Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

Novel Serotype of Epizootic Hemorrhagic Disease Virus, China.

In 2018, a strain of epizootic hemorrhagic disease virus (EHDV), named YNDH/V079/2018, was isolated from a sentinel calf in Mangshi County, Yunnan Province, China. Nucleotide sequencing and neutralization tests indicated that the virus belongs to a novel serotype of EHDV that had not been reported previously.

Suicoccus acidiformans gen. nov., sp. nov., isolated from a sick pig.

A Gram-stain-positive, non-spore-forming, catalase-positive and facultatively anaerobic coccus, designated ZY16052T, was isolated from mesenteric lymph nodes of a sick piglet in Kunming, Yunnan Province, PR China and its taxonomic position was studied by following a polyphasic approach. Optimal growth was observed at 37 °C, pH 8.0 and 2 % NaCl (w/v) on Columbia agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY16052T formed a separated evolutionary lineage from recognized genera of the family Aerococcaceae and shared low similarity to its closest related species Facklamiasourekii (93.8 %) and Ignavigranum ruoffiae (93.4 %). Phylogenetic analysis based on the housekeeping gene recA indicated that strain ZY16052T represented a deep and distinct evolutionary lineage, and was well separated from all genera in the family Aerococcaceae, with very low sequence similarity(≤73.2 %). Sequence analysis based on the housekeeping gene rpoA indicated that strain ZY16052T shared very low similarity ≤77.0 % to related genera. The genomic OrthoANI values between strain ZY16052T and type species of related genera in the family Aerococcaceae and species in the genus Facklamia were ≤67.77 and ≤68.11 %, respectively. The genomic G+C content was 42.3 mol%. The predominant fatty acids (>5 %) were C16 : 0, C18 : 1ω9c, C14 : 0 and summed feature 5 (C18 : 2ω6,9c and/or C18 : 0 ante). The major polar lipids were digalactosyldiacylglycerol, phosphatidylglycerol, diacylglycerols, triacylglycerol and phosphatidic acid. The peptidoglycan contained the amino acids lysine, glycine, alanine and glutamic acid, which is characteristic of peptidoglycan type A1a. Based on the phylogenetic and phenotypic evidence, we propose that the unknown bacterium be classified as Suicoccus acidiformans gen. nov., sp. nov. The type strain of Suicoccus acidiformans is ZY16052T (=CCTCC AB 2017017T=DSM 105755T).

Full genome sequence of the first bluetongue virus serotype 21 (BTV-21) isolated from China: evidence for genetic reassortment between BTV-21 and bluetongue virus serotype 16 (BTV-16).

Bluetongue (BT) is one of the most important insect-borne, non-contagious viral diseases of ruminants and can cause severe disease and death in sheep. Its pathogen, bluetongue virus (BTV) has a double-stranded RNA genome consisting of 10 segments that provides an opportunity for field and vaccine strains of different serotypes to reassort whilst simultaneously infecting the same animal. For the first time, we report the full-length genome sequence of a BTV strain of serotype 21 (5149E) isolated from sentinel cattle in Guangxi Province in China in 2015. Sequence analysis suggested that the isolate 5149E had undergone a reassortment incident and acquired seg-6 from an isolate of BTV-16 which originated from Japan. This study aims to provide more understanding as to the origin and epidemiology of BTV.

Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China.

BACKGROUND: Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China. METHODS: Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests. RESULTS: We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3-3-3-1 pattern, similar to the migrating bands of Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell (T2, and outer core (T13) proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock. CONCLUSIONS: A new strain of TIBOV was isolated from Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock.

Complete genome sequence of a Chuzan virus strain isolated for the first time in mainland China.

Chuzan virus (CHUV) belongs to the Palyam serogroup, causes bovine congenital disease, and is prevalent in Asia. To date, only one full Palyam virus (PALV) genome sequence, that of Japanese CHUV strain K47, has been reported. Sequence analysis indicates that PALV strains isolated from different geographical regions show significant diversity, which is mainly shaped by geographically independent evolution and genetic reassortment. Our understanding of the genetic characteristics of PALV is hampered by a very limited genomic sequence database. In this study, we report the complete genome sequence of CHUV strain SZ187, which was isolated for the first time in 2012 in mainland China. Sequence alignment and phylogenetic analysis demonstrate that SZ187 is closely related to other CHUV strains isolated in Taiwan and Japan, indicating that they may share a common ancestor. This new full-length CHUV genome sequence could help in the design of broader assays for epidemiological studies and facilitate the identification of new CHUV isolates in the future.

Complete genome sequence of the first bluetongue virus serotype 7 isolate from China: evidence for entry of African-lineage strains and reassortment between the introduced and native strains.

Bluetongue virus (BTV) mainly infects sheep but can be transmitted to other domestic and wild ruminants, resulting in a considerable financial burden and trade restriction. Our understanding of the origin, movement, and distribution of BTV has been hindered by the fact that this virus has a segmented genome with the possibility of reassortment, the existence of 27 identified serotypes, and a lack of complete sequences of viruses isolated from different parts of the world. BTV serotype 7 is one of the prevalent BTV serotypes in Asia. Nonetheless, no complete genomic sequence of an Asian isolate of this serotype is available. In an effort to understand the molecular epidemiology of BTV infection in China, for the first time, we report here the complete genome sequence of a BTV serotype 7 strain, GDST008, which was isolated in 2014 in China. This sequence also represents the first complete genome sequence of a BTV serotype 7 from Asia and the third one in the world. Sequence analysis suggests that GDST008 consists of segments from BTV viruses of African lineage as well as those from China. Together, these results improve our understanding of the origin, emergence/re-emergence, and movement of BTV and thus can be applied in the development of vaccines and diagnostics.

Isolation and characterization of two novel serotypes of Tibet orbivirus from Culicoides and sentinel cattle in Yunnan Province of China.

Tibet orbivirus (TIBOV), a new candidate of Orbivirus genus, was initially isolated from mosquitoes in Tibet in 2009 and subsequently from both Culicoides and mosquitoes in several provinces of China and Japan. Little is known about the origin, genetic diversity, dissemination and pathogenicity of TIBOV, although its potential threat to animal health has been acknowledged. In this study, two viruses, V290/YNSZ and V298/YNJH, were isolated from the Culicoides and sentinel cattle in Yunnan Province. Their genome sequences, cell tropism in mammalian and insect cell lines along with pathogenicity in suckling mice were determined. Genome phylogenetic analyses confirmed their classification as TIBOV species; however, OC1 proteins of the V290/YNSZ and V298/YNJH shared maximum sequence identities of 31.5% and 33.9% with other recognized TIBOV serotypes (TIBOV-1 to TIBOV-4) and formed two monophyletic branches in phylogenetic tree, indicating they represented two novel TIBOV serotypes which were tentatively designated as TIBOV-5 and TIBOV-6. The viruses replicated robustly in BHK, Vero and C6/36 cells and triggered overt clinical symptoms in suckling mice after intracerebral inoculation, causing mortality of 100% and 25%. Cross-sectional epidemiology analysis revealed silent circulation of TIBOV in Yunnan Province with overall prevalence of 16.4% (18/110) in cattle, 10.8% (13/120) in goats and 5.5% (6/110) in swine. The prevalence patterns of four investigated TIBOV serotypes (TIBOV-1, -2, -5 and 6) differed from each one another, with their positive rates ranging from 8.2% (9/110) for TIBOV-2 in cattle to 0.9% (1/110) for TIBOV-1 and TIBOV-5 in cattle and swine. Our findings provided new insights for diversity, pathogenicity and epidemiology of TIBOV and formed a basis for future studies addressing the geographical distribution and the zoonotic potential of TIBOV.

Bluetongue virus non-structural protein 3 (NS3) and NS4 coordinatively antagonize type â interferon signaling by targeting STAT1.

Previous studies have pointed out that bluetongue virus (BTV) down-regulates the expression levels of type Ⅰ interferon (IFN-Ⅰ) and inhibits IFN-Ⅰ signaling by targeting on the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription protein (STAT) pathway. However, individual viral protein could not effectively block IFN-Ⅰ signaling. There is a need to explore the underlying mechanisms by which viral proteins of BTV coordinate to antagonize the IFN-Ⅰ signaling. We investigated the coordinative role of BTV-1 nonstructural protein 3 (NS3) and NS4 in counteracting IFN-Ⅰ signaling in the JAK-STAT pathway by directly interacting with STAT1. The NS3 and NS4 targeted the SH2 domain of STAT1 to inhibit its phosphorylation, heterodimerization, nuclear translocation, as well as activation of downstream genes of the JAK-STAT pathway. NS3 and NS4 impaired STAT1 phosphorylation induced by IFN-Ⅰ in a dose dependent manner. Overall, this study confirmed that NS3 and NS4 of BTV participate in interfering with IFN-Ⅰ signaling process. Also, a new mechanism employed by BTV to evade host innate immune responses was revealed.

Novel putative bluetongue virus serotype 29 isolated from inapparently infected goat in Xinjiang of China.

Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus causing bluetongue (BT) in sheep, bovine and other ruminants. Twenty-four serotypes and several atypical serotypes of BTV were identified worldwide. In present study, a novel strain of BTV (V196/XJ/2014) was isolated from an asymptomatic sentinel goat in Yuli County, Xinjiang of China. Serotype identification of this isolate exhibited uniform negative results by serotype-specific conventional RT-PCR and real-time RT-PCR for BTV-1 to BTV-27, and virus neutralization tests using reference sera of BTV-1 to BTV-24. Genomic analysis showed V196/XJ/2014 grouped with atypical serotypes of BTV-25 to BTV-28, BTV-X/XJ1407, BTV-X/ITL2015 and BTV-Y/TUN2017, while segment 2 and VP2 protein of V196/XJ/2014 shared <63.4%/61.4% nucleic acids and amino acids sequence identities with other recognized BTV serotypes and its segment 2 formed a separate 'nucleotype' in phylogenetic tree. These results indicated V196/XJ/2014 does not belong to any reported serotypes of BTV. Further studies of infectivity and pathogenicity showed that goats infected with V196/XJ/2014 did not exhibit observed clinical symptoms, but high level of virus amplification and homologous neutralization antibodies were detected post-infection. Our studies suggested a novel putative serotype of BTV-29 was isolated in Xinjiang of China, which expands our knowledge about the diversity of BTV.

Complete Genome Sequence of a Bluetongue Virus Serotype 15 Strain Isolated from China in 1996.

The full-genome sequence of bluetongue virus serotype 15 (BTV-15) strain B105/YN/1996 isolated in China was determined for the first time. The virus was isolated from sentinel cattle in Yunnan Province, China, in 1996. The total size of the BTV-15 strain B105/YN/1996 genome is 19,161 bp in length. Phylogenetic analyses demonstrate that it belongs to the major eastern BTV topotype. This work is the first to document the complete genomic sequence of a BTV-15 strain from China. The sequence information will help determine the geographic origin of Chinese BTV-15 and provide data to facilitate future analyses of the genetic diversity and phylogenetic relationships of BTV strains.

Isolation and Genetic Characterization of Mangshi Virus: A Newly Discovered Seadornavirus of the Reoviridae Family Found in Yunnan Province, China.

BACKGROUND: Seadornavirus is a genus of viruses in the family Reoviridae, which consists of Banna virus, Kadipiro virus, and Liao ning virus. Banna virus is considered a potential pathogen for zoonotic diseases. Here, we describe a newly discovered Seadornavirus isolated from mosquitos (Culex tritaeniorhynchus) in Yunnan Province, China, which is related to Banna virus, and referred to as Mangshi virus. METHODS AND RESULTS: The Mangshi virus was isolated by cell culture in Aedes albopictus C6/36 cells, in which it replicated and caused cytopathic effects, but not in mammalian BHK-21 or Vero cells. Polyacrylamide gel analysis revealed a genome consisting of 12 segments of double-stranded RNA, with a "6-4-2" pattern in which the migrating bands were different from those of the Banna virus. Complete genome sequencing was performed by full-length amplification of cDNAs. Sequence analysis showed that seven highly conserved nucleotides and three highly conserved nucleotides were present at the ends of the 5'- and 3'-UTRs in each of 12 genome segments. The amino acid identities of Mangshi virus shared with Balaton virus varied from 27.3% (VP11) to 72.3% (VP1) with Banna virus varying from 18.0% (VP11) to 63.9% (VP1). Phylogenetic analysis based on amino acid sequences demonstrated that Mangshi virus is a member of the genus Seadornavirus and is most closely related to, but distinct from, Balaton virus and Banna virus in the genus Seadornavirus of the family Reoviridae. CONCLUSION: Mangshi virus isolated from mosquitoes (C. tritaeniorhynchus) was identified as a newly discovered virus in the genus Seadornavirus and is phylogenetically close to Banna virus, suggesting that there is genetic diversity of seadornaviruses in tropical and subtropical areas of Southeast Asia.

Genomic Changes in an Attenuated ZB Strain of Foot-and-Mouth Disease Virus Serotype Asia1 and Comparison with Its Virulent Parental Strain.

The molecular basis of attenuation of foot-and-mouth disease virus (FMDV) serotype Asia1 ZB strain remains unknown. To understand the genetic changes of attenuation, we compared the entire genomes of three different rabbit-passaged attenuated ZB strains (ZB/CHA/58(att), ZBRF168, and ZBRF188) and their virulent parental strains (ZBCF22 and YNBS/58). The results showed that attenuation may be brought about by 28 common amino acid substitutions in the coding region, with one nucleotide point mutation in the 5'-untranslated region (5'-UTR) and another one in the 3'-UTR. In addition, a total of 21 nucleotides silent mutations had been found after attenuation. These substitutions, alone or in combination, may be responsible for the attenuated phenotype of the ZB strain in cattle. This will contribute to elucidation of attenuating molecular basis of the FMDV ZB strain.

Genome analysis and development of infectious cDNA clone of a virulence-attenuated strain of foot-and-mouth disease virus type Asia 1 from China.

The RNA genome sequence of the rabbit passage-attenuated strain of foot-and-mouth disease virus (FMDV) Asia 1, ZB/CHA/58(att), was determined to be 8165 nt in length excluding the poly(C) tract in the 5' UTR and the poly(A) tail at the 3' end. ZB/CHA/58(att) was most similar to the vaccine strain Asia 1/YNBS/58 in genome sequence and there were no deletions or insertions within the deduced polyprotein between ZB/CHA/58(att) and YNBS/58, but there were a total of 25 substitutions at the amino acid level and an extra 19-nt stretch in the 5' UTR was found in ZB/CHA/58(att). An infectious full-length cDNA clone of ZB/CHA/58(att) was developed. Infectious virus could be recovered in BHK-21 cells transfected with the synthetic viral RNA transcribed in vitro. The plaque morphology, growth kinetics and antigenic profile of the infectious clone-derived virus (termed tZB) were indistinguishable from those induced by the parental virus. Furthermore, the virulence properties of ZB/CHA/58(att) and tZB were found to be highly similar in the mouse model. The availability of genome sequence information and infectious cDNA clone of the FMDV ZB/CHA/58(att) lays a new ground for further investigation of FMDV virulence determinants and development of new potent vaccine to FMD.