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The picornavirus Aichi virus (AiV) is a non-enveloped RNA virus that causes acute gastroenteritis symptoms, such as diarrhea, abdominal pain, nausea, vomiting, and fever. Antiviral host defense involves the fast response of type I interferon (IFN) and the secretion of inflammatory cytokines against pathogens. However, the intestinal inflammatory and antiviral response to AiV infection is poorly understood. This study evaluated the antiviral activity of intestinal epithelial cells (IECs), which form a single-cell layer separating the bowel wall from pathogens. Isolated primary mouse IECs were subjected to AiV infection and virion production, inducing the mRNA expression of type I/type III IFNs and inflammatory cytokines. The mechanism involved induced the expression of phospho-IFN regulatory factor 3 and mitochondrial antiviral-signaling protein of type I IFN signaling. These findings were also observed in AiV-infected human colon carcinoma cells. In summary, a viral productive and pathogenic infection of AiV in primary murine IECs is validated.

Background Canine Kobuvirus (CaKoV) has been detected both in healthy and diarrheic dogs and in asymptomatic wild carnivores. In this study, we conducted a survey of CaKoV at small animal hospitals in Bangkok and vicinity of Thailand during September 2016 to September 2018. Results Three hundred and seven rectal swab samples were collected from healthy dogs (n = 55) and dogs with gastroenteritis symptoms (n = 252). Of 307 swab samples tested by using one-step RT-PCR specific to 3D gene, we found CaKoV positivity at 17.59% (54/307). CaKoVs could be detected in both sick (19.44%) and healthy (9.09%) animals. In relation to age group, CaKoV could be frequently detected in younger dogs (25.45%). Our result showed no seasonal pattern of CaKoV infection in domestic dogs. In this study, we characterized CaKoVs by whole genome sequencing (n = 4) or 3D and VP1 gene sequencing (n = 8). Genetic and phylogenetic analyses showed that whole genomes of Thai CaKoVs were closely related to Chinese CaKoVs with highest 99.5% amino acid identity suggesting possible origin of CaKoVs in Thailand. Conclusions In conclusion, this study was the first to report the detection and genetic characteristics of CaKoVs in domestic dogs in Thailand. CaKoVs could be detected in both sick and healthy dogs. The virus is frequently detected in younger dogs. Thai CaKoVs were genetically closely related and grouped with Chinese CaKoVs. Our result raises the concerns to vet practitioners that diarrhea in dogs due to canine Kobuvirus infection should not be ignored. Electronic supplementary material The online version of this article (10.1186/s12917-019-1994-6) contains supplementary material, which is available to authorized users.

In this study, the full length genomes of three phylogenetically distant picornaviruses (family Picornaviridae) belonging to the genus Rosavirus (rat08/rRoB/HUN, MN116648), Kobuvirus (rat08/rAiA/HUN, MN116647), and Cardiovirus (rat08/rCaB/HUN, MN116646) were obtained from a single faecal sample of a free-living Norway rat (Rattus norvegicus) in Hungary using viral metagenomics and RT-PCR/Sanger sequencing. The acquired complete genomes were in silico analyzed in detail revealing the presence of a second minor open reading frame encoding an alternative Leader peptide (L*) in rat08/rCaB/HUN and a ca. 222 nt-long sequence repeat with compact secondary RNA structure in the 3′ UTR of rat08/rRoB/HUN. The studied rat picornaviruses were frequently detectable by RT-PCR with relatively high viral loads ranged between 8.99E+02 and 1.29E+06 copies/ml in rat faecal samples collected from five geographically distant locations throughout Hungary. The VP1 sequence-based phylogenetic analyses show the presence of multiple, mostly location-specific lineages for all three picornaviruses. Rat rosavirus and rat cardiovirus were identified in spleen while rat cardiovirus was also detected in liver, muscle and kidney samples with variable copy numbers (6.42E+01–1.90E+05 copies/μg total RNA) suggesting extra-intestinal dissemination. Both viruses were also prevalent (70.0% and 18.2%) among two populations of laboratory rats (“Wistar-type” and “hooded-type”) held in different, isolated laboratory animal units.

Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornavirus stability., Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses are not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for interhost transmission, since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 [CVB3]), Kobuvirus (Aichi virus), and Cardiovirus (mengovirus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while mengovirus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and mengovirus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria, since viruses bound to bacteria in a pulldown assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses. IMPORTANCE Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornavirus stability.

Enteric viral diseases of swine are one of the most frequent disorders causing huge economic losses in pork production. After the reappearance of an emerging enteropathogen, porcine epidemic diarrhoea virus (PEDV) in Hungary in 2016, an extensive survey was initiated in an attempt to identify diarrhoea-related porcine viruses, including adeno-, astro-, boca-, calici-, circo-, corona-, kobu-, rota- and Torque teno viruses. A total of 384 faecal samples collected during a twoyear period from diarrhoeic and asymptomatic pigs of various ages in 17 farms were screened by conventional and real-time PCR methods. Half of the samples contained at least one examined virus with the dominance of kobuvirus (55.1%) followed by bocaviruses (33.2%) and rotavirus groups A and C together (20.9%), while coronaviruses including PEDV were not found in this set of samples. Statistical analysis showed a highly significant difference (P < 0.0001) in the frequency of single infections compared to mixed ones with the exception of weaned pigs, in which group additionally most viruses were detected. The results of this study suggest that the complexity of this disease may vary with age, which makes the prevention of diarrhoea a challenge, especially in weaned pigs.

Background In recent years, Aichi virus (AiV) has been involved in acute viral gastroenteritis outbreaks. However, the common pathogenesis of AiV releases more in subclinical infections underestimating the impact of AiV in human health. Objectives The present study describes the presence and genetic diversity of AiV in patients with gastroenteritis in Northwestern Spain. Study design: A total of 2667 stool samples, obtained between July 2010 and June 2011, from diarrheic outpatients were studied for detection and molecular characterization of AiV using PCR techniques followed by sequencing and phylogenetic analyses. Results The virus was detected in 124 (5.0%) of the samples among all age groups. Coinfections were also detected, from the 124 positive samples, 72 (58.1%) were positive only for AiV, whereas mixed contaminations with Norovirus genogroup I or genogroup II, Sapovirus, or other enteric pathogens were detected in 52 (41.9%) samples. A total of 70 positive samples could be genotyped, being characterized as genotype A (58.6%) or B (41.4%). AiV was detected from August to April, being the highest number of AiV positive samples detected during autumn and winter seasons. Conclusions This survey remarks the importance of emerging enteric viruses in patients who require medical assistance, and offers more information about the real importance of AiV as gastroenteritis agent.

The progress of sequencing technologies has facilitated metagenomics projects on environmental samples like sewage water. The present study concerned the analysis of sewage samples collected from 3 locations in Xinjiang Uygur Autonomous Region in China. The analysis focused on RNA viruses known to infect humans and identified viruses from 10 families. The proportion of human virus species in the sewage samples was relatively stable with an average of 17%. Thirty virus species known to infect humans were identified and they belonged to 6 families: Picornaviridae (12), Astroviridae (11), Reoviridae (3), Caliciviridae (2), Papillomaviridae (1) and Picobirnaviridae (1). A total of 16 full-length genomes were generated from Astroviridae, Picornaviridae (Salivirus and Kobuvirus) and Picobirnaviridae. Astroviruses appeared to be the most present viruses and were detected in all sewage samples. Analyzing the virome of sewage samples should help to monitor any potential risks to public health.

This study presents the pathological, immunohistochemical, and molecular findings associated with the extra-intestinal detection of canine kobuvirus (CaKV) in a 5-month-old Chihuahua puppy, that had a clinical history of bloody-tinged feces. Principal pathological findings were interstitial pneumonia, necrotizing bronchitis, and parvovirus-induced enteritis. Molecular diagnostic methods identified CaKV within the cerebellum, cerebrum, lung, tonsil, and liver. CaKV and rotavirus were not identified within the feces and intestine. Immunohistochemistry (IHC) assays detected antigens of CDV and CAdV-1 in the lungs. These results confirmed the extra-intestinal detection of CaKV in this puppy and represent the first extra-intestinal detection of CaKV in a dog.

By screening 139 rectal swabs collected from either asymptomatic or diarrhoeic goats in Italy, we identified kobuvirus RNA in eight samples (5.8 %). Higher positivity rates were observed in diarrhoeic goats (6.5 %, 3/46) than in asymptomatic animals (5.4 %, 5/93), although the difference was not statistically significant. Based on the analysis of a portion of the 3D gene, four strains were found to share the highest nucleotide (nt) sequence identity with bovine kobuviruses (95.0-98.0 %), which had been detected previously in calves in the UK and Korea. Interestingly, two strains were genetically related to the newly discovered caprine kobuviruses (83.0-97.0 % nt sequence identity), which had been identified in black goats in Korea and in roe deer in Italy. Taken together, these findings demonstrate that kobuviruses are common enteric viruses of goats, although their clinical relevance remains to be investigated.

Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV.

Kobuvirus is a newly discovered virus that belongs to the Kobuvirus genus in Picornaviridae family, which comprised of three species including Aichivirus A, Aichivirus B, and Aichivirus C. The kobuvirus isolated from human has been classified as Aichi virus 1 and belongs to Aichivirus A species. The present study aimed to assess the epidemiology and to perform molecular characterization of Aichi virus 1 in children admitted to hospitals with acute gastroenteritis in Chiang Mai, Thailand. A total of 923 fecal specimens collected from January, 2011 to December, 2013 were screened for the presence of Aichi virus 1 by RT semi-nested PCR. Out of 923 fecal specimens tested, Aichi virus 1 was detected with the prevalence of 2.6% (24/923). Of these, 0.3% (3/923) was genotype A and 2.3% (21/923) were genotype B. It is interesting to note that the genotype A showed the nucleotide sequence closely related to the Aichi virus reference strain isolated from sewage in Tunisia, while genotype B was most closely related to other human Aichi virus B reference strains. The results suggest that Aichi virus 1 of both genotypes A and B are circulating in pediatric patients in Thailand. J. Med. Virol. 89:234-238, 2017. © 2016 Wiley Periodicals, Inc.

The aims of this study were to examine the removal of bacteria and viruses by household point-of-use (POU) treatments and to apply a previously developed large-volume virus concentration method (∼20 L). First, the removal of microbes by household POU treatment was investigated in the laboratory. Second, the prevalence of viruses in drinking water sources for households and the removal efficiency of microbes by POU treatments in two suburban communities in Hanoi, Vietnam, were investigated. Indigenous pepper mild mottle virus (PMMoV) was used as the main target together with adenovirus, Aichi virus, enterovirus, F-specific bacteriophage genogroup 1, and Escherichia coli to investigate the removal efficiency of household treatments. The results from laboratory and field survey were compared. From the laboratory study, ceramic membranes were not effective for removing viruses and bacteria from water; pathogen reduction was less than 1.5 log10. By contrast, reverse osmosis (RO) devices reduced microbes by 3 to5 log10. In a field study, PMMoV was found to be the most prevalent waterborne virus. Household sand filtration was ineffective for removing E. coli, total coliforms and PMMoV; the reduction was less than 1 order of magnitude. Boiling the water and then filtering it with a ceramic membrane reduced E. coli by 3 orders of magnitude, but this was not effective for removing PMMoV. RO filtration was one of the promising methods for removing E. coli, total coliforms and PMMoV to below their detection limits in most of the samples studied. The removal of E. coli, total coliforms and PMMoV was2.3,4 and3 log10, respectively. The laboratory results of virus removal efficiency by POU devices agreed with the field study. Due to the prevalence and characteristics of PMMoV, it is a strong candidate for an indigenous indicator to investigate the viral removal efficiency of household POU treatments.

Aqueous Hibiscus sabdariffa extracts possess antimicrobial properties with limited information available on their antiviral effects. Aichi virus (AiV) is an emerging foodborne pathogen that causes gastroenteritis. Vaccines are currently unavailable to prevent their disease transmission. The objective of this study was to determine the antiviral effects of aqueous H. sabdariffa extracts against AiV. AiV at ~5 log PFU/ml was incubated with undiluted (200 mg/ml), 1:1 (100 mg/ml) or 1:5 (40 mg/ml) diluted aqueous hibiscus extract (pH 3.6), phosphate-buffered saline (pH 7.2 as control), or malic acid (pH 3.0, acid control) at 37 °C over 24 h. Treatments were stopped by serially diluting in cell-culture media containing fetal bovine serum and titers were determined using plaque assays on confluent Vero cells. Each treatment was replicated thrice and assayed in duplicate. AiV did not show any significant reduction with 1:1 (100 mg/ml) or 1:5 (40 mg/ml) diluted aqueous hibiscus extracts or malic acid after 0.5, 1, or 2 h at 37 °C. However, AiV titers were reduced to non-detectable levels after 24 h with all the three tested concentrations, while malic acid showed only 0.93 log PFU/ml reduction after 24 h. AiV was reduced by 0.5 and 0.9 log PFU/ml with undiluted extracts (200 mg/ml) after 2 and 6 h, respectively. AiV treated with 1:1 (100 mg/ml) and 1:5 (40 mg/ml) diluted extracts showed a minimal ~0.3 log PFU/ml reduction after 6 h. These extracts show promise to reduce AiV titers mainly through alteration of virus structure, though higher concentrations may have improved effects.

Molecular replacement and noncrystallographic symmetry averaging were used to detwin a data set affected by perfect hemihedral twinning. The noncrystallographic symmetry averaging of the electron-density map corrected errors in the detwinning introduced by the differences between the molecular-replacement model and the crystallized structure., Hemihedral twinning is a crystal-growth anomaly in which a specimen is composed of two crystal domains that coincide with each other in three dimensions. However, the orientations of the crystal lattices in the two domains differ in a specific way. In diffraction data collected from hemihedrally twinned crystals, each observed intensity contains contributions from both of the domains. With perfect hemihedral twinning, the two domains have the same volumes and the observed intensities do not contain sufficient information to detwin the data. Here, the use of molecular replacement and of noncrystallographic symmetry (NCS) averaging to detwin a 2.1 Å resolution data set for Aichi virus 1 affected by perfect hemihedral twinning is described. The NCS averaging enabled the correction of errors in the detwinning introduced by the differences between the molecular-replacement model and the crystallized structure. The procedure permitted the structure to be determined from a molecular-replacement model that had 16% sequence identity and a 1.6 Å r.m.s.d. for Cα atoms in comparison to the crystallized structure. The same approach could be used to solve other data sets affected by perfect hemihedral twinning from crystals with NCS.

Canine kobuviruses (CaKVs) are newly recognized picornaviruses that have been recently detected in dogs in the U.S.A., Italy, U.K., the Republic of Korea and Tanzania. To trace the evolution of CaKV strains, a total of 201 fecal samples from rectal swabs of diarrheic dogs, which were obtained from May 2014 to April 2015 in northeast China, were detected by reverse transcription-PCR targeting a partial (504 bp) fragment of the 3D gene. Furthermore, a phylogenetic analysis of the CaKV strains identified in northeast China was conducted based on the partial 3D gene sequence. The results indicated that 36 fecal samples (17.91%, 36/201) were positive for CaKV, in which the co-infection rates of canine coronavirus, canine parvovirus-2 and canine bocavirus were 58.33%, 41.67%, and 11.11%, respectively. Sequence comparison of the partial 3D gene revealed nucleotide homologies of 94.4-100%, 95.6-98.6%, 94.3-97.6%, 94.4-96.3% and 93.3-95.1% within the 36 Chinese CaKV strains, and between the 36 Chinese CaKV strains and four CaKV reference strains from South Korea, Italy, U.S.A. and Tanzania, respectively. A phylogenetic tree revealed that the 36 Chinese CaKV strains formed one specific CaKV lineage with CaKVs that have recently been identified in other countries. The 36 Chinese CaKV strains were closely related to CaKV reference strains from Asia and Europe, but differed genetically from CaKV reference strains from North America and Africa. This study provides evidence that CaKVs circulate in diarrhoetic dogs in China and that they exhibit substantial genetic diversity and high co-infection rates with other enteric viruses.

Picornaviruses (family Picornaviridae) are small, non-enveloped viruses with positive sense, single-stranded RNA genomes. The numbers of the novel picornavirus species and genera are continuously increasing. Picornaviruses infect numerous vertebrate species from fish to mammals, but have not been identified in a member of the Lagomorpha order (pikas, hares and rabbits). In this study, a novel picornavirus was identified in 16 (28.6%) out of 56 faecal samples collected from clinically healthy rabbits (Oryctolagus cuniculus var. domestica) in two (one commercial and one family farms) of four rabbit farms in Hungary. The 8364 nucleotide (2486 amino acid) long complete genome sequence of strain Rabbit01/2013/HUN (KT325852) has typical picornavirus genome organization with type-V IRES at the 5′UTR, encodes a leader (L) and a single 2AH-box/NC proteins, contains a hepatitis-A-virus-like cis-acting replication element (CRE) in the 2A, but it does not contain the sequence forming a “barbell-like” secondary structure in the 3′UTR. Rabbit01/2013/HUN has 52.9%, 52% and 57.2% amino acid identity to corresponding proteins of species Aichivirus A (genus Kobuvirus): to murine Kobuvirus (JF755427) in P1, to canine Kobuvirus (JN387133) in P2 and to feline Kobuvirus (KF831027) in P3, respectively. The sequence and phylogenetic analysis indicated that Rabbit01/2013/HUN represents a novel picornavirus species possibly in genus Kobuvirus. This is the first report of detection of picornavirus in rabbit. Further study is needed to clarify whether this novel picornavirus plays a part in any diseases in domestic or wild rabbits., Highlights • Rabbit picornavirus was detected in rabbit faecal samples collected from lapine farms in Hungary. • The complete genome of the first rabbit picornavirus was determined. • The rabbit picornavirus represents a novel species possibly in genus Kobuvirus.

Highlights • The first development a SYBR Green I-based real-time PCR assay to detect canine kobuvirus. • Canine kobuvirus (CaKoV) was detected at a rate of 8.7% in the Anhui province. • This method has implications for the prevention and control of CaKoV-infect in dogs., Canine kobuvirus (CaKoV) is a causative agent of gastroenteritis in dogs. Rapid detection of CaKoV is important for preventing and controlling this condition. In this study, an SYBR Green I-based quantitative real-time PCR assay was established for CaKoV detection. Specific primers targeting a highly conserved region of the CaKoV 3D gene were developed. After optimization, the method detected a minimum of 1 × 101 copies/µL with high specificity, stability, and repeatability. Moreover, the entire process only required approximately 1.5 h for completion. Our results were supported by those obtained for clinical samples, in which our developed method was successfully applied. The newly established real-time PCR is a rapid, sensitive, specific, and repeatable method for the quantitative detection of CaKoV and can, therefore, be used in epidemiological studies.

Caprine kobuvirus (CKoV), a member of the genus Kobuvirus, has only been identified in South Korea and Italy until now. In this study, 24 goat diarrheic fecal samples were collected from 3 farms in Sichuan province, China, and 87.5% (21/24) samples were detected as CKoV positive by RT-PCR. Meanwhile, full-length VP0, VP3, and VP1 genes were simultaneously cloned from 17 clinical samples. Phylogenetic analysis showed that all CKoV strains were most closely related to porcine kobuvirus based on amino acid (aa) sequences of VP0 and VP3 proteins, but CKoV strains were closely related to with Aichivirus B strains (ferret, bovine, and sheep kobuvirus) based on aa sequences of the VP1 protein. Interestingly, compared with known CKoV strains in the GenBank database, Chinese CKoV strains have unique amino acid changes in VP0 and VP1 proteins. Moreover, the first Chinese CKoV nearly complete genome was successfully obtained from a diarrheic fecal sample, named SWUN/F11/2019. Compared with the two known CKoV strains, five aa mutations (S60A, L252I, V267T, I, V 306 L, V331I) were found in the VP0 gene and 7 aa mutations (S57N, G, T243A, V244I, T, A248V, L, S251A, R252H, and M255L) were found in VP1 in the SWUN/F11/2019 genome. This was the first report of the detection and molecular characteristics of CKoV from goats in China, which could be helpful for improving the understanding of the prevalence and genetic evolution of CKoV.

Aichi virus 1 (AiV-1) has a worldwide distribution and is associated with gastroenteritis in humans. In this study, raw sewage and mussel samples were analyzed for the presence of AiV-1 using reverse transcription-PCR (RT-PCR). Amplification and sequencing of the 3CD and VP1 genomic regions followed by phylogenetic analysis using selected genome sequences revealed the presence of AiV-1, genotype B. The results highlight the importance of further screening to evaluate the prevalence and epidemiology of this clinically important virus in South Africa.

Background Surveillance and characterization of pig enteric viruses such as transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), rotavirus, astrovirus (PAstV), sapovirus (PSaV), kobuvirus and other agents is essential to evaluate the risks to animal health and determination of economic impacts on pig farming. This study reports the detection and genetic characterization of PAstV, PSaV in healthy and diarrheic domestic pigs and PEDV and TGEV in diarrheic pigs of different age groups. Results The presence of PAstV and PSaV was studied in 411 rectal swabs collected from healthy (n = 251) and diarrheic (n = 160) pigs of different age categories: suckling (n = 143), weaned (n = 147) and fattening (n = 121) animals on farms in Slovakia. The presence of TGEV and PEDV was investigated in the diarrheic pigs (n = 160). A high presence of PAstV infections was detected in both healthy (94.4%) and diarrheic (91.3%) pigs. PSaV was detected less often, but also equally in clinically healthy (8.4%) and diarrheic (10%) pigs. Neither TGEV nor PEDV was detected in any diarrheic sample. The phylogenetic analysis of a part of the RdRp region revealed the presence of all five lineages of PAstV in Slovakia (PAstV-1 – PAstV-5), with the most frequent lineages being PAstV-2 and PAstV-4. Analysis of partial capsid genome sequences of the PSaVs indicated that virus strains belonged to genogroup GIII. Most of the PSaV sequences from Slovakia clustered with sequences originating from neighbouring countries. Conclusions Due to no significant difference between healthy and diarrheic pigs testing of the presence of PAstV and PSaV provides no diagnostic value. Genetic diversity of PAstV was very high as all five lineages were identified in pig farms in Slovakia. PSaV strains were genetically related to the strains circulating in Central European region.

Ultrafiltration (UF) membranes are increasingly being used for wastewater reclamation treatment for their high removal of pathogens and suspended solids. However, breakage of UF membrane fibers could allow leakage of pathogens into the permeate and create health risks in the use of reclaimed water. Here, we assessed the log10 reduction value (LRV) of human enteric viruses and microbial indicators of new and aged UF modules in a pilot-scale UF process to evaluate the influence of fiber breakage. Norovirus genotypes I and II, Aichi virus, and Escherichia coli were not detected in any permeate samples of intact UF modules, but were detected in samples of damaged UF modules. LRVs of all microorganisms assayed decreased as fiber breakage of new UF modules increased, with maximum decreases of > 3.3 log10. Fiber breakage in the aged UF modules did not decrease LRVs of somatic coliphages and MS2, but breakage in the new UF modules did decrease them. Intact new UF modules gave higher LRVs than intact aged UF modules. When the LRV of intact UF module was assumed to be 1 or 2 log10, increasing fiber breakage did not significantly decrease the predicted LRV, but when it was ≥ 3 log10, it did decrease LRV, in good agreement with measured LRVs in the degraded UF modules. These results suggest that the LRV of intact UF modules affects the decrease in LRV and confirm the leakage of human enteric viruses following fiber breakage in UF modules of different ages in the UF process of wastewater reclamation.

Viruses are the leading cause of acute gastroenteritis in children worldwide. Understanding of the occurrence and genetic diversity of these viruses can help to prevent infections.The present study describes the presence, genetic diversity and possible recombination of five enteric viruses in children with gastroenteritis in Southwestern Nigeria.From August 2012 to December 2013, stool samples and sociodemographic data of 103 diarrheic children5 years were collected to detect and characterize rotavirus A, norovirus, human astrovirus, aichivirus and sapovirus using PCR techniques followed by sequencing and phylogenetic analyses.At least one virus was identified in 58.3% (60/103) of the stool samples. Rotavirus, norovirus and astrovirus were detected in 39.8% (41/103), 10.7% (11/103), and 6.8% (7/103) respectively. Notably, aichivirus was detected for the first time in Nigeria (1/103; 0.97%). Sapovirus was not detected in the study. Co-infections with rotavirus were observed in eight samples either with norovirus or astrovirus or aichivirus. Phylogenetic analyses of different genome regions of norovirus positive samples provided indication for recombinant norovirus strains. A novel astrovirus strain closely related to canine astrovirus was identified and further characterized for the first time.Viruses are the common cause of acute gastroenteritis in Nigerian infants with rotavirus as most frequently detected pathogen. New norovirus recombinants and a not yet detected zoonotic astrovirus were circulating in Southwestern Nigeria, providing new information about emerging and unusual strains of viruses causing diarrhea.

Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses specifically is not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for inter-host transmission since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 (CVB3)), Kobuvirus (Aichi virus) and Cardiovirus (Mengo virus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while Mengo virus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and Mengo virus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria since viruses bound to bacteria in a pull-down assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses.IMPORTANCERecent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effect of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles on picornavirus stability.

Molecular assays are currently the most widely used method to quantify pathogenic viruses in water; however, their performance is often disrupted by matrices present in environmental samples. The present study used ferrihydrite (Fh) treatment to mitigate inhibition of RT-qPCR virus detection from environmental water concentrates. Fh treatment was performed to improve the detection of spiked Aichi virus 1 (AiV) and Qβ bacteriophage (Qβ) in commercial humic acids and seawater concentrates. The optimal Fh doses were found to be 200, 500, and 1000 mgFe/L for humic acid concentrates at UV254 of 1.5, 3.0, and 6.0 cm-1 respectively, whereas this value was 1000 mgFe/L for the seawater concentrates at UV254 of 1.5 cm-1. At these optimal doses, the recoveries of spiked AiV and Qβ ranged from 2% to 12% in the humic acid and seawater concentrates, respectively. In addition, high levels of indigenous viruses (including AiV ≥ 2.81 log10 copies/mL and pepper mild mottle virus ≥ 2.77 log10 copies/mL) were detected from seawater concentrates after Fh treatment, while none were detected without Fh treatment. Fh treatment effectively mitigated the inhibitive effects from environmental water samples.

Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (106.42–107.01 copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.

Feline astrovirus (FeAstV) which belonged to the genus Mamastrovirus was first identified in the feces of kittens with diarrhea in the USA in 1981 by electron microscopy, and had been reported in many countries. Presently, there are no any reports of the circulation of FeAstV in mainland China. We performed this study to investigate the apparent prevalence and genetic variability of FeAstV infected in cats in mainland China for the first time. We tested fecal samples of 105 cats with diarrhea and 92 asymptomatic cats in five cities in northeast China by RT-PCR targeting RNA-dependent RNA polymerase (RdRp) gene of FeAstV, and analyzed sequences variability and phylogenetic evolution based on the complete capsid gene of FeAstV strains obtained from positive samples. The overall prevalence of FeAstV was 23.4% (46/197) of which 38 were tested in cats with diarrhea (36.2%, 38/105) and 8 were in asymptomatic cats (8.7%, 8/92). Mixed infection with other enteroviruses including feline parvovirus (FPV), feline bocavirus (FBoV) and feline kobuvirus (FeKoV) was found in 38 FeAstV-positive samples. Phylogenetic analysis based on the complete capsid gene revealed all FeAstV strains were divided into two different groups with a 0.454±0.016 of mean amino acid genetic distance between two groups, suggesting that FeAstVs should be classified into two different genotype species. This study provided the first molecular evidence that FeAstV with considerable genetic diversity was circulating in northeast China, and analyzed genetic variability and classification of FeAstVs for the first time.

Canine kobuvirus (CaKoV) is a newly emerging virus in dogs, which relates to the diarrhea of dogs. To investigate the CaKoV infection in dog population, fecal samples of dogs were collected from three provinces of China in 2015. The results of genetic analysis based on the complete VP1 gene showed that six CaKoVs isolates in this study were closely related with the Chinese canine originated isolate CH1 (90.6%-91.9% nucleotide identities). The phylogenetic analysis demonstrated that the Chinese isolates clustered into a unique branch compared with isolates from other countries. The present study suggested that the CaKoVs had established infection in Chinese dog population. The systematic epidemiological investigation should be further carried out to evaluate the prevalence of the CaKoV infection in China.

Cross-species transmission of viruses poses a sustained threat to public health. Due to increased contact between humans and other animal species the possibility exists for cross-species transmissions and ensuing disease outbreaks. By using conventional PCR amplification and next generation sequencing, we obtained 130 partial or full genome kobuvirus sequences from humans in a sentinel cohort in Vietnam and various mammalian hosts including bats, rodents, pigs, cats, and civets. The evolution of kobuviruses in different hosts was analysed using Bayesian phylogenetic methods. We estimated and compared time of origin of kobuviruses in different host orders; we also examined the cross-species transmission of kobuviruses within the same host order and between different host orders. Our data provide new knowledge of rodent and bat kobuviruses, which are most closely related to human kobuviruses. The novel bat kobuviruses isolated from bat roosts in Southern Vietnam were genetically distinct from previously described bat kobuviruses, but closely related to kobuviruses found in rodents. We additionally found evidence of frequent cross-species transmissions of kobuviruses within rodents. Overall, our phylogenetic analyses reveal multiple cross-species transmissions both within and among mammalian species, which increases our understanding of kobuviruses genetic diversity and the complexity of their evolutionary history.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

Objective: Since its discovery, Aichivirus (AiV) A has been detected, with an incidence of 0.9–4.1%, primarily when studying outbreaks of diarrhea in children or young adults. In this paper, we report the first detection of AiV in Piedmont, Italy, in pediatric patients. Methods: A total of 159 fecal specimens (from 96 males and 63 females) previously screened for rotaviruses, adenoviruses, noroviruses, human parechoviruses, saliviruses, and sapoviruses were collected from infants and children with acute gastroenteritis. Results: The most commonly detected virus was norovirus GII (33.80%), fol lowed by rotavirus (21.30%), astrovirus (18.87%), boca virus (13.92%), sapovirus (10.90%), parechovirus (8%), norovirus GI (6.70%), adenovirus (1%), and salivirus (0.52%). Real-time polymerase chain reaction detected AiV A in 1 (0.62%) case subjects. AiV A was detected in monoinfection only in January. Conclusions: Our results indicate that AiV may be associated with a limited number of diarrhea cases in pediatric patients.

Highlights • Porcine astrovirus type 4 was prevalent in the 49 European farms with a high viral load. • Rotaviruses were mainly found in diarrheic pigs. • Kobuvirus and porcine circovirus 2 were ubiquitous., Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

During surveillance for bovine diarrhea of unknown causes in Japanese black cattle in Kagoshima Prefecture, Japan, we found two types of novel kobu-like viruses in fecal samples of calves. Sequence analyses revealed that they had L protein and 2A protein with H-box/NC sequence motif, which are present in kobuviruses. Phylogenetic analysis revealed that they were related to kobuviruses; however, they clustered apart from other kobuviruses. In the prevalence study of two types of novel kobu-like viruses, 16.9% and 10.4% prevalence of these viruses were observed in the feces of diarrheal calves in this area.

Porcine kobuvirus has been detected from pig fecal samples in the USA, but there is still no information on the full-length genomes. In this study, we characterized the first complete genomic sequence of a US porcine kobuvirus strain OH/RV50/2011. The viral genome is 8123 nucleotides (nt) long, including a 576-nt 5'-untranslated region (UTR), a 7380-nt polyprotein encoding sequence, and a 167-nt 3'-UTR. A complete genome sequence alignment suggested that two types of porcine kobuviruses were found based on whether a 30-aa deletion existed in the 2B encoding region. Furthermore, several conserved motifs that can be used for the design of universal kobuvirus or porcine kobuvirus-specific primers were verified in non-structural protein genes. Phylogenetic analysis based on the complete genome sequence showed that RV50 was grouped with other porcine kobuviruses and more closely related to Chinese strains. Secondary structure analysis of the 5'-UTR showed that RV50 has three stem-loop domains in the first 108 nt and has a potential hepacivirus-/pestivirus-like type IV group-B-like internal ribosomal entry site, like the porcine kobuvirus prototype strain S-1. Codon usage analysis showed that the most preferred usage tends to be C or U at the end of a codon in a porcine kobuvirus genome. These results will be useful in understanding the evolution of porcine kobuviruses .

Aichi virus 1 (AiV-1), belonging to the genus Kobuvirus in the family Picornaviridae, has been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. AiV-1 is globally distributed and has been detected in various types of environmental samples, such as sewage, river water, groundwater, and shellfish. Recent environmental studies revealed that this virus could be detected in higher frequency and greater abundance than other human enteric viruses. These findings suggest that AiV-1 could potentially be an appropriate indicator of viral contamination in the environment because of its high prevalence in water environments as well as structural and genetic similarity with some of the other important enteric viruses. Further studies on the occurrence and fate of AiV-1 in environments, even in combination with clinical studies of many regions, are needed for a better understanding of their epidemiology, temporal and geographical distribution, environmental stability, and potential health risks to humans.

Two-hundred eight swine fecal samples from six Italian farms were tested using a kobuvirus-specific RT-PCR with primers that amplify a region within the 3D gene. All farms were kobuvirus positive, with prevalence rates ranging between 24 % and 84 %. Overall, 57.5 % of asymptomatic pigs and 49.7 % of animals with diarrhea were positive for kobuvirus. Sequence analysis showed a different predominant strain circulating on each farm and indicated that the strains detected were related to both European and Asiatic strains. A possible pathogenic role of kobuvirus should be investigated further, since infections with this virus occur frequently in pigs of different ages.

Highlights • Kobuviruses were found in cats with diarrhoea. • Kobuviruses were not detected in asymptomatic cats. • The complete genome sequence of one such strains was determined. • Kobuviruses resembling the newly described feline kobuviruses were identified. • First evidence on the circulation of feline kobuviruses outside the Asian continent., Kobuviruses have been identified in the enteric tract of several mammalian species but their role as enteric pathogens is still not defined. In this study, feline kobuviruses were found in 13.5% of cats with diarrhoea, but not in asymptomatic animals. In the full-length genome, one such strains, TE/52/13/ITA, displayed the highest nucleotide identity (96.0%) to the prototype strain FK-13. These results provide firm evidence that kobuviruses are common constituents of feline enteric viroma and that they are not geographically restricted to the Asian continent, where they were first signalled.

Background Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. Methods The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015–2016 were analyzed. Results A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. Conclusion These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae.

This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.

AiV-1 is considered an emerging human enteric pathogens and foodborne transmission has been documented as an important source of exposure for humans, chiefly in relation to non-safe, risky food habits. We surveyed the presence of AiV-1 in retail shellfish, including oysters and mussles, identifying the virus in 3/170 (1.8%) of the analysed samples. The AiV-1 positive samples were of different geographic origin. Upon sequence analysis of a portion of the 3CD junction region, two AiV strains identified from harvesting areas in Northern Italy were characterised as genotype B and displayed 99–100% identity at the nucleotide level to other AiV-1 strains detected in sewages in Central Italy in 2012, suggesting that such strains are stably circulating in Italian ecosystems. Interestingly, a strain identified from mussles harvested in Southern Italy could not be characterised firmly, as inferred in the Bayesian analysis and by sequence comparison, indicating that different AiV strains are also circulating in Italy. Viral contamination in retail shellfish challenges the microbiological guidelines for food control and requires the development and optimization of additional diagnostic and prevention strategies.

Background Porcine Deltacoronavirus (PDCoV) is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. Results In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR). This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), and porcine circovirus type 2 (PCV2). By screening a panel of clinical specimens (N = 192), this method presented a similar sensitivity with nested RT-PCR and was 1–2 log more sensitive than conventional RT-PCR in detection of PDCoV. Conclusions The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology. Electronic supplementary material The online version of this article (doi:10.1186/s40659-017-0135-6) contains supplementary material, which is available to authorized users.

In this study, a multiplex RT-PCR method was developed for detection of seven diarrhea-associated porcine viruses, including porcine teschovirus (PTV), porcine sapovirus (PSV), porcine deltacornavirus (PDCoV), porcine kobuvirus (PKV), porcine sapovirus (PSaV), porcine astrovirus (PAstV) and porcine torovirus (PToV). A total of 419 samples were screened by this method and results showed that PKV had the highest positive rate of 26.98%?45.79% and its mixed infection rate reached 9.52%-18.54%. On account of high positive rate of PKV and its important role in diarrhea disease, complete genomic sequences of three PKV positive samples were further sequenced. Three PKV labeled as PD-PKV, JS-PKV and CM-PKV were classified into porcine kobuvirus genus and had far genetic distance with other kobuviruses. The complete genome homologies among them were 88.1%-89.1%. CM-PKV had the highest identity with the Chinese strain JS-02a-CHN/2013 reported in 2013 while JS-PKV and PD-PKV were most closed to the K-30-HUN/2008/HUN strain reported in Hungary in 2008. This illustrates the significant genetic differences of the different PKV isolates in Shanghai while its relationship with the viral pathogenicity still needs to be explored. This research provides references for further understanding the prevalence of PKV and its role in swine diarrhea.为了解引起猪腹泻疫病的病毒性病原种类及感染现状，本研究首先针对7种报道较少但可引起猪腹泻的病毒性病原建立了多重RT-PCR检测方法，包括猪捷申病毒 (PTV)、猪萨佩罗病毒 (PSV)、猪丁型冠状病毒 (PDCoV)、猪嵴病毒 (PKV)、猪札幌病毒 (PSaV)、猪星状病毒 (PAstV)和猪环曲病毒 (PToV)。利用该方法对419份临床腹泻粪便样品进行筛检，结果显示PKV检出率最高，阳性率达26.98%–45.79%；PKV和其他病原混合感染率达9.52%–18.54%。基于PKV的高检出率及其在腹泻病中扮演重要角色，本试验进一步选取3个PKV阳性样品进行全基因组序列测定及遗传进化分析。测序结果表明，这3个阳性样品感染的PKV均归属于猪嵴病毒属，且与其他动物嵴病毒遗传距离较远，分别标记为CM-PKV、JS-PKV和PD-PKV；它们的全基因组同源性为88.1%–89.1%；CM-PKV与2013年报道的中国株JS-02a-CHN/2013同源性最高，而JS-PKV和PD-PKV则与2008年报道的匈牙利株K-30-HUN/2008/HUN同源性最高。这说明上海不同地区猪群中存在的PKV有明显的遗传差异，这种差异与病毒致病力的关系还有待深入研究。本研究为深入了解PKV的流行情况及探讨其在猪腹泻疫情中的作用提供了参考。.

Background Large-scale outbreaks of porcine epidemic diarrhea (PED) have re-emerged in China in recent years. However, little is known about the genetic diversity and molecular epidemiology of field strains of PED virus (PEDV) in China in 2016–2017. To address this issue, in this study, 116 diarrhea samples were collected from pig farms in 6 Chinese provinces in 2016–2017 and were detected using PCR for main porcine enteric pathogens, including PEDV, porcine deltacoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV) and porcine kobuvirus (PKV). In addition, the complete S genes from 11 representative PEDV strains were sequenced and analyzed. Results PCR detection showed that 52.6% (61/116) of these samples were positive for PEDV. Furthermore, sequencing results for the spike (S) genes from 11 of the epidemic PEDV strains showed 93–94% nucleotide identity and 92–93% amino acid identity with the classical CV777 strain. Compared with the CV777 vaccine strain, these strains had an insertion (A133), a deletion (G155), and a continuous 4-amino-acid insertion (56NNTN59) in the S1 region. Phylogenetic analysis based on the S gene indicated that the 11 assessed PEDV strains were genetically diverse and clustered into the G2 group. These results demonstrate that the epidemic strains of PEDV in China in 2016–2017 are mainly virulent strains that belong to the G2 group and genetically differ from the vaccine strain. Importantly, this is the first report that the samples collected in Hainan Province were positive for PEDV (59.2%, 25/42). Conclusions To our knowledge, this article presents the first report of a virulent PEDV strain isolated from Hainan Island, China. The results of this study will contribute to the understanding of the epidemiology and genetic characteristics of PEDV in China.