Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A.

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Recombinant Viruses from the Picornaviridae Family Occurring in Racing Pigeons.

Viruses from Picornaviridae family are known pathogens of poultry, although the information on their occurrence and pathogenicity in pigeons is scarce. In this research, efforts are made to broaden the knowledge on Megrivirus B and Pigeon picornavirus B prevalence, phylogenetic relationship with other avian picornaviruses and their possible connection with enteric disease in racing pigeons. As a result of Oxford Nanopore Sequencing, five Megrivirus and two pigeon picornavirus B-like genome sequences were recovered, among which three recombinant strains were detected. The recombinant fragments represented an average of 10.9% and 25.5% of the genome length of the Pigeon picornavirus B and Megrivirus B reference strains, respectively. The phylogenetic analysis revealed that pigeons are carriers of species-specific picornaviruses. TaqMan qPCR assays revealed 7.8% and 19.0% prevalence of Megrivirus B and 32.2% and 39.7% prevalence of Pigeon picornavirus B in the group of pigeons exhibiting signs of enteropathy and in the group of asymptomatic pigeons, respectively. In turn, digital droplet PCR showed a considerably higher number of genome copies of both viruses in sick than in asymptomatic pigeons. The results of quantitative analysis leave the role of picornaviruses in enteropathies of pigeons unclear.

Longitudinal analysis of the enteric virome in paediatric subjects from the Free State Province, South Africa, reveals early gut colonisation and temporal dynamics.

The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut.

Use of FTA card for the detection of two RNA (CSFV and SV-A) and two DNA viruses (ASFVand SuHV-1) of importance in veterinary medicine.

The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary.

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) - as a novel variant - could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs. Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well. Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

Isolation, Characterization, and Molecular Detection of Porcine Sapelovirus.

Porcine sapelovirus (PSV) is an important emerging pathogen associated with a wide variety of diseases in swine, including acute diarrhoea, respiratory distress, skin lesions, severe neurological disorders, and reproductive failure. Although PSV is widespread, serological assays for field-based epidemiological studies are not yet available. Here, four PSV strains were recovered from diarrheic piglets, and electron microscopy revealed virus particles with a diameter of ~32 nm. Analysis of the entire genome sequence revealed that the genomes of PSV isolates ranged 7569-7572 nucleotides in length. Phylogenetic analysis showed that the isolated viruses were classified together with strains from China. Additionally, monoclonal antibodies for the recombinant PSV-VP1 protein were developed to specifically detect PSV infection in cells, and we demonstrated that isolated PSVs could only replicate in cells of porcine origin. Using recombinant PSV-VP1 protein as the coating antigen, we developed an indirect ELISA for the first time for the detection of PSV antibodies in serum. A total of 516 swine serum samples were tested, and PSV positive rate was 79.3%. The virus isolates, monoclonal antibodies and indirect ELISA developed would be useful for further understanding the pathophysiology of PSV, developing new diagnostic assays, and investigating the epidemiology of the PSV.

Recombinase polymerase amplification assay for rapid detection of Seneca Valley Virus.

Seneca Valley virus (SVV) is related to vesicular disease in pigs, and its clinical symptoms are indistinguishable from other notifiable clinical symptoms of vesicular disease such as foot-and-mouth disease. The rapid and accurate detection of SVV is essential to confirm the pathogenic factors and initiate the implementation of control measures. The development of a rapid, simple, convenient, and low-cost molecular (nucleic acid amplification) test that can be used at the sample collection point has been identified as a key component for controlling SVV. This study describes the development and demonstration of recombinase polymerase amplification (RPA) test targeting the conserved regions of SVV for detection of SVV. The Primers and probes designed by us have shown good sensitivity and specificity in RPA test, which is helpful for RPA to be an effective tool for rapid diagnosis of SVV.

First Human Cosavirus Detection From Cerebrospinal Fluid in Hospitalized Children With Aseptic Meningitis and Encephalitis in Iran.

OBJECTIVE: Human cosavirus (HCosV) is a newly recognized virus that seems to be partly related to nonpolio flaccid paralysis and acute gastroenteritis in pediatric patients. However, the relationship between HCosV and diseases in humans is unclear. To assess an investigation for the occurrence of HCosV among pediatric patients involved in meningitis and encephalitis, we implemented a real-time quantitative polymerase chain reaction assay for detection and quantification of HCosV in stool specimens. MATERIALS AND METHODS: In this study, a total of 160 cerebrospinal fluid samples from September 2019 to October 2020 were collected from presenting pediatric patients with meningitis and encephalitis in a Karaj hospital, Iran. After viral RNA extraction, the real-time quantitative polymerase chain reaction was performed to amplify the 5'Un-Translated Region region of the HCosV genome and viral load was analyzed. RESULTS: Of the 160 samples tested, the HCosV genomic RNA was detected in 2/160 (1.25%) of samples. The minimum viral load of HCosV was 3.5 × 103 copies/mL from 4 years male patient. The maximum viral load was determined to be 2.4 × 105 copies/mL in one sample obtained from 3.5 years female patient. CONCLUSIONS: This is the first documentation of HCosV detection in cerebrospinal fluid samples that better demonstrates relation of HCosV with neurologic diseases including meningitis and encephalitis. Also, these results indicate that HCosV has been circulating among Iranian pediatric patients.

A Novel Picornavirus Discovered in White Leg Shrimp Penaeus vannamei.

Global shrimp farming is increasingly threatened by various emerging viruses. In the present study, a novel picornavirus, Penaeus vannamei picornavirus (PvPV), was discovered in moribund White leg shrimp (Penaeus vannamei) collected from farm ponds in China in 2015. Similar to most picornaviruses, PvPV is non-enveloped RNA virus, with a particle diameter of approximately 30 nm. The sequence of the positive single-stranded RNA genome with a length of 10,550 nts was characterized by using genome sequencing and reverse transcription PCR. The existence of PvPV related proteins was further proved by confirmation of viral amino acid sequences, using mass spectrometry analysis. Phylogenetic analysis based on the full-length genomic sequence revealed that PvPV was more closely related to the Wenzhou shrimp virus 8 than to any other dicistroviruses in the order Picornavirales. Genomic sequence conservative domain prediction analysis showed that the PvPV genome encoded a large tegument protein UL36, which was unique among the known dicistroviruses and different from other dicistroviruses. According to these molecular features, we proposed that PvPV is a new species in the family Dicistroviridae. This study reported the first whole-genome sequence of a novel and distinct picornavirus in crustaceans, PvPV, and suggests that further studies of PvPV would be helpful in understanding its evolution and potential pathogenicity, as well as in developing diagnostic techniques.

Novel Picornavirus Detected in Wild Deer: Identification, Genomic Characterisation, and Prevalence in Australia.

The use of high-throughput sequencing has facilitated virus discovery in wild animals and helped determine their potential threat to humans and other animals. We report the complete genome sequence of a novel picornavirus identified by next-generation sequencing in faeces from Australian fallow deer. Genomic analysis revealed that this virus possesses a typical picornavirus-like genomic organisation of 7554 nt with a single open reading frame (ORF) encoding a polyprotein of 2225 amino acids. Based on the amino acid identity comparison and phylogenetic analysis of the P1, 2C, 3CD, and VP1 regions, this novel picornavirus was closely related to but distinct from known bopiviruses detected to date. This finding suggests that deer/bopivirus could belong to a novel species within the genus Bopivirus, tentatively designated as "Bopivirus C". Epidemiological investigation of 91 deer (71 fallow, 14 sambar and 6 red deer) and 23 cattle faecal samples showed that six fallow deer and one red deer (overall prevalence 7.7%, 95% confidence interval [CI] 3.8-15.0%) tested positive, but deer/bopivirus was undetectable in sambar deer and cattle. In addition, phylogenetic and sequence analyses indicate that the same genotype is circulating in south-eastern Australia. To our knowledge, this study reports for the first time a deer-origin bopivirus and the presence of a member of genus Bopivirus in Australia. Further epidemiological and molecular studies are needed to investigate the geographic distribution and pathogenic potential of this novel Bopivirus species in other domestic and wild animal species.

Identification and genome analysis of a novel picornavirus from captive belugas (Delphinapterus leucas) in China.

The discovery of new viruses is important for predicting their potential threats to the health of humans and other animals. A novel picornavirus was identified from oral, throat, and anal swab samples collected from belugas (Delphinapterus leucas), from Dalian Sun Asia Tourism Holding Co., China, between January and December 2018, using a metagenomics approach. The genome of this novel PicoV-HMU-1 strain was 8197 nucleotides (nt) in length, with a open reading frame (from 1091 to 8074 nt) that encoded a polyprotein precursor of 2328 amino acids. Moreover, the genomic length and GC content of PicoV-HMU-1 were within the ranges found in other picornaviruses, and the genome organization was also similar. Nevertheless, PicoV-HMU-1 had a lower amino acid identity and distinct host species compared with other members of the Picornaviridae family. Phylogenetic trees were constructed based on the P1 and 3D amino acid sequences of PicoV-HMU-1 along with representative members of the Picornaviridae family, which showed that PicoV-HMU-1 was related to unclassified bat picornaviruses groups. These findings suggest that the PicoV-HMU-1 strain represents a potentially novel genus of picornavirus. These data can enhance our understanding of the picornavirus genetic diversity and evolution.

Description of the first isolates of guinea fowl corona and picornaviruses obtained from a case of guinea fowl fulminating enteritis.

Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B. The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis".RESEARCH HIGHLIGHTSFirst isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.

Identification of boosepivirus B in U.S. calves.

Bovine enteric disease has a complex etiology that can include viral, bacterial, and parasitic pathogens and is a significant source of losses due to morbidity and mortality. Boosepivirus was identified in calves with enteric disease with unclear etiology in Japan in 2009 and has not been reported elsewhere. Metagenomic sequencing and PCR here identified boosepivirus in bovine enteric disease diagnostic submissions from six states in the USA with 98% sequence identity to members of the species Boosepivirus B. In all cases, boosepivirus was identified as a coinfection with the established pathogens bovine coronavirus, bovine rotavirus, and cryptosporidia. Further research is needed to determine the clinical significance of boosepivirus infection.

Viral Metagenomic Analysis of Japanese Quail (Coturnix japonica) with Enteritis in the Republic of Korea.

We performed viral metagenomics analysis of Japanese quail affected with enteritis to elucidate the viral etiology. Metagenomics generated 21,066,442 sequence reads via high-throughput sequencing, with a mean length of 136 nt. Enrichment in viral sequences suggested that at least three viruses were present in quail samples. Coronavirus and picornavirus were identified and are known as pathogens causing quail enteritis that match the observed morphology. Abundant reads of coronavirus from quail samples yielded four fragment sequences exhibiting six genomes of avian coronavirus. Sequence analysis showed that this quail coronavirus was related to turkey coronavirus and chicken infectious bronchitis virus. Quail picornavirus 8177 bp in size was identified and was similar to the QPV1/HUN/01 virus detected in quails without clinical symptoms in Hungary with 84.6% nucleotide and 94.6% amino acid identity. Our results are useful for understanding the genetic diversity of quail viruses. Further studies must be performed to determine whether quail coronavirus and quail picornavirus are pathogens of the digestive tract of quails.

Artículo regular­Análisis metagenómico viral de la codorniz japonesa (Coturnix japonica) con enteritis en la República de Corea. Se realizó un análisis de metagenómica viral de codornices japonesas afectadas con enteritis para dilucidar la etiología viral. La metagenómica generó 21,066,442 lecturas de secuencia mediante secuenciación de alto rendimiento, con una longitud media de 136 nucleótidos. El enriquecimiento en secuencias virales sugirió que al menos tres virus estaban presentes en las muestras de codorniz. Se identificaron coronavirus y picornavirus que son conocidos como patógenos que causan enteritis de codornices que coinciden con la morfología observada. Las lecturas abundantes de coronavirus de muestras de codorniz produjeron cuatro secuencias de fragmentos que exhibían seis genomas de coronavirus aviar. El análisis de secuencia mostró que este coronavirus de codorniz estaba relacionado con el coronavirus del pavo y con el virus de la bronquitis infecciosa del pollo. Se identificó un picornavirus de codorniz de 8177 pares de bases de tamaño y fue similar al virus QPV1/HUN/01 detectado en codornices sin signos clínicos en Hungría con 84.6% de nucleótidos y 94.6% de identidad de aminoácidos. Estos resultados son útiles para comprender la diversidad genética de los virus de la codorniz. Se deben realizar más estudios para determinar si el coronavirus y el picornavirus de las codornices son patógenos del tracto digestivo de las codornices.

New Parvoviruses and Picornavirus in Tissues and Feces of Foals with Interstitial Pneumonia.

Six foals with interstitial pneumonia of undetermined etiology from Southern California were analyzed by viral metagenomics. Spleen, lung, and colon content samples obtained during necropsy from each animal were pooled, and nucleic acids from virus-like particles enriched for deep sequencing. The recently described equine copiparvovirus named eqcopivirus, as well as three previously uncharacterized viruses, were identified. The complete ORFs genomes of two closely related protoparvoviruses, and of a bocaparvovirus, plus the partial genome of a picornavirus were assembled. The parvoviruses were classified as members of new ungulate protoparvovirus and bocaparvovirus species in the Parvoviridae family. The picornavirus was classified as a new species in the Salivirus genus of the Picornaviridae family. Spleen, lung, and colon content samples from each foal were then tested for these viral genomes by nested PCR and RT-PCR. When present, parvoviruses were detected in both feces and spleen. The picornavirus, protoparvovirus, and eqcopivirus genomes were detected in the lungs of one animal each. Three foals were co-infected with the picornavirus and either a protoparvovirus, bocaparvovirus, or eqcopivirus. Two other foals were infected with a protoparvovirus only. No viral infection was detected in one animal. The complete ORFs of the first equine protoparvoviruses and bocaparvovirus, the partial ORF of the third equine picornavirus, and their detection in tissues of foals with interstitial pneumonia are described here. Testing the involvement of these viruses in fatal interstitial pneumonia or other equine diseases will require larger epidemiological and/or inoculation studies.

Novel picornavirus (family Picornaviridae) from freshwater fishes (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) in Hungary.

In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fish species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete genome of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5'UTRIRES-?/P1(VP0-VP3-VP1)/P2(2A1NPG↓P-2A2H-box/NC-2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3'UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fish-origin species in an unassigned genus in the family Picornaviridae.

Four novel picornaviruses detected in Magellanic Penguins (Spheniscus magellanicus) in Chile.

Members of the Picornaviridae family comprise a significant burden on the poultry industry, causing diseases such as gastroenteritis and hepatitis. However, with the advent of metagenomics, a number of picornaviruses have now been revealed in apparently healthy wild birds. In this study, we identified four novel viruses belonging to the family Picornaviridae in healthy Magellanic penguins, a near threatened species. All samples were subsequently screened by RT-PCR for these new viruses, and approximately 20% of the penguins were infected with at least one of these viruses. The viruses were distantly related to members of the genera Hepatovirus, Tremovirus, Gruhelivirus and Crahelvirus. Further, they had more than 60% amino acid divergence from other picornaviruses, and therefore likely constitute novel genera. Our results demonstrate the vast undersampling of wild birds for viruses, and we expect the discovery of numerous avian viruses that are related to hepatoviruses and tremoviruses in the future.

Isolation and Characterization of Hepatitis E Virus Subtype 4b Using a PLC/PRF/5 Cell-Derived Cell Line Resistant to Porcine Sapelovirus Infection.

The human hepatocarcinoma cell line PLC/PRF/5 is susceptible to hepatitis E virus (HEV) infection and is used for HEV isolation. It is difficult to use the cell line for this purpose directly from fecal specimens of swine or wild boar contaminated with porcine sapelovirus (PSV) because PSV infection results in rapid and extensive cytopathic effects in PLC/PRF/5 cells, interrupting the growth of HEV. Herein, we used a PSV infection-resistant cell line, N1380, derived from PLC/PRF/5 cells, and successfully isolated a HEV-4b strain from a PSV-positive swine fecal specimen. Our results indicated that N1380 cells are a useful tool for the isolation of HEV from swine or wild boar fecal specimens, even when the cells are co-infected with PSV.

POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.