMed]

93. Zhang, C.; Niu, P.; Hong, Y.; Wang, J.; Zhang, J.; Ma, X. A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria. *J. Microbiol. Methods* **2015**, *118*, 93–98. [CrossRef] [PubMed]

94. Chau, M.L.; Hartantyo, S.H.P.; Yap, M.; Kang, J.S.L.; Aung, K.T.; Gutiérrez, R.A.; Ng, L.C.; Tam, C.C.; Barkham, T. Diarrheagenic pathogens in adults attending a hospital in Singapore. *BMC Infect. Dis.* **2016**, *16*, 32. [CrossRef] [PubMed]
95. Jiang, Y.; Fang, L.; Shi, X.; Zhang, H.; Li, Y.; Lin, Y.; Qiu, Y.; Chen, Q.; Li, H.; Zhou, L.; et al. Simultaneous detection of five enteric viruses associated with gastroenteritis by use of a PCR assay: A single real-time multiplex reaction and its clinical application. *J. Clin. Microbiol.* **2014**, *52*, 1266–1268. [CrossRef] [PubMed]

96. Nicholson, M.R.; van Horn, G.T.; Tang, Y.-W.; Vinjé, J.; Payne, D.C.; Edwards, K.M.; Chappell, J.D. Using Multiplex Molecular Testing to Determine the Etiology of Acute Gastroenteritis in Children. *J. Pediatr.* **2016**, *176*, 50–56.e2. [CrossRef] [PubMed]

97. Phan, T.G.; Nguyen, T.A.; Yan, H.; Yagyu, F.; Kozlov, V.; Kozlov, A.; Okitsu, S.; Müller, W.E.G.; Ushuijma, H. Development of a novel protocol for RT-multiplex PCR to detect diarrheal viruses among infants and children with acute gastroenteritis in Eastern Russia. *Clin. Lab.* **2005**, *51*, 429–435. [PubMed]

98. Saikruang, W.; Khamrin, P.; Suantai, B.; Okitsu, S.; Hayakawa, S.; Ushijima, H.; Maneekarn, N. Detection of diarrheal viruses circulating in adult patients in Thailand. *Arch. Virol.* **2014**, *159*, 3371–3375. [CrossRef] [PubMed]

99. Shigemoto, N.; Fukuda, S.; Tanizawa, Y.; Kuwayama, M.; Ohara, S.; Seno, M. Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers. *Microbiol. Immunol.* **2011**, *55*, 369–372. [CrossRef] [PubMed]

100. Siah, S.P.; Merif, J.; Kaur, K.; Nair, J.; Huntington, P.G.; Karagiannis, T.; Stark, D.; Rawlinson, W.; Olma, T.; Thomas, L.; et al. Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels. *Pathology (Philadelphia)* **2014**, *46*, 53–59. [CrossRef] [PubMed]

101. Tolentino-Ruiz, R.; Montoya-Varela, D.; García-Espitia, M.; Salas-Benito, M.; Gutiérrez-Escolano, A.; Gómez-García, C.; Figueroa-Arredondo, P.; Salas-Benito, J.; de Nova-Ocampo, M. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children. *Curr. Microbiol.* **2012**, *65*, 361–368. [CrossRef] [PubMed]

102. Van Maarseveen, N.M.; Wessels, E.; de Brouwer, C.S.; Vossen, A.C.T.M.; Claas, E.C.J. Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays. *J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol.* **2010**, *49*, 205–210. [CrossRef] [PubMed]

103. Chen, L.; Ma, M.; Zhang, R.; Xu, Q.; Si, X.; Wang, Y.; Xie, Z.; Jiang, S. Simultaneous detection of duck hepatitis A virus types 1 and 3, and of duck astrovirus type 1, by multiplex RT-PCR. *Virol. Sin.* **2014**, *29*, 196–198. [CrossRef] [PubMed]

104. Day, J.M.; Spackman, E.; Pantin-Jackwood, M. A multiplex RT-PCR test for the differential identification of turkey astrovirus type 1, turkey astrovirus type 2, chicken astrovirus, avian nephritis virus, and avian rotavirus. *Avian Dis.* **2007**, *51*, 681–684. [CrossRef]

105. Jindal, N.; Chander, Y.; Patnayak, D.P.; Mor, S.K.; Ziegler, A.F.; Goyal, S.M. A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys. *Avian Dis.* **2012**, *56*, 592–596. [CrossRef] [PubMed]

106. Sellers, H.S.; Koci, M.D.; Linnemann, E.; Kelley, L.A.; Schultz-Cherry, S. Development of a multiplex reverse transcription-polymerase chain reaction diagnostic test specific for turkey astrovirus and coronavirus. *Avian Dis.* **2004**, *48*, 531–539. [CrossRef] [PubMed]

107. Spackman, E.; Kapczynski, D.; Sellers, H. Multiplex real-time reverse transcription-polymerase chain reaction for the detection of three viruses associated with poult enteritis complex: turkey astrovirus, turkey coronavirus, and turkey reovirus. *Avian Dis.* **2005**, *49*, 86–91. [CrossRef] [PubMed]

108. Buss, S.N.; Leber, A.; Chapin, K.; Fey, P.D.; Bankowski, M.J.; Jones, M.K.; Rogatcheva, M.; Kanack, K.J.; Bourzac, K.M. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. *J. Clin. Microbiol.* **2015**, *53*, 915–925. [CrossRef] [PubMed]

109. McAuliffe, G.N.; Anderson, T.P.; Stevens, M.; Adams, J.; Coleman, R.; Mahagamasekera, P.; Young, S.; Henderson, T.; Hofmann, M.; Jennings, L.C.; et al. Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples. *J. Infect.* **2013**, *67*, 122–129. [CrossRef] [PubMed]

110. Wolffs, P.F.G.; Bruggeman, C.A.; van Well, G.T.J.; van Loo, I.H.M. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. *J. Clin. Microbiol.* **2011**, *49*, 1926–1931. [CrossRef] [PubMed]

111. Rand, K.H.; Tremblay, E.E.; Hoidal, M.; Fisher, L.B.; Grau, K.R.; Karst, S.M. Multiplex gastrointestinal pathogen panels: implications for infection control. *Diagn. Microbiol. Infect. Dis.* **2015**, *82*, 154–157. [CrossRef] [PubMed]
112. Onori, M.; Coltella, L.; Mancinelli, L.; Argentieri, M.; Menichella, D.; Villani, A.; Grandin, A.; Valentini, D.; Raponi, M.; Russo, C. Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients. *Diagn. Microbiol. Infect. Dis.* **2014**, *79*, 149–154.
[CrossRef] [PubMed]

113. Gu, Z.; Zhu, H.; Rodriguez, A.; Mhaissen, M.; Schultz-Cherry, S.; Adderson, E.; Hayden, R.T. Comparative Evaluation of Broad-Panel PCR Assays for the Detection of Gastrointestinal Pathogens in Pediatric Oncology Patients. *J. Mol. Diagn.* **2015**, *17*, 715–721.
[CrossRef] [PubMed]

114. Ishii, S.; Kitamura, G.; Segawa, T.; Kobayashi, A.; Miura, T.; Sano, D.; Okabe, S. Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples. *Appl. Environ. Microbiol.* **2014**, *80*, 7505–7511.
[CrossRef] [PubMed]

115. Coudray-Meunier, C.; Fraisse, A.; Martin-Latil, S.; Delannoy, S.; Fach, P.; Perelle, S. A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System. *PLoS ONE* **2016**, *11*, e0147832.
[CrossRef] [PubMed]

116. Jääskeläinen, A.J.; Maunula, L. Applicability of microarray technique for the detection of noro- and astroviruses. *J. Virol. Methods* **2006**, *136*, 210–216.
[CrossRef] [PubMed]

117. Brown, D.W.; Gunning, K.B.; Henry, D.M.; Awdeh, Z.L.; Brinker, J.P.; Tzipori, S.; Herrmann, J.E. A DNA oligonucleotide microarray for detecting human astrovirus serotypes. *J. Virol. Methods* **2008**, *147*, 86–92.
[CrossRef] [PubMed]

118. Kim, J.-M.; Kim, S.Y.; Park, Y.B.; Kim, H.J.; Min, B.S.; Cho, J.-C.; Yang, J.M.; Cho, Y.-H.; Ko, G. Simultaneous detection of major enteric viruses using a combimatrix microarray. *J. Microbiol. Seoul Korea* **2012**, *50*, 970–977.
[CrossRef] [PubMed]

119. Martínez, M.A.; de Soto-Del Río, M.L.D.; Gutiérrez, R.M.; Chiu, C.Y.; Greninger, A.L.; Contreras, J.F.; López, S.; Arias, C.F.; Isa, P. DNA microarray for detection of gastrointestinal viruses. *J. Clin. Microbiol.* **2015**, *53*, 136–145.
[CrossRef] [PubMed]

120. Stenglein, M.D.; Velazquez, E.; Greenacre, C.; Wilkes, R.P.; Ruby, J.G.; Lankton, J.S.; Ganem, D.; Kennedy, M.A.; DeRisi, J.L. Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis. *Virol. J.* **2012**, *9*, 216.
[CrossRef] [PubMed]

121. Lecuit, M.; Eloit, M. The potential of whole genome NGS for infectious disease diagnosis. *Expert Rev. Mol. Diagn.* **2015**, *15*, 1517–1519.
[CrossRef] [PubMed]

122. Amimo, J.O.; El Zowalaty, M.E.; Githae, D.; Wamalwa, M.; Djikeng, A.; Nasrallah, G.K. Metagenomic analysis demonstrates the diversity of the fecal virome in asymptomatic pigs in East Africa. *Arch. Virol.* **2016**, *161*, 887–897.
[CrossRef] [PubMed]

123. Chen, X.; Zhang, B.; Yue, H.; Wang, Y.; Zhou, F.; Zhang, Q.; Tang, C. A novel astrovirus species in the gut of yaks with diarrhoea in the Qinghai–Tibetan Plateau, 2013. *J. Gen. Virol.* **2015**, *96*, 3672–3680.
[CrossRef] [PubMed]

124. Li, L.; Shan, T.; Wang, C.; Côté, C.; Kolman, J.; Onions, D.; Gulland, F.M.D.; Delwart, E. The fecal viral flora of California sea lions. *J. Virol.* **2011**, *85*, 9909–9917.
[CrossRef] [PubMed]

125. Nagai, M.; Omatsu, T.; Aoki, H.; Otomaru, K.; Uto, T.; Koizumi, M.; Minami-Fukuda, F.; Takai, H.; Murakami, T.; Masuda, T.; et al. Full genome analysis of bovine astrovirus from fecal samples of cattle in Japan: identification of possible interspecies transmission of bovine astrovirus. *Arch. Virol.* **2015**, *160*, 2491–2501.
[CrossRef] [PubMed]

126. Ng, T.F.F.; Kondov, N.O.; Deng, X.; van Eenennaam, A.; Neibergs, H.L.; Delwart, E. A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. *J. Virol.* **2015**, *89*, 5340–5349.
[CrossRef] [PubMed]

127. Ng, T.F.F.; Mesquita, J.R.; Nascimento, M.S.J.; Kondov, N.O.; Wong, W.; Reuter, G.; Knowles, N.J.; Vega, E.; Esona, M.D.; Deng, X.; et al. Feline fecal virome reveals novel and prevalent enteric viruses. *Vet. Microbiol.* **2014**, *171*, 102–111.
[CrossRef] [PubMed]

128. Ng, T.F.F.; Marine, R.; Wang, C.; Simmonds, P.; Kapusinszky, B.; Bodhidatta, L.; Oderinde, B.S.; Wommack, K.E.; Delwart, E. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. *J. Virol.* **2012**, *86*, 12161–12175.
[CrossRef] [PubMed]

129. Padmanabhan, A.; Hause, B.M. Detection and characterization of a novel genotype of porcine astrovirus 4 from nasal swabs from pigs with acute respiratory disease. *Arch. Virol.* **2016**, *161*, 2575–2579.
[CrossRef] [PubMed]
130. Pankovics, P.; Boros, Á.; Kiss, T.; Delwart, E.; Reuter, G. Detection of a mammalian-like astrovirus in bird, European roller (Coracias garrulus). *Infect. Genet. Evol.* **J. Mol. Epidemiol. Evol. Genet. Infect. Dis.** **2015**, *34*, 114–121. [CrossRef] [PubMed]

131. Phan, T.G.; Nordgren, J.; Ouermi, D.; Simpore, J.; Nitiema, L.W.; Deng, X.; Delwart, E. New astrovirus in human feces from Burkina Faso. *J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol.* **2014**, *60*, 161–164. [CrossRef] [PubMed]

132. Phan, T.G.; Kapusinszky, B.; Wang, C.; Rose, R.K.; Lipton, H.L.; Delwart, E.L. The fecal viral flora of wild rodents. *PLoS Pathog.* **2011**, *7*, e1002218. [CrossRef] [PubMed]

133. Reuter, G.; Pankovics, P.; Delwart, E.; Boros, Á. Identification of a novel astrovirus in domestic sheep in Hungary. *Arch. Virol.* **2012**, *157*, 323–327. [CrossRef] [PubMed]

134. Zhang, B.; Tang, C.; Yue, H.; Ren, Y.; Song, Z. Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China. *J. Gen. Virol.* **2014**, *95*, 1603–1611. [CrossRef] [PubMed]

135. Zhang, W.; Li, L.; Deng, X.; Kapusinszky, B.; Pesavento, P.A.; Delwart, E. Faecal virome of cats in an animal shelter. *J. Gen. Virol.* **2014**, *95*, 2553–2564. [CrossRef] [PubMed]

136. Bouzalas, I.G.; Wüthrich, D.; Selimovic-Hamza, S.; Drögemüller, C.; Bruggmann, R.; Seuberlich, T. Full-genome based molecular characterization of encephalitis-associated bovine astroviruses. *Infect. Genet. Evol.* **J. Mol. Epidemiol. Evol. Genet. Infect. Dis.** **2016**, *44*, 162–168. [CrossRef] [PubMed]

137. Bouzalas, I.G.; Wüthrich, D.; Walland, J.; Drögemüller, C.; Zurbriggen, A.; Vandevelde, M.; Oevermann, A.; Bruggmann, R.; Seuberlich, T. Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe. *J. Clin. Microbiol.* **2014**, *52*, 3318–3324. [CrossRef] [PubMed]

138. Li, L.; Diab, S.; McGraw, S.; Barr, B.; Traslavina, R.; Higgins, R.; Talbot, T.; Blanchard, P.; Rimoldi, G.; Fahsbender, E.; et al. Divergent astrovirus associated with neurologic disease in cattle. *Emerg. Infect. Dis.* **2013**, *19*, 1385–1392. [CrossRef] [PubMed]

139. Schlottau, K.; Schulze, C.; Bilk, S.; Hanke, D.; Höper, D.; Beer, M.; Hoffmann, B. Detection of a Novel Bovine Astrovirus in a Cow with Encephalitis. *Transbound. Emerg. Dis.* **2016**, *63*, 253–259. [CrossRef] [PubMed]

140. Seuberlich, T.; Wüthrich, D.; Selimovic-Hamza, S.; Drögemüller, C.; Oevermann, A.; Bruggmann, R.; Bouzalas, I. Identification of a second encephalitis-associated astrovirus in cattle. *Emerg. Microbes Infect.* **2016**, *5*, e5. [CrossRef] [PubMed]

141. Wüthrich, D.; Boujon, C.L.; Truchet, L.; Selimovic-Hamza, S.; Oevermann, A.; Bouzalas, I.G.; Bruggmann, R.; Seuberlich, T. Exploring the virome of cattle with non-suppurative encephalitis of unknown etiology by metagenomics. *Virology* **2016**, *493*, 22–30. [CrossRef] [PubMed]

<figure><img src="image_3.png" /></figure>

© 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).