VP1 Sequences Research Articles

Identification and assessment of pathogenicity of a naturally reassorted infectious bursal disease virus from Henan, China

Infectious bursal disease virus (IBDV) is still a vital etiological agent in poultry farms. IBDV outbreaks occasionally occur due to the presence of very virulent, reassortment or variant strains. Vaccine immunization has played crucial roles in IBD control for decades. However, survival pressure of IBDV from the vaccine immunization also increases the reassortments of circulating viruses. In this study, an IBDV strain was isolated from several broiler farms in Henan Province, central part of China, and named IBDV HN strain. Based on the results of RT-PCR, sequencing and phylogenic analyses of VP1 and VP2 genes, the IBDV HN strain is a novel reassortment strain in the Henan region. Segment A of this strain appears to originate from the very virulent IBDV strain, while segment B comes from the other field reassortment strains. This may be the result of natural reassortant of virus circulating in the field. About 60% (6/10) of experimentally infected specific pathogen-free chickens died after 3 to 5 d post-infection with typical symptom and pathological lesions. The IBDV HN strain was prone to horizontal transmission, which poses a serious threat to the chicken industry. Further investigation on the prevalence, virulence, and evolution of HN strain IBDV will provide a foundation for the prevention and control of the disease in this region.

Molecular and evolutionary characterization of norovirus GII.17 in the northern region of Brazil

BackgroundCurrently, norovirus (NoV) is associated with one-fifth of all acute gastroenteritis (AGE) cases worldwide. The NoV GII.17_2014 variant has been associated with gastroenteritis outbreaks in several Asian countries, replacing the previously dominant Sydney 2012 variant. There is limited data about circulation of this new strain in Brazil. This study aimed to describe the phylogenetic and evolutionary characteristics of the GII.17_2014 strains in the Northern region of Brazil.MethodsNoV was detected by enzyme immunoassay (EIA) in 645 stool samples of AGE cases that were reported in Pará and Amazonas states during 2015–2016. All positive samples were tested for NoV GI and GII by reverse transcription polymerase chain reaction (RT-PCR) and the amplicons were subjected to genome sequencing. The GII.17-positive samples were retested by PCR using different sets of designed primers, which target a highly conserved capsid gene region. Next, the amplicons were sequenced and phylogenetically analyzed using Bayesian inferences.ResultsOf the 645 samples tested, 208 (32.2%) tested were positive for NoV by EIA, among which 95 (45.7%) were genotyped. Among the genotyped samples, 12 (12.6%) were characterized as GII.17_2014 with the first case detected in November 2015 (1/30, 3.3%) and the others in 2016 (11/65, 16.9%). All strains found in our study were clustered in clade D (epidemic strain). The uncorrelated log-normal model estimations calculated the rate of evolution for GII-17 strains as 1.95 × 10− 3 (1.28 × 10− 3–2.63 × 10− 3). In total, 36 nucleotide changes were observed after analyzing the VP1 sequence, among which 28 occurred in the P2 region.ConclusionsThese data demonstrate the evolutionary dynamics in NoV GII.17_2014 strains, which indicated high mutation rates with nucleotide substitutions and indels that are related to the elevated levels of antigenic diversity. This partly explains the increase in viral prevalence.

Typing of Human Cosaviruses by sequencing of full VP1: Update on global genetic diversity and identification of possible new genotypes circulating in Tunisia, North Africa

Human Cosaviruses (HCoSVs) are relatively newly characterized picornaviruses; they have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. Previous studies showed HCoSV circulation in Tunisia and only six genotypes circulating in the country were reported. In the present study, we sequenced 27 complete VP1 genomic region from HCoSV isolates in human feces from healthy individuals and patients with acute flaccid paralysis in Tunisia. Most of the Tunisian sequences belong to species A (78%, 21 out of 27). Three sequences belong to species B, two to species E and one sequence to species D. The Tunisian sequences belonged to genotype A6, A7, A8, A10, A1, A17 and E2. Based on genetic distance criteria for assigning genotypes corresponding to neutralization serotypes in enteroviruses we also identified 4 new HCoSV genotypes named A25, B2, B3 and D6. Our study updates the genetic classification of HCoSVs, proposes new genotypes within species A, B and D and contributes to a better knowledge of the HCoSV circulation throughout the world.

Inactivation of foot-and-mouth disease virus in epithelium samples for safe transport and processing in low-containment laboratories

During a foot-and-mouth disease (FMD) outbreak, transport and testing of potentially infectious samples, including epithelium from suspect lesions, presents a biosafety risk, particularly in FMD-free countries. Therefore, treatment to inactivate virus prior to transport is important. Tongue epithelium from cattle infected with FMD virus (FMDV) serotype O (O ALG/3/2014 – Lineage O/ME-SA/Ind-2001d) or A (A IRN/22/2015 – Lineage A/ASIA/G-VII) was incubated in RNAlater, RNA Shield or phosphate-buffered saline (pH 7.4) at room temperature for 2, 6, 24 or 48 h. After incubation, tissues were homogenised and tested by virus titration. Viral RNA in the homogenate was quantified by RT-qPCR, used for sequencing, and transfected into LFBKαVβ6 cells to recover infectious virus.RNAlater reduced A IRN/22/2015 titres by 4 log10 after 24 h, and completely after 48 h incubation. While O ALG/3/2014 was detected by VI after 2, 6 and 24 h, titration yielded no infectious virus, likely as a result of freeze-thawing. RNA Shield was cytotoxic at high concentrations but was effective at inactivating both strains after 24 h. Regardless of reagent or inactivation period, RT-qPCR, VP1 sequencing, and transfection of RNA to recover infectious virus were possible. RNA Shield appears a better choice for FMDV inactivation in tissues, however 24 h incubation is recommended.

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin. In a phylogenetic tree, nucleotide sequences of segment A and B formed a common branch with those vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of deduced amino acid sequences segment B, confirmed the presence of the conserved TDN tripeptide found in all of the vvIBDV genotype and revealed the presence of 2 substitutions I472L and E688D specific for the vvIBDV Moroccan isolates. The deduced amino acid sequences of segment A genes showed the presence of the “signature” typical of the vvIBDV genotype and revealed the presence of 7 aa substitutions specific for the vvIBDV Moroccan strains. The evolution rate for IBDV VP2 gene was estimated at 5.875 × 10−4 substitutions/site/year. The estimation of the time to most common recent ancestor of Moroccan vvIBDV based on the VP2 sequences available was 31 years, corresponding to 3 years earlier than the first vvIBDV case detection in layers in the country.

Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt

Infectious bursal disease virus (IBDV), the agent of an immunosuppressive and sometimes lethal disease in chickens, is causing recurrent outbreaks in broiler chickens in Egypt. In particular, an antigenically modified isolate of very virulent IBDV (vvIBDV) called 99323 was detected in Egypt nearly twenty years ago; this isolate was shown to be experimentally controlled by an antigenically classical live vaccine. However, acute IBD is still reported, even in vaccinated flocks, and little is known about the genetic and antigenic properties of viruses currently circulating in Egypt. In the present study, ten samples collected in Egyptian broiler farms in 2015 as well as five samples collected in 2001 were analyzed. Genetic analyses of partial VP2 sequences revealed that 8 isolates clustered with vvIBDV strains, and 5 with tissue culture adapted and vaccine strains. Similar results were observed for partial VP1 sequences with the exception of isolate 160019, for which VP2 clustered with the vaccine strain Bursine while VP1 clustered with vvIBDV, suggesting reassortment. For isolates genetically related to vvIBDV, antigenic profiling revealed two patterns: while some isolates exhibited typical European vvIBDV reactivity with lack of binding of mAbs 5, other revealed extensive antigenic modifications, with lack of binding of mAbs 3, 5, 6, 8 and 9, similar to isolate 99323. These different patterns were associated with a single amino acid mutation at position 321 of VP2 that is located within peak PHI. Full genome sequencing was performed for three isolates, among which two were representative of the two antigenic patterns observed for vvIBDV as well as the reassortant isolate 160019. This study highlights the co-circulation of both antigenically typical and modified vvIBDV during the last fifteen years in Egypt.

National Epidemiology and Evolutionary History of Four Hand, Foot and Mouth Disease-Related Enteroviruses in China from 2008 to 2016.

Hand, foot and mouth disease (HFMD) is a major public health concern in China. The most predominant enteroviruses that cause HFMD have traditionally been attributed to enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16). Since its first large outbreak in 2008, the dominant HFMD pathogens are constantly changing. In 2013 and 2015, CVA6 exceeded both EVA71 and CVA16 to become the leading cause of HFMD in some provinces. However, there still lacks a comprehensive overview on the molecular epidemiology and evolution of HFMD-related enteroviruses at the national level. In this study, we performed systematic epidemiological analyses of HFMD-related enteroviruses using the data of 64 published papers that met the inclusion criteria, and conducted phylogenetic analyses based on 12,080 partial VP1 sequences identified in China before 31st June 2018. We found that EVA71 prevalence has decreased sharply but other enteroviruses have increased rapidly from 2008 to 2016 and that one subtype of each enterovirus is represented during the epidemic. In addition, four genotypes EVA71_C4, CVA16_B1, CVA6_D and CVA10_C are the most predominant enterovirus strains and collectively they cause over 90% of all HFMD cases in China according to the phylogenetic trees using representative partial VP1 sequences. These four major enterovirus genotypes have different geographical distributions, and they may co-circulate with other genotypes and serotypes. These results suggest that more molecular epidemiological studies should be performed on several enteroviruses simultaneously, and such information should have implications for virological surveillance, disease management, vaccine development and policy-making on the prevention and control of HFMD.Electronic supplementary materialThe online version of this article (10.1007/s12250-019-00169-2) contains supplementary material, which is available to authorized users.

2635. Outbreak of Enterovirus D68 Among Children in Japan and Simultaneous Circulation of Clade B3 in Europe

BackgroundEnterovirus D68 (EV-D68) is a re-emerging, high-morbidity pathogen that causes severe respiratory infection and acute flaccid myelitis in children. EV-D68 is phylogenetically divided into 4 major groups—clade A through D—and each clade has subclades defined by genetic variant. Outbreaks of all strains have been reported in more than 10 countries. However, no study has compared the viral genomic characteristics of EV-D68 in contemporaneous outbreaks in different countries.MethodsAn outbreak of EV-D68 in children younger than 15 years occurred in Niigata, Japan, from October through November 2018. The patients were admitted to hospital with respiratory distress and wheezing episodes. RNA extracted from nasopharyngeal samples was tested by EV-D68-specific PCR. Clade was determined by semi-nested PCR analysis of the VP1 region, and the phylogenetic tree of the VP1 sequence was constructed. To clarify viral genomic characteristics, we compared the clade to those of outbreaks occurring during 2014–2018 in other countries.ResultsEV-D68 testing of 47 patients yielded positive results for 22 (47%) (median age 4.6 years; IQR, 2.9–6.7), and 15 (69%) had a past medical history of allergic disease. No patient developed acute flaccid myelitis. The VP1 sequences from all isolates belonged to clade B3, the same clade detected during the 2015 outbreak in Japan (figure). Interestingly, EV-D68 outbreaks were reported in France and Italy (clades D1/B3 and D1, respectively) during this period. Although the EV-D68 clades responsible for outbreaks in 2014 differed by region, clade B3 is now the dominant clade and has caused concurrent EV-D68 outbreaks in children since 2015 (table).ConclusionIn 2018, an EV-D68 clade B3 outbreak occurred among children in Niigata, Japan. This clade is now dominant and periodically circulates around the world. Because EV-D68 can cause simultaneous outbreaks worldwide and is associated with high morbidity, detailed real-time epidemiological surveillance of EV-D68 is warranted. Disclosures All authors: No reported disclosures.

Emergence of novel lineage of foot-and-mouth disease virus serotype Asia1 BD-18 (G-IX) in Bangladesh.

Foot-and-mouth disease virus (FMDV) is a highly evolutionary divergent pathogen causing great economic havoc in many countries. Among its seven existing serotypes, Asia1 is the least divergent with a single topotype both genetically and antigenically. It is reported sporadically in Indian subcontinent and was classified under lineage G-VIII. In 2018, serotype Asia1 re-emerged in Bangladesh after 2013, along with circulation of a novel serotype Asia1 BD-18 (G-IX) lineage. VP1 phylogeny and sequence variation clearly demonstrated the novel strains which was estimated to have at least>5% nucleotide divergence with distinct clade formation. Also, the Bayesian phylogeographic inferences traced back to the origin time of lineage G-IX in early 2017 and a possible origin in Bangladesh. Mutational analysis considering established eight lineages revealed that the virus strains belonged to lineage G-IX contained a unique mutation at 44 position in the B-C loop region of VP1. Inappropriate vaccination and inefficient outbreak surveillance possibly contributed to the current episode of emergence. Therefore, active surveillance and continued vigilance are essential to assess and timely detect the occurrence, extent and distribution of this novel Asia1 strains in Bangladesh and theneighbouring countries.

Characterization of the FMDV-serotype-O isolates collected during 1962 and 1997 discloses new topotypes, CEY-1 and WCSA-1, and six new lineages

The genetic diversity of the FMD viruses collected from the outbreaks during the second half of the 20th Century in Sri Lanka was assessed in the present study. We sequenced the VP1 genomic region of the samples collected during FMDV epidemics caused by serotype O in Sri Lanka during 1962 and 1997. For comparison, we sequenced the VP1 of the related viral isolates collected from other Asian countries. We analyzed the VP1 sequences of the viral strains using the UPGMA method with uncorrected pairwise distances. Nucleotide divergence (ND) thresholds of 15%–20% and 5%–<15% were used to differentiate topotypes and lineages, respectively. We calibrated the divergence times and lineage-specific substitution rates using Bayesian-skyline models. Based on the ND estimations and phylogenetic relationships, we identified and named two new topotypes [CEYLON 1 (CEY-1) and WEST, CENTRAL AND SOUTH ASIA 1 (WCSA-1)] and six new lineages (Syr-62, Srl-77, Tur-69, May-78, Tai-87 and Bur-77) of serotype O. We believe that the novel topotypes and lineages named may have disappeared although they have similar substitution rates for epizootic outbreaks. Because the amino acid selection analysis revealed that the two topotypes and six lineages identified were under purifying selection during the outbreaks.

Vaccineassociated paralytic poliomyelitis and acute flaccid paralysis on some territories of Russia during 20 years

Aim: Analysis of the morbidity of vaccine-associated paralytic poliomyelitis and acute flaccid paralysis and the results of virological investigation of the patients on 14 territories of the Russian Federation in 1998-2017. Materials and methods: We investigated nearly 3000 stool samples from paralytic patients and contact persons. Isolation and identification of polioviruses were performed according to WHO recommendations with the help of cell lines RD and L20B. We conducted the sequencing of the genome fragments VP3-VP1, VP1-2A and full sequencing of genome region VP1 of 45 poliovirus strains. Results: From 1998 till 2017 1257 cases of acute flaccid paralysis were registered on 14 territories of Russia, 15 cases of which (1,2%) were classified as vaccine-associated paralytic poliomyelitis. From these patients 9 children were non vaccinated and 6 children received from one to four doses of oral poliomyelitis vaccine. The percentage of the detection of polioviruses from the patients and contact persons in different years was not equal and constituted from 3, 4±0,89% to 9, 5±0,79%. All in all from the patients with acute flaccid paralysis and contact persons we isolated 191 polioviruses, 60 of them belonged to type 1, 55 polioviruses were identified as types 2 and 76 as type 3. Some cases of vaccine-associated paralytic poliomyelitis are described in the article; polioviruses were isolated from all these patients. The sequencing of the genome fragments of 45 poliovirus strains showed that the majority of them had the nucleotide substitutions including neurovirulent substitutions. Conclusion:In order to prevent the risk of the appearance of vaccine-associated paralytic poliomyelitis it is necessary to maintain the high quality of surveillance of poliomyelitis and acute flaccid paralysis, to ensure the 95% coverage of children with poliomyelitis vaccine, to minimize the cases of groundless delays of vaccination according to medical recommendations and parents’ refusals to vaccinate children against poliomyelitis and to respect strictly the National calendar of vaccination.

Epidemiological and genetic characteristics of EV71 strains circulating in Jiangsu Province from 2009 to 2018

Objective To analyze the molecular epidemiology, genetic variations and evolution of enterovirus 71 (EV71) strains isolated in Jiangsu Province from 2009 to 2018. Methods Statistical methods were used to analyze the data about epidemiological characteristics and results of pathogen detection in cases with EV71 infection in Jiangsu Province from 2009 to 2018. The complete VP1 sequences of 80 EV71 strains were amplified and sequenced for analysis of diversity and phylogenesis. Results A total of 41 858 enterovirus-positive hand, foot and mouth disease cases were reported in Jiangsu Province from 2009 to 2018. EV71 was the predominant pathogen, accounting for 36.52%, and responsible for most of the severe cases. However, the percentage of EV71 among all pathogens gradually decreased over time. EV71 infection reached the peak in April to June and mainly occurred in children aged six months to five years old with higher incidence in males than in females. In terms of regional distribution, EV71 infections were characterized by area clustering in Jiangsu Province, mainly detected in Nanjing, Suzhou, Wuxi and Lianyungang. The 80 EV71 isolates belonged to C4a genotype. Nucleotide differences between them and three vaccine strains (H07, FY23 and FY7VP5)were 0.6%-5.5%, 0.8%-5.7% and 1.9%-6.9% and amino acid difference were 0-1.4%, 0.3%-2.0% and 0.3%-2.0%, respectively. Amino acid mutations in the epitopes of the 80 EV71 strains did not marked by years or regions. Conclusions EV71 strains showed obvious epidemiological characteristics in time, population and regional distribution in Jiangsu Province from 2009 to 2018.All of the 80 EV71 isolates belonged to C4a subgenotype. The nucleotide sequences between them and the vaccine strains varied greatly, but the homology of amino acids was relatively high, indicating the existence of some synonymous mutations and no risk of antigenic drift. This study would provide reference for EV71 vaccination in Jiangsu Province. Key words: Hand, foot and mouth disease; Enterovirus 71; Epidemiology; Genetic characteristics

Emerging recombination of the C2 sub-genotype of HFMD-associated CV-A4 is persistently and extensively circulating in China

Sporadic outbreaks caused by coxsackievirus A4 (CV-A4) have been reported worldwide. To further elucidate the detailed genetic characteristics and evolutionary recombination events of CV-A4, virus samples from nationwide hand, foot and mouth disease (HFMD) surveillance, encompassing 27 out of the 31 provinces in China, were investigated. Comprehensive and systematic phylogenetic analyses were performed by using 29 complete genomes, 142 complete CV-A4 VP1 sequences. Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) were re-identified based on the complete VP1 sequences. C2 is the predominant sub-genotype of CV-A4 associated with HFMD and has evolved into 3 clusters. Cluster 1 is a major cluster that has been persistently and extensively circulating in China since 2006 and has been associated with all severe cases. All the sequences showed high homology with the CV-A4 prototype in the P1 region, while higher identities with CV-A5, CV-14 and CV-16 in the P2 and P3 regions. Recombination analysis revealed that C2 had two specific genetic recombination patterns with other EV-A prototypes in the 5′-UTR and 3D region compared with C5. These recombination patterns might be associated with the increased transmissibility of C2 viruses, which were obtained due to their persistent and extensive circulation in populations.

Characterization of bluetongue virus serotype 28

Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV-28. Phylogenetic analysis of BTV-28 segment 2 (Seg-2) showed that it is related to Seg-2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%-66% identity in nucleotide sequences (nt) and 59%-62% in amino acid (aa) sequences of BTV VP2. BTV-28 Seg-6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg-5 was most closely related to a South African BTV-4 strain, and all other segments showed close similarity to BTV-26. Experimental infection by injection of 6-month-old ewes caused clinical signs in all injected animals, lasting from 2 to 3days to several weeks post-infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV-injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact-infected animals. These findings demonstrate that BTV-28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.

Prevalence and complete genome of bovine norovirus with novel VP1 genotype in calves in China

Bovine norovirus (BNoV) is a diarrhea-causing pathogen of calves. In this study, 211 diarrheic fecal samples were collected from 25 farms across six provinces in China, between November 2017 and September 2018. 20.4% of the samples were detected as BNoV-positive by RT-PCR. Phylogenetic analyses based on RdRp, VP1, and VP2 fragments revealed these BNoV strains had unique evolutionary characteristics. The complete genome of strain Bo/BET–17/18/CH was successfully sequenced. It was 7321 nucleotides (nt) in length, shared 79.4–80.9% nt identity with all five BNoV genomes, clustered on a separate branch of the phylogenetic tree, suggesting that strain Bo/BET–17/18/CH could represent a novel BNoV strain. Two interesting characteristics were found in the genome: (i) the VP1 sequence differed greatly from known BNoV VP1 sequences; (ii) a recombination event is predicted within the ORF1–ORF2 overlap. Moreover 16.3% (7/43) of the BNoV were identified as the novel VP1 genotype, which were distributed on four farms across two provinces, indicating that the novel VP1 genotype strain has spread. To our knowledge, this is first description of the molecular and genomic characteristics of BNoV in China. These findings extend our understanding of the genetic evolution and epidemics of BNoV.

Reliable detection, sequencing, and transfection of foot-and-mouth disease virus RNA from badly preserved vesicular epithelium.

Epithelium or fluid from vesicular lesions are the preferred samples to confirm foot-and-mouth disease virus (FMDV) infection in livestock. A pH-neutral buffered transport medium is recommended for optimal preservation of epithelial samples, but may not be necessary for all circumstances based on the results of our study. Pieces of epithelium were collected from FMDV-infected cattle (isolates O/FRA/1/2001 and A/IRN/22/2015) and stored at room temperature in sealed tubes without any liquid or preservatives. Using RNA extracted from the severely decayed epithelium up to 3 wk after collection, FMDV was successfully detected by RT-rtPCR, and the viral strain was identified by sequencing of capsid protein VP1. Direct isolation of the virus in cell culture was only possible for vesicular material stored for up to 2-5 d, depending on the serotype, but, for both serotypes, infectious virus was recovered by transfection of RNA extracted from epithelium after 3 wk of storage at room temperature. Specialized transport medium will give optimal results, particularly for low-titer samples, but is not required for the reliable detection and characterization of FMDV in highly positive vesicular epithelium by molecular methods.

Rotavirus A Associated with Clinical Disease and Hepatic Necrosis in California Pigeons (Columba livia domestica).

Retrospective analysis of pigeon necropsy submissions to the California Animal Health and Food Safety Laboratory System from 2000 to 2018 revealed 14 submissions diagnosed with rotavirus A hepatic necrosis or "reoviruslike" viral hepatitis. Nine of the 14 submissions (64%) occurred in 2018. Submissions were racing pigeons and squab breeders from flocks with increased mortality. Juvenile and adult pigeons were submitted with a history of depression, diarrhea, regurgitation, labored breathing, and weakness. Flock morbidity peaked at 80% and mortality at 28%. The most consistent findings on postmortem examination were variably congested, mottled, and enlarged livers and spleens. Microscopically, mild to severe hepatic necrosis was observed with variable bile duct hyperplasia, sinusoidal congestion, hemosiderosis, and portal lymphoplasmacytic inflammation. Rotavirus A was detected in hepatocytes and inflammatory cells by immunohistochemistry. Negative-stain electron microscopy identified viral particles consistent with a member of Reoviridae in all negatively stained liver homogenates. Eleven cases were analyzed by reverse transcriptase-PCR targeting rotavirus A viral protein (VP) 6 and VP7 genes. Subsequent phylogenetic analysis of the VP6 and VP7 sequences compared to published Chinese, Nigerian, and German rotavirus A VP6 and VP7 sequences demonstrated the formation of two and three distinct clades, respectively. To the authors' knowledge, rotavirus A hepatic necrosis in pigeons has not been previously reported in the United States and represents a significant emerging disease for the pigeon industry due to the potential for high flock mortality and lost production.

First detection of neboviruses in yak (Bos grunniens) and identification of a novel neboviruses based on complete genome

Neboviruses (NeVs) is an important causative agent of calf diarrhea. Here, 354 diarrhoeic samples were collected from yak on 55 farms in the Qinghai-Tibet Plateau, China. 22.0% of the diarrhoeic samples were detected as NeVs-positive by RT–PCR assay. Phylogenetic analysis of 78 NeVs RdRp fragments showed that 69 strains were closely related to NB-like strains, and the remaining 9 strains were clustered into an independent branch, which may represent a novel RdRp genotype. Two complete NeVs genomes (YAK/NRG-17/17/CH and YAK/HY1-2/18/CH) were successfully sequenced with 7459 nt and 7460 nt in length, respectively. The genomes of the two strains only shared 68.1%–69.3% nt identity with all six known NeVs genomes, and phylogenetic trees based on its genome, VP1, RdRp, VP2, P34, NTPase, P30, VPg and 3CLpro proteins suggested that the two strains may represent a novel NeVs strain with novel VP1 genotype and novel RdRp genotype. Notably, 11.5% NeVs strains were screened as the novel NeVs strains based VP1 and RdRp sequences. These novel NeVs strains were detected from 6 farms in two counties, indicating that the novel NeVs has spread in local region. To best of our knowledge, this is the first detection of NeVs in yak. Moreover, a novel NeVs strain was identified based on complete genome. These results contribute to further understand the prevalence and genetic evolution of NeVs.

The resurgence of the norovirus GII.4 variant associated with sporadic gastroenteritis in the post-GII.17 period in South China, 2015 to 2017

BackgroundHuman norovirus is regarded as the leading cause of nonbacterial acute diarrhea in developing and developed countries. Among all genotypes, GII.4 has been the predominant genotype, but in East Asia, it was replaced by the GII.17 in 2014/2015. However, after the prevalence of new GII.17 variant in South China, a sharply increase in the number of norovirus infections associated with sporadic acute diarrhea was detected. In this study, we would investigate the prevalence and genetic diversity of noroviruses in the sporadic acute gastroenteritis cases in the post-GII.17 period in South China.MethodsNorovirus was screened from 217 patients with sporadic acute gastroenteritis from August 2015 to October 2017 by reverse transcription-polymerase chain reaction. Then, two regions including the partial RNA polymerase and the capsid gene of positive samples were amplified and sequenced. Phylogenetic analyses were performed to determine norovirus genotypes. Complete VP1 sequences of GII.4 strains detected in this study were also amplified and subjected into evolutionary tracing analyses.ResultsA total of 43 (19.82%) norovirus samples were confirmed from 217 stool specimens, and it was found that GII.4 resurged as the new predominant variant, accounting for 76.74% (33/43) of positive samples. Only one local strain GZ2015-L550 was clustered with the contemporary GII.P16/GII.4–2012 recombinant variant, and other 32 local strains belonged to the clade with the GII.Pe/GII.4–2012 variant. Other genotypes including GII.17 (n = 4), GII.3 (n = 4), GII.8 (n = 1) and GI. 6 (n = 1) were also detected. Furthermore, all GII.4 strains were phylogenetic analyzed based on their capsid P2 subdomains. Combined with other reported 754 strains, the GII.4–2012 variant could be divided into two clades. Most GII.4 strains collected in 2016 and 2017 in this study (7/8) formed a new cluster A in Clade II with additional 103 contemporaneous strains. In addition, evolutionary tracing of the capsid P2 subdomain of this variant was also analyzed, and one specific amino acid substitutions (N373) was identified for Cluster A.ConclusionIn summary, this study confirmed a norovirus infection peak in the post-GII.17 period in South China, which was caused by the resurgence of the GII.4 variant.

Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region\u2019s genetic variability

Fragmented data are available on the human polyomaviruses (HPyVs) prevalence in the gastrointestinal tract. Rearrangements in the non-coding control region (NCCR) of JCPyV and BKPyV have been extensively studied and correlated to clinical outcome; instead, little information is available for KIPyV, WUPyV and MCPyV NCCRs. To get insights into the role of HPyVs in the gastrointestinal tract, we investigated JCPyV, BKPyV, KIPyV, WUPyV and MCPyV distribution among hematological patients in concomitance with gastrointestinal symptoms. In addition, NCCRs and VP1 sequences were examined to characterize the strains circulating among the enrolled patients. DNA was extracted from 62 stool samples and qPCR was carried out to detect and quantify JCPyV, BKPyV, KIPyV, WUPyV and MCPyV genomes. Positive samples were subsequently amplified and sequenced for NCCR and VP1 regions. A phylogenetic tree was constructed aligning the obtained VP1 sequences to a set of reference sequences. qPCR revealed low viral loads for all HPyVs searched. Mono and co-infections were detected. A significant correlation was found between gastrointestinal complications and KIPyV infection. Archetype-like NCCRs were found for JCPyV and BKPyV, and a high degree of NCCRs stability was observed for KIPyV, WUPyV and MCPyV. Analysis of the VP1 sequences revealed a 99% identity with the VP1 reference sequences. The study adds important information on HPyVs prevalence and persistence in the gastrointestinal tract. Gastrointestinal signs were correlated with the presence of KIPyV, although definitive conclusions cannot be drawn. HPyVs NCCRs showed a high degree of sequence stability, suggesting that sequence rearrangements are rare in this anatomical site.