Astrovirus induced nonpurulent encephalomyelitis in sheep: First report from Türkiye by high-throughput sequencing.

BACKGROUND: This study presents the case of non-purulent encephalomyelitis associated with astrovirus infection in a sheep from Eastern Anatolia, Türkiye. METHODS: A necropsy was performed on a sheep showing nervous signs. Afterwards, brain tissue samples were taken and examined with histopathological, immunohistochemical and molecular techniques. RESULTS: Neuropathologic changes included neuronal degeneration, diffuse gliosis, multifocal perivascular cuffing, neuronophagy and neuronal necrosis in the cerebrum, the cerebellum and the cervical spinal cord. Aerobic and anaerobic bacterial culture, selective culture for Listeria monocytogenes, and PCR analysis for rabies virus, tick-borne encephalitis virus, Türkiye encephalitis virus, small ruminant lentiviruses and border disease virus were negative. However, the presence of astrovirus RNA in cerebral, cerebellar and spinal cord samples was demonstrated by a pan-astrovirus RT-PCR. Immunohistochemical examinations revealed astrovirus antigens within the neuronal cytoplasm. High-throughput sequencing techniques identified the causative agent as a member of the genotype species Mamastrovirus 13 but representing a distinct genetic lineage with similarity to ovine astrovirus 1 in the open-reading frames (ORF)1ab region and muskox astrovirus in the ORF2 region. CONCLUSION: This report provides evidence that astroviruses are potentially encephalitis-causing pathogens in ovine populations in Türkiye, featuring an astrovirus strain distinct from those previously identified in sheep.

The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Investigation of caliciviruses and astroviruses in Gabonese rodents: A possible influence of national and international trade on the spread of enteric viruses.

Caliciviruses (Caliciviridae) and astroviruses (Astroviridae) are among the leading cause of non-bacterial foodborne disease and gastroenteritis in human. These non-enveloped RNA viruses infect a wide range of vertebrate species including rodents. Rodents are among the most important hosts of infectious diseases globally and are responsible for over 80 zoonotic pathogens that affect humans. Therefore, screening pathogens in rodents will be is necessary to prevent cross-species transmission to prevent zoonotic outbreaks. In the present study, we screened caliciviruses and astroviruses in order to describe their diversity and whether they harbor strains that can infect humans. RNA was then extracted from intestine samples of 245 rodents and retrotranscribed in cDNA to screen caliciviruses and astroviruses by PCRs. All the samples tested negative for caliciviruses and while astroviruses were detected in 18 (7.3%) samples of Rattus rattus species. Phylogenetic analyses based on the RdRp gene showed that all the sequences belonged to Mamastrovirus genus in which they were genetically related to R. rattus related AstVs previously detected in Gabon or in Rattus spp. AstV from Kenya and Asia. These findings suggested that transportation such as land and railway, as well national and international trade, are likely to facilitate spread of AstVs by the dissemination of rodents.

Isolation, Identification, and Pathogenicity of a Goose Astrovirus Genotype 1 Strain in Goslings in China.

Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3-98.4%; ORF1b: 92.3-99.1%; ORF2: 95.8-98.8%) and amino acid sequences (ORF1a: 99.4-99.5%; ORF1b: 98.2-98.8%; ORF2: 97.0-99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese.

Bovine astrovirus and its role in lymphocytic encephalitis in cattle in Ontario, Canada, 1988-2019.

Astroviruses have been found in cattle and other species with encephalitis. Our objective was to determine the frequency of neurotropic bovine astrovirus (BoAstV) in cases of encephalitis in cattle ≥ 4-mo-old. Of 56 cases of idiopathic lymphocytic encephalitis examined retrospectively (1988-2019), fixed brain from 11 cases (19%) tested positive by semi-quantitative RT-PCR for BoAstV CH13/NeuroS1. None of the control cases tested positive, including 32 with other forms of encephalitis and 40 with no neurologic disease. Most astrovirus-positive cases were 1-2-y-old, with a range of 7 mo to 7 y, and affected both beef and dairy breeds with wide geographic distribution. BoAstV-positive cases had acute onset of neurologic signs of 12 h to 7 d before death or euthanasia. Affected cattle had lymphocytic inflammation throughout the brain including cerebrum, thalamus, midbrain, cerebellum, medulla oblongata, and spinal cord, and affecting gray and white matter. Further PCR testing identified a possible cause in 9 of the 45 (20%) remaining idiopathic cases of lymphocytic encephalitis, including eastern equine encephalitis virus, Listeria monocytogenes, bovine viral diarrhea virus, bovine alphaherpesvirus 1, and ovine gammaherpesvirus 2 (malignant catarrhal fever); we found no cases of infection by West Nile virus, rabies virus, or Chlamydia spp. No cause was identified in 36 of 56 (64%) cases of lymphocytic encephalitis. We frequently identified neurotropic BoAstV in cases of lymphocytic encephalitis that had no previously identified cause. Neurotropic BoAstV infections had gone undetected for decades, but the frequency of BoAstV infections has not increased among contemporary cases.

Enteric viruses other than rotavirus and norovirus in children under 5 years of age with gastroenteritis in Argentina, 2010-2021. A descriptive study. / Virus entéricos distintos de rotavirus y norovirus en menores de 5 años con gastroenteritis en Argentina, 2010-2021. Estudio descriptivo.

Introduction. Data on the frequency of enteric adenoviruses, sapoviruses, and astroviruses in cases of sporadic acute gastroenteritis in Argentina are scarce. Methods. Descriptive design of a selection of fecal samples of children with diarrhea younger than 5 years referred between 2010 and 2021, with a previous negative result for rotavirus and norovirus. The presence of enteric adenovirus, sapovirus, and astrovirus was tested by molecular methods, with subsequent genotyping of positive samples. Results. At least 1 of the tested viruses was detected in 226 (39.4%) of the 574 selected samples. Specifically, adenovirus, sapovirus, and astrovirus were detected in 30.7%, 5.6%, and 3.1% of the samples, respectively. The most frequent viruses detected were adenovirus 41, sapoviruses GI.1 and GI.2, and astrovirus 1. Non-classic astroviruses were detected in 2 samples. Conclusions. Despite being less frequent, these enteropathogens are responsible for a large number of sporadic diarrhea events. Therefore, their study and surveillance contribute significantly to reduce the gap of undiagnosed cases.

Introducción. Los datos de frecuencia de los adenovirus entéricos, sapovirus y astrovirus en casos de gastroenteritis aguda esporádica en Argentina son escasos. Métodos. Diseño descriptivo sobre una selección de muestras de heces de menores de 5 años con diarrea remitidas durante el período 2010-2021, con resultado previo negativo para rotavirus y norovirus. Se estudió la presencia de adenovirus entéricos, sapovirus y astrovirus por métodos moleculares, con posterior genotipificación de las muestras positivas. Resultados. De 574 muestras seleccionadas, en 226 (39,4 %) se identificó al menos uno de los virus estudiados. En particular, se detectaron adenovirus, sapovirus y astrovirus en el 30,7 %, el 5,6 % y el 3,1 %, respectivamente. El adenovirus 41, los sapovirus GI.1 y GI.2, y el astrovirus 1 fueron los más frecuentemente detectados. Se identificaron dos muestras con astrovirus no clásicos. Conclusiones. A pesar de ser menos frecuentes, estos enteropatógenos son responsables de un número considerable de episodios de diarrea esporádica. Por lo tanto, su estudio y vigilancia contribuye significativamente a reducir la brecha de casos no diagnosticados.

Aminoguanidine alleviates gout in goslings experimentally infected with goose astrovirus-2 by reducing kidney lesions.

Goose astrovirus (GAstV)-2, a novel pathogen identified in 2018, mainly causes visceral gout in goslings, leading to approximately 50% mortality. At present, no commercial veterinary products are available to prevent and treat the disease. Our previous studies showed that nitric oxide (NO) and inducible NO synthase (iNOS) were markedly higher in the kidney and spleen of goslings infected with GAstV-2, but their effects during GAstV-2 infection remain unclear. In the present study, goslings were intraperitoneally injected with aminoguanidine (AG)-an iNOS inhibitor-to examine the role of NO during GAstV-2 infection. AG significantly decreased the serum NO concentration and iNOS mRNA expression in the kidney. Moreover, AG reduced the mortality, serum uric acid and creatinine content, and urate deposition in visceral organs and joints. Histopathological analysis demonstrated that AG reduced renal tubular cell necrosis, inflammatory cell infiltration, glycogen deposition in glomerular mesangium, and interstitial fibrosis, suggesting alleviation of kidney lesions. Furthermore, AG decreased the expression of renal injury markers such as KIM-1 and desmin; inflammatory cytokine-related genes such as IL-1ß, IL-8, and MMP-9; and autophagy-related genes and proteins such as LC3II, ATG5, and Beclin1. However, quantitative real-time PCR and immunohistochemistry showed that treatment with AG did not affect the kidney and liver viral load. These findings suggest that AG decreases the mortality rate and kidney lesions in goslings infected with GAstV-2 through mechanisms associated with autophagy and inhibition of inflammatory cytokine production in the kidney but not with GAstV-2 replication.

Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/µL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Characterization of natural co-infection with goose astrovirus genotypes I and II in gout affected goslings.

RESEARCH HIGHLIGHTS: Urate tophi were found in the kidneys, liver, spleen and lungs.IFA confirmed the co-expression of GoAstV-I and II antigens in the same kidney.

High prevalence of Enterovirus E, Bovine Kobuvirus, and Astrovirus revealed by viral metagenomics in fecal samples from cattle in Central Colombia.

Livestock plays a crucial role in ensuring food security and driving the global economy. However, viral infections can have far-reaching consequences beyond economic productivity, affecting the health of cattle, as well as posing risks to human health and other animals. Identifying viruses present in fecal samples, a primary route of pathogen transmission, is essential for developing effective prevention, control, and surveillance strategies. Viral metagenomic approaches offer a broader perspective and hold great potential for detecting previously unknown viruses or uncovering previously undescribed agents. Ubaté Province is Colombia's dairy capital and a key center for livestock production in the country. Therefore, the purpose of this study was to characterize viral communities in fecal samples from cattle in this region. A total of 42 samples were collected from three municipalities in Ubaté Province, located in central Colombia, using a convenient non-probabilistic sampling method. We utilized metagenomic sequencing with Oxford Nanopore Technologies (ONT), combined with diversity and phylogenetic analysis. The findings revealed a consistent and stable viral composition across the municipalities, primarily comprising members of the Picornaviridae family. At the species level, the most frequent viruses were Enterovirus E (EVE) and Bovine Astrovirus (BoAstV). Significantly, this study reported, for the first time in Colombia, the presence of viruses with veterinary importance occurring at notable frequencies: EVE (59%), Bovine Kobuvirus (BKV) (52%), and BoAstV (19%). Additionally, the study confirmed the existence of Circular replicase-encoding single-stranded (CRESS) Virus in animal feces. These sequences were phylogenetically grouped with samples obtained from Asia and Latin America, underscoring the importance of having adequate representation across the continent. The virome of bovine feces in Ubaté Province is characterized by the predominance of potentially pathogenic viruses such as BoAstV and EVE that have been reported with substantial frequency and quantities. Several of these viruses were identified in Colombia for the first time. This study showcases the utility of using metagenomic sequencing techniques in epidemiological surveillance. It also paves the way for further research on the influence of these agents on bovine health and their frecuency across the country.

Bastroviruses (Astroviridae): genetic diversity and potential impact on human and animal health.

INTRODUCTION: Bastroviruses were discovered in the Netherlands in 2016 in human stool samples and show partial genetic similarities to astroviruses and hepatitis E viruses. Their association with disease onset has not yet been established. MATERIALS AND METHODS: Metagenomic sequencing of fecal samples of Nyctalus noctula bats collected in the Russian Federation in 2023 was performed. Two almost complete genomes of bastroviruses were assembled. The zoonotic potential of these viruses was assessed using machine learning methods, their recombination was studied, and phylogenetic trees were constructed. RESULTS: A nearly complete bastrovirus genome was de novo assembled in one of the samples, and it was used to assemble another genome in another sample. The zoonotic potential of the virus from one of these samples was estimated as high. The existence of recombination between structural and non-structural polyproteins was demonstrated. CONCLUSION: Two bastrovirus genomes were assembled, phylogenetic and recombination analyses were performed, and the zoonotic potential was evaluated.

Identification of Astrovirus in the virome of the upper and lower respiratory tracts of calves with acute signs of bronchopneumonia.

IMPORTANCE: Astroviruses (AstV) are known suspects of enteric disease in humans and livestock. Recently, AstV have been linked to encephalitis in immunocompromised patients and other animals, such as cattle, minks, and swine. In our study, we also identified AstV in the respiratory samples of calves with signs of bronchopneumonia, suggesting that their tropism could be even broader. We obtained one bovine AstV (BAstV) complete genome sequence by next-generation sequencing and showed that respiratory and enteric AstV from different species formed a divergent genetic cluster with AstV isolated from encephalitis cases, indicating that tropism might be strain-specific. These data provide further insight into understanding the biology of these understudied pathogens and suggest BAstV as a potential new candidate for bovine respiratory disease.

Detection and Molecular Characterization of Animal Adenovirus and Astrovirus from Western Maharashtra, India.

Astroviruses (AstV) and adenoviruses (AdV) are associated with diarrhoea in young animals. However, the epidemiology and genetic diversity of AstVs and AdVs in animals is not well studied. Hence, the present study was conducted to detect and characterize AstVs and AdVs in calves, piglets and puppies from Western Maharashtra, India. Out of the processed porcine (48), canine (80), and bovine (65) faecal samples, the porcine AstV (PAstV), bovine AstV (BAstV), canine AstV (CAstV), and porcine AdV (PAdV) were detected in 12.5%, 7.69%, 3.75% and 4.1% of samples, respectively. In the RNA-dependent RNA polymerase region-based phylogenetic analysis, the detected BAstV strains grouped with MAstV-28, MAstV-33, and MAstV-35, CAstV strains belonged to MAstV-5; PAstV strains belonged to MAstV-24, MAstV-26, and MAstV-31. However, in hexon gene-based phylogeny, both the detected PAdV were of genotype 3, exhibiting 91.9-92.5% nucleotide identity with Ivoirian and Chinese strains. The study reports first-time BAstVs from calves and PAdV-3 from piglets in India. The study revealed diversity in the circulation of AstVs in tested animals and AdVs in pigs, and suggested that they alone might be associated with other diarrhoea or in combination with other enteric pathogens, thus highlighting the necessity of extensive epidemiological investigations to develop diagnostic tools and control measures.

Genetic characteristics of classical astroviruses in Shenzhen, China, 2016-2019.

Human astrovirus (HAstV) is a single-stranded, positive-sense RNA virus and is the leading cause of viral gastroenteritis. However, despite its prevalence, astroviruses still remain one of the least studied enteroviruses. In this study, we sequenced 11 classical astrovirus strains from clinical samples collected in Shenzhen, China from 2016 to 2019, analyzed their genetic characteristics, and deposited them into GenBank. We conducted phylogenetic analysis using IQ-TREE software, with references to astrovirus sequences worldwide. The phylogeographic analysis was performed using the Bayesian Evolutionary Analysis Sampling Trees program, through Bayesian Markov Chain Monte Carlo sampling. We also conducted recombination analysis with the Recombination Detection Program. The newly sequenced strains were categorized as HAstV genotype 1, which is the predominant genotype in Shenzhen. Phylogeographic reconstruction indicated that HAstV-1 may have migrated from the United States to China, followed by frequent transmission between China and Japan. The recombination analysis revealed recombination events within and across genotypes, and identified a recombination-prone region that produced relatively uniform recombination breakpoints and fragment lengths. The genetic analysis of HAstV strains in Shenzhen addresses the current lack of astrovirus data in the region of Shenzhen and provides key insights to the evolution and transmission of astroviruses worldwide. These findings highlight the importance of improving surveillance of astroviruses.

Detection and Genetic Characterization of Astroviruses in Brain Tissues of Wild Raccoon Dogs.

Astroviruses (AstVs) have been detected in a wide range of animal species, including mammals and birds. Recently, a novel AstV associated with neurological symptoms has been detected in the brains of some mammals. Raccoon dog AstV has been reported recently in China. However, there have been no reports in South Korea. Therefore, the present study aimed to detect and genetically characterize AstVs in the intestine and brain tissues of 133 wild raccoon dogs collected in Korea between 2017 and 2019. Of the seven raccoon dogs, AstVs were detected in six intestinal tissues and four brain tissues. Analysis of the capsid protein amino acid sequences of raccoon dog AstVs detected in Korea revealed a high similarity to canine AstVs, suggesting possible interspecies transmission between raccoon dogs and dogs. Phylogenetic and capsid protein amino acid sequence analysis of raccoon dog AstVs detected in the brain the 17-148B strain belonging to the HMO clade and exhibiting conserved sequences found in neurotropic AstVs (NT-AstVs), indicating their potential as NT-AstVs. However, the pathogenicity and transmission routes of the raccoon dog AstV detected in Korea have not yet been elucidated, so further research and continued surveillance for AstV in wild raccoon dogs are needed.

Identification of new astroviruses in synanthropic squamates.

Astroviruses have been identified in a wide variety of animal species and are associated with gastro-intestinal disease in humans. Pathologies due to extra-intestinal localization are known in different hosts. We report the detection of astroviruses in synanthropic squamate reptile species (Podercis siculus and Tarentola mauritanica). Fecal samples were collected from 100 squamates from urban and peri-urban areas of three regions in South Italy and tested for the presence of astroviruses using a broadly reactive (pan-astrovirus) RT-PCR protocol targeting the RNA-dependent RNA polymerase. Astrovirus RNA was detected in 11% of the samples and for six strains a 3 kb-long fragment at the 3' end of the genome was sequenced, obtaining information on the complete capsid-encoding ORF2 sequence. Viral RNA was also detected in the brain of one of the positive animals. The sequences generated from the astrovirus strains shared low nucleotide identities in the ORF2 (< 43.7%) with other known reptilian astrovirus sequences, hinting to the massive genetic diversity of members of this viral family. Based on the partial RdRp gene of the sequenced strains, however, we observed species-specific patterns, regardless of the geographic origin of the animals, and we also identified a possible inter-species transmission event between geckoes and lizards.

Genome Sequences and Characterization of Chicken Astrovirus and Avian Nephritis Virus from Tanzanian Live Bird Markets.

The enteric chicken astrovirus (CAstV) and avian nephritis virus (ANV) are the type species of the genus Avastrovirus (AAstV; Astroviridae family), capable of causing considerable production losses in poultry. Using next-generation sequencing of a cloacal swab from a backyard chicken in Tanzania, we assembled genome sequences of ANV and CAstV (6918 nt and 7318 nt in length, respectively, excluding poly(A) tails, which have a typical AAstV genome architecture (5'-UTR-ORF1a-ORF1b-ORF2-'3-UTR). They are most similar to strains ck/ANV/BR/RS/6R/15 (82.72%) and ck/CAstV/PL/G059/14 (82.23%), respectively. Phylogenetic and sequence analyses of the genomes and the three open reading frames (ORFs) grouped the Tanzanian ANV and CAstV strains with Eurasian ANV-5 and CAstV-Aii viruses, respectively. Compared to other AAstVs, the Tanzanian strains have numerous amino acid variations (substitutions, insertions and deletions) in the spike region of the capsid protein. Furthermore, CAstV-A has a 4018 nt recombinant fragment in the ORF1a/1b genomic region, predicted to be from Eurasian CAstV-Bi and Bvi parental strains. These data should inform future epidemiological studies and options for AAstV diagnostics and vaccines.

First Discovery of Phenuiviruses within Diverse RNA Viromes of Asiatic Toad (Bufo gargarizans) by Metagenomics Sequencing.

Most zoonotic pathogens originate from mammals and avians, but viral diversity and related biosafety risk assessment in lower vertebrates also need to be explored. Amphibians are an important group of lower vertebrates that played a momentous role in animal evolution. To elucidate the diversity of RNA viruses in one important species of amphibians, the Asiatic toad (Bufo gargarizans), we obtained 44 samples including lung, gut, liver, and kidney tissues from Asiatic toads in Sichuan and Jilin provinces, China, for viral metagenomics sequencing. More than 20 novel RNA viruses derived from the order Bunyavirales and 7 families of Astroviridae, Dicistroviridae, Leviviridae, Partitiviridae, Picornaviridae, Rhabdoviridae, and Virgaviridae were discovered, which were distinct from previously described viruses and formed new clusters, as revealed by phylogenetic analyses. Notably, a novel bastrovirus, AtBastV/GCCDC11/2022, of the family Astroviridae was identified from the gut library, the genome of which contains three open reading frames, with the RNA-dependent RNA polymerase (RdRp) coded by ORF1 closely related to that of hepeviruses, and ORF2 encoding an astrovirus-related capsid protein. Notably, phenuiviruses were discovered for the first time in amphibians. AtPhenV1/GCCDC12/2022 and AtPhenV2/GCCDC13/2022 clustered together and formed a clade with the group of phenuiviruses identified from rodents. Picornaviruses and several invertebrate RNA viruses were also detected. These findings improve our understanding of the high RNA viral diversity in the Asiatic toad and provide new insights in the evolution of RNA viruses in amphibians.

A Novel Coronavirus and a Broad Range of Viruses in Kenyan Cave Bats.

BACKGROUND AND METHODS: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. RESULTS: Viruses belonging to the Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. CONCLUSIONS: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Unusual "Asian-origin" 2c to 2b point mutant canine parvovirus (Parvoviridae) and canine astrovirus (Astroviridae) co-infection detected in vaccinated dogs with an outbreak of severe haemorrhagic gastroenteritis with high mortality rate in Hungary.

In this study, the aetiological background of an outbreak of severe haemorrhagic gastroenteritis (HGE) in a colony of purebred Jack Russell Terriers vaccinated against CPV-2 in Hungary was investigated. Canine parvovirus 2 (CPV-2, Parvoviridae) and canine astrovirus (CaAstV, Astroviridae) co-infection was identified by viral metagenomics and next-generation sequencing (VM-NGS) methods from a rectal swab of an affected 7-week-old puppy. The complete coding sequence of CPV-2 strain FR1/CPV2-2021-HUN (ON733252) and the complete genome of CaAstV strain FR1/CaAstV-2021-HUN (ON733251) were determined by VM-NGS and PCR methods. Results of sequence and phylogenetic analyses showed that CPV-2 strain FR1/CPV2-2021-HUN was different from the applied vaccine strains and previously identified strains from Hungary but showed high sequence identity (> 99.8%) and close phylogenetic relationship to recently described "Asian-origin" CPV-2c strains from Italy. But, based on the single amino acid difference on position 426 of VP2 (Glu/Asp) between the study strain and the closest relatives, FR1/CPV2-2021-HUN belonged to the 2b antigenic type rather than 2c. The CaAstV strain FR1/CaAstV-2021-HUN showed close relationship with a CaAstV strain identified previously from a diarrhoeic dog in Hungary. Both viruses were continuously detectable by PCR in additional enteric samples, and the CPV-2 could also be detected in several (n = 32) tissue samples from 9 affected deceased puppies. Further comparative studies are necessary to confirm the role of the point mutation causing the change in the antigenic type of this "Asian-origin" CPV-2 and/or the role of CaAstV co-infection in the development and/or severity of (haemorrhagic) gastroenteritis among dogs vaccinated against CPV-2.