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.

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome.

A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20-30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.

An outbreak of neonatal enteritis in buffalo calves associated with astrovirus.

BACKGROUND: Enteritis of an infectious origin is a major cause of productivity and economic losses to cattle producers worldwide. Several pathogens are believed to cause or contribute to the development of calf diarrhea. Astroviruses (AstVs) are neglected enteric pathogens in ruminants, but they have recently gained attention because of their possible association with encephalitis in humans and various animal species, including cattle. OBJECTIVES: This paper describes a large outbreak of neonatal diarrhea in buffalo calves (Bubalus bubalis), characterized by high mortality, which was associated with an AstV infection. METHODS: Following an enteritis outbreak characterized by high morbidity (100%) and mortality (46.2%) in a herd of Mediterranean buffaloes (B. bubalis) in Italy, 16 samples from buffalo calves were tested with the molecular tools for common and uncommon enteric pathogens, including AstV, kobuvirus, and torovirus. RESULTS: The samples tested negative for common enteric viral agents, including Rotavirus A, coronavirus, calicivirus, pestivirus, kobuvirus, and torovirus, while they tested positive for AstV. Overall, 62.5% (10/16) of the samples were positive in a single round reverse transcription polymerase chain reaction (PCR) assay for AstV, and 100% (16/16) were positive when nested PCR was performed. The strains identified in the outbreak showed a clonal origin and shared the closest genetic relationship with bovine AstVs (up to 85% amino acid identity in the capsid). CONCLUSIONS: This report indicates that AstVs should be included in a differential diagnosis of infectious diarrhea in buffalo calves.

Identification of a novel astrovirus in goats in China.

In this study, a total of 143 fecal samples (107 diarrheic and 36 non-diarrheic) were collected from 11 goat farms in southwest China, and 3.7% of diarrheic and 8.3% of non-diarrheic samples were detected as astrovirus-positive by RT-PCR. A nearly complete astrovirus genomic sequence (SWUN/F4/2019) of 6278 nucleotides (nt), which contained a 6186 bp open reading frame, was successfully obtained. The genome of strain SWUN/F4/2019 shared the highest nt identity (77.0%) and the closest genetic relationship with CapAstV-G5.1. It is worth noting that in the nonstructural protein 1ab, strain SWUN/F4/2019 shared the highest amino acid (aa) identity (93.8%) with strain CapAstV-G5.1; however, its capsid protein shared the highest aa identity (72.7%) with the Sichuan takin astrovirus strain LLT03 and only shared 23.1-64.8% aa identities with all available ovine and caprine astrovirus strains. Interestingly, a region recombination event was predicted in the ORF2 gene of strain SWUN/F4/2019, with CapAstV-G5.1 as the putative major parental strain and CcAstV/roe_deer/SLO/D5-14/2014 as the possible minor parental strain. According to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV), SWUN/F4/2019 may represent a novel astrovirus in goats. To our knowledge, this is the first detection of astrovirus in goats in China and a novel astrovirus strain was identified in goats. These findings increase the understanding of the epidemic and the genetic evolution of astroviruses.

Astrovirus in Reunion Free-Tailed Bat (Mormopterus francoismoutoui).

Astroviruses (AstVs) are RNA viruses infecting a large diversity of avian and mammalian species, including bats, livestock, and humans. We investigated AstV infection in a free-tailed bat species, Mormopterus francoismoutoui, endemic to Reunion Island. A total of 380 guano samples were collected in a maternity colony during 38 different sampling sessions, from 21 June 2016 to 4 September 2018. Each sample was tested for the presence of the AstV RNA-dependent RNA-polymerase (RdRp) gene using a pan-AstV semi-nested polymerase chain reaction assay. In total, 27 guano samples (7.1%) tested positive, with high genetic diversity of the partial RdRp gene sequences among positive samples. Phylogenetic analysis further revealed that the detected viruses were genetically related to AstVs reported in rats, reptiles, dogs, and pigs, but did not cluster with AstVs commonly found in bats. Although more investigations need to be conducted to assess the prevalence of infected bats in the studied population, our findings show that Reunion free-tailed bats are exposed to AstVs, and suggest that cross-species transmission may occur with other hosts sharing the same habitat.

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera-i.e., Pedacovirus, Nyctacovirus, and Myotacovirus-that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus-present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Neurotropic Astroviruses in Animals.

Astrovirus infections are among the main causes of diarrhea in children, but their significance for animal health has remained underestimated and largely unknown. This is changing due to the increasing amount of newly identified neurotropic astroviruses in cases of nonsuppurative encephalitis and neurological disease in humans, pigs, ruminant species and minks. Neurological cases in ruminants and humans usually occur sporadically and as isolated cases. This contrasts with the situation in pigs and minks, in which diseases associated with neurotropic astroviruses are endemic and occur on the herd level. Affected animals show neurological signs such as mild ataxia to tetraplegia, loss of orientation or trembling, and the outcome is often fatal. Non-suppurative inflammation with perivascular cuffing, gliosis and neuronal necrosis are typical histological lesions of astrovirus encephalitis. Since astroviruses primarily target the gastrointestinal tract, it is assumed that they infect the brain through the circulatory system or retrograde following the nerves. The phylogenetic analysis of neurotropic astroviruses has revealed that they are genetically closely related, suggesting the presence of viral determinants for tissue tropism and neuroinvasion. In this review, we summarize the current knowledge on neurotropic astrovirus infections in animals and propose future research activities.

RT-qPCR assay for detection of mink astrovirus in outbreaks of diarrhea on Danish mink farms.

Diarrhea in mink kits is a major cause of disease and mortality in the mink production. The etiology remains unknown in most outbreaks due to a lack of diagnostic assays. In the current study we present an RT-qPCR method to detect mink astrovirus in fecal samples from mink kits with diarrhea. All sampled animals were classified based on age and patoanatomical evaluation as having pre-weaning diarrhea, diarrhea in the growth period or as having no macroscopic signs of diarrhea. Fecal samples were analyzed for MiAstV with RT-qPCR, next generation sequencing and electron microscopy in parallel. Mink astrovirus was detected with RT-qPCR in 92 out of 203 samples. This detection was confirmed by next generation sequencing in a high proportion of samples (22/27), and by visualization of astrovirus particles with EM in some of the samples. Mink astrovirus was highly prevalent (68%) among kits in the outbreaks of pre-weaning diarrhea, in particular outbreaks from May, while less prevalent in outbreaks in June. Mink astrovirus was detected in outbreaks of diarrhea in the growth period, though in a much lesser extent than in the pre-weaning period. The role of mink astrovirus in the diarrhea disease complex of mink remain to be investigated, and for that purpose this sensitive and robust RT-qPCR can be a valuable tool in the future.

Genome characterization, prevalence and tissue distribution of astrovirus, hepevirus and norovirus among wild and laboratory rats (Rattus norvegicus) and mice (Mus musculus) in Hungary.

Rodents including rats are reservoir of several pathogens capable of affecting human health. In this study, faecal and different organ specimens from free-living Norway rats (Rattus norvegicus) (N = 18) and faecal samples from laboratory rodents (rats N = 21 and mice N = 20) collected from different geographic areas in Hungary between 2017 and 2020 were investigated by viral metagenomics and conventional RT-PCR methods. The complete genome of three different RNA viruses, rat astrovirus, rat norovirus and rat hepevirus were characterized and analysed in detail. Rat norovirus was detected in faecal (17.6%, 3/17) and kidney (7.1%, 1/14) samples; rat astrovirus in faecal (23.5%, 4/17) and spleen (13.3%, 2/15) samples, and rat hepevirus in 43% to 67% the faecal, liver, kidney, lung, heart, muscle, brain and blood samples from Norway rats, respectively. Rat norovirus was also identifiable in 5% (1/21) of laboratory rats and rat astrovirus in 40% (8/20) of faecal samples from laboratory mice. Co-infections were found in 28% (5/18) wild Norway rats. The highest RNA viral load of astrovirus (1.81 × 108 copy/g) and norovirus (3.49 × 107 copy/g) were measured in faecal samples; while the highest RNA viral load of hepevirus (1.16 × 109 copy/g) was found in liver samples of Norway rats, respectively. This study confirms the wide geographic distribution and high prevalence of astrovirus, norovirus and hepevirus among wild rats in Hungary with confirmation of different organ involvement of as well as the detection of norovirus and astrovirus in laboratory rats and mice, respectively. This finding further strengthens the role of rodents in the spread of viral pathogens especially infecting human.

Detection of mink astrovirus in Poland and further phylogenetic comparison with other European and Canadian astroviruses.

Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein (length of sequencing product 170 bp) from all samples. The presence of astrovirus RNA was detected in each of the samples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples. Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other countries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe.

Causes of neonatal calf diarrhea and mortality in pasture-based dairy herds in Uruguay: a farm-matched case-control study.

Neonatal calf diarrhea (NCD) and mortality cause significant losses to the dairy industry. The preweaning dairy calf mortality risk in Uruguay is high (15.2%); however, causes for these losses are largely unknown. This study aimed to assess whether various pathogens were associated with NCD and death in Uruguayan dairy calves and whether these infections, diarrhea, or deaths were associated with the failure of transfer of passive immunity (FTPI). Contemporary diarrheic (n = 264,) and non-diarrheic (n = 271) 1- to 30-day-old calves from 27 farms were sampled. Feces were analyzed by antigen-capture ELISA for Cryptosporidium spp., rotavirus, bovine coronavirus, and Escherichia coli F5+, RT-PCR for bovine astrovirus (BoAstV), and bacterial cultures for Salmonella enterica. Blood/serum was analyzed by RT-PCR or antigen-capture ELISA for bovine viral diarrhea virus (BVDV). Serum of ≤ 8-day-old calves (n = 95) was assessed by refractometry to determine the concention of serum total proteins (STP) as an indicator of FTPI. Whether the sampled calves died before weaning was recorded. At least one pathogen was detected in 65.4% of the calves, and this percentage was significantly higher in diarrheic (83.7%) versus non-diarrheic (47.6%) calves. Unlike the other pathogens, Cryptosporidium spp. and rotavirus were associated with NCD. Diarrheic calves, calves infected with any of the pathogens, and calves infected with rotavirus had significantly lower concentrations of STP. Diarrheic calves had higher chances of dying before weaning than non-diarrheic calves. Diarrheic calves infected with S. enterica were at increased risk of mortality. Controlling NCD, salmonellosis, cryptosporidiosis, and rotavirus infections, and improving colostrum management practices would help to reduce calf morbi-mortality in dairy farms in Uruguay.

Molecular survey of parvovirus, astrovirus, coronavirus, and calicivirus in symptomatic dogs.

Gastrointestinal disorders caused by enteric viruses are frequently reported in dogs worldwide, with significant mortality rates in unvaccinated individuals. This study reports the identification and molecular characterization of Canine parvovirus (CPV-2), Canine coronavirus (CcoV), Canine astrovirus (AstV), and Canine calicivirus (CcaV) in a panel of dogs showing severe enteric clinical signs sampled in a typical Mediterranean environment (Sardinia, Italy). At least one of these viral species was detected in 92.3% samples. CPV-2 was the most frequently detected virus (87.2%), followed by AsTv (20.5%), CCoV-IIa (18%), and CCoV-I (10.3%). CCoV-IIb and CaCV were not detected in any sample. Single infection was detected in 24 samples (66.7%), mainly related to CPV-2 (91.7%). Coinfections were present in 33.3% samples with constant detection of CPV-2. Canine coronavirus was present only in coinfected animals. The VP2 sequence analysis of CPV-2 positive samples confirmed the presence of all variants, with CPV-2b most frequently detected. Phylogeny based on the CcoV-IIa spike protein (S) gene allowed to identify 2 different clades among Sardinian isolates but failed to distinguish enteric from pantropic viruses. Study on presence and prevalence of enteroviruses in dogs increase our knowledge about the circulation of these pathogens in the Mediterranean area and highlight the need for dedicated routine vaccine prophylaxis. Molecular analyses of enteric viruses are fundamental to avoid failure of vaccines caused by frequent mutations observed in these enteroviruses.

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan.

Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.

The origin and past demography of murine astrovirus 1 in laboratory mice.

Astroviruses are non-enveloped, positive-sense, ssRNA viruses and often associated with gastrointestinal diseases. Murine astrovirus (MuAstV) was first confirmed in a laboratory mouse colony in 2011. Although infected mice do not present significant clinical symptoms, the virus might interfere with research results. A recent surveillance has shown that MuAstV is highly prevalent in laboratory mice. The aims of the present study were to identify and characterize MuAstV strains as well as to investigate the prevalence rate of viral RNA in laboratory mice in Taiwan, and to estimate the origin and past population demography of MuAstVs. Based on molecular surveillance, MuAstV RNA was detected in 45.7 % of laboratory mice (48/105) from seven of nine colonies. Three fully sequenced MuAstV strains, MuAstV TW1, TW2 and TW3, exhibited 89.1-94.4 % and 89.1-90.0 % nucleotide identities with the reference strains MuAstV STL1 and STL2, respectively. Phylogenetic analyses of the partial regions of the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP) genes of 18 Taiwan strains along with other astroviruses revealed that there are three distinct lineages of mouse astrovirus, MuAstV1, MuAstV2 and mouse astrovirus JF755422. The mutation rates of MuAstV1 were 2.6×10-4 and 6.2×10-4 substitutions/site/year for the RdRp and CP regions, respectively. Based on the above molecular clock, the colonization of MuAstV1 in laboratory mice was between 1897 and 1912, in good agreement with the establishment of 'modern' laboratory mouse facilities. Since its initial infection, the population size of MuAstV1 has increased 15-60-fold, probably consistent with the increased use of laboratory mice. In conclusion, MuAstV1 has been associated with modern laboratory mice since the beginning, and its influence on research results may require further investigation.

Isolation and Characterization of Porcine Astrovirus 5 from a Classical Swine Fever Virus-Infected Specimen.

Many new astroviruses have been identified in humans and other animals in recent years, but only a few have been successfully isolated for extensive biological study. Here, we report an unusual isolation of a porcine astrovirus 5 (PAstV5) strain from a clinical classical swine fever virus (CSFV)-infected tissue sample. Incubation of porcine PK-15 cells with an extract of the CSFV-positive tissue resulted in unexpected cytopathic effects (CPEs), and high-throughput viromic sequencing identified PAstV5 and porcine circovirus type 2 (PCV2) as well as CSFV in the culture. After clearance of CSFV and PCV2, a pure PAstV5 strain, named PAstV5-AH29-2014, was obtained. Analysis revealed virus of typical astroviral morphology with a genome of 6,448 nucleotides, sharing 84.3 to 88.9% nucleotide identity with previously published PAstV5 strains. A mechanistic study showed that CSFV coinfection was likely an important factor for successful isolation by significantly enhancing PAstV5 replication in PK-15 cells via suppression of a type I interferon response. Altogether, PAstV5-AH29-2014, as the first isolated PAstV5 strain, will provide critical material for the investigation of the biological and pathogenic properties of this virus as well as for future development of relevant biological and diagnostic reagents.IMPORTANCE Porcine astroviruses are mainly associated with gastroenteritis and neurological diseases in pigs, and five genotypes have been identified (PAstV1-5). However, the clinical manifestations of genotypes other than PAstV1 have not yet been determined because of the failure of in vitro virus isolation. Here, we report a surprising isolation of a PAstV5 strain from a clinical classical swine fever virus (CSFV)-infected tissue sample, which can stably passage in PK-15 cells, and coinfection with CSFV significantly enhanced the replication of PAstV5, possibly through suppression of beta interferon production. Thus, the first isolated PAstV5 strain will be useful for investigating the biological and pathogenic properties of this virus, and the findings obtained in this study provide new insights into defining the interaction mechanism between CSFV and PAstV5.

Comprehensive Analysis of Codon Usage on Porcine Astrovirus.

Porcine astrovirus (PAstV), associated with mild diarrhea and neurological disease, is transmitted in pig farms worldwide. The purpose of this study is to elucidate the main factors affecting codon usage to PAstVs. Phylogenetic analysis showed that the subtype PAstV-5 sat at the bottom of phylogenetic tree, followed by PAstV-3, PAstV-1, PAstV-2, and PAstV-4, indicating that the five existing subtypes (PAstV1-PAstV5) may be formed by multiple differentiations of PAstV ancestors. A codon usage bias was found in the PAstVs-2,3,4,5 from the analyses of effective number of codons (ENC) and relative synonymous codon usage (RSCU). Nucleotides A/U are more frequently used than nucleotides C/G in the genome CDSs of the PAstVs-3,4,5. Codon usage patterns of PAstV-5 are dominated by mutation pressure and natural selection, while natural selection is the main evolutionary force that affects the codon usage pattern of PAstVs-2,3,4. The analyses of codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) showed the codon usage similarities between the PAstV and animals might contribute to the broad host range and the cross-species transmission of astrovirus. Our results provide insight into understanding the PAstV evolution and codon usage patterns.

Rapid and visual detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription loop-mediated isothermal amplification.

To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription-quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.

Metatranscriptomic Analysis of Virus Diversity in Urban Wild Birds with Paretic Disease.

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spillover to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species, and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased metatranscriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds, including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case, the presence of the virus was confirmed by reverse transcription (RT)-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular, and neuropathology in birds of the Corvidae and Artamidae families and neuropathology in members of the Psittaculidae The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock, and human health.IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing, we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free-ranging wildlife and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

Genomic and phylogenetic characteristics of a novel goose astrovirus in Anhui Province, Central-Eastern China.

Goose astrovirus (GAstV) causes a novel disease characterized by urate deposition in the viscera and joints in goslings in many provinces of China, leading to huge economic losses in the goose industry. To better understand the genetic diversity of GAstV in the Anhui Province, Central-Eastern China, 48 kidney samples from goslings with gout were subjected to reverse-transcription polymerase chain reaction (RT-PCR) analysis for detecting GAstV, and phylogenetic analysis of whole genomes and ORFs was performed. Thirty-five samples were GAstV-positive, indicating that the virus is a frequent cause of gout. The whole genomes of 5 GAstV strains were successfully sequenced and named AHAU1-5. The sequenced genomes and those of reference GAstV strains in GenBank displayed 97.4-99.8% similarity. The isolates had high nucleotide sequence similarity with the GAstV reference strain SDPY. A phylogenetic analysis showed that AHAU1 and AHAU4 were closely related to the reference strain SDPY; AHAU2, AHAU3, and AHAU5 formed separate branches. Furthermore, recombination analysis revealed putative recombination sites in the Jiangsu strains that originated from strains in the Anhui and Shandong Provinces, accompanied by the recombination of different strains in the Anhui Province. This study is the first to carry out systematic phylogenetic analysis of GAstV isolated in the Anhui Province, Central-Eastern China. By improving our understanding of the diversity of GAstV in the Anhui Province, these results provide a basis for the prevention and control of its spread.

Prevalence of murine astrovirus in laboratory animal facilities in Japan.

To investigate the prevalence of murine astrovirus (MuAstV) in mice in laboratory animal facilities in Japan, a polymerase chain reaction (PCR) test targeting the RNA-dependent RNA polymerase (RdRP) gene was performed on the cecum contents of 1,212 mice (1,183 immunocompetent mice and 29 immunodeficient mice) from 226 facilities. The results showed that 118 (52.2%) of the 226 facilities were positive for MuAstV. Out of the 1,212 mice, 424 (35.0%) were positive. No gross lesions were observed in any of the mice examined. A phylogenetic analysis for 15 selected strains revealed that 13 strains formed one cluster, while two were genetically distant from that cluster. These results suggest that multiple strains are prevalent in laboratory mice in Japan.