The presence of duck Tembusu virus in Thailand since 2007: A retrospective study

Duck Tembusu virus (DTMUV ), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT ‐PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV , which was the most closely related DTMUV . It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUV s, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUV s; however, it was genetically distinctive to any of the currently circulating DTMUV s. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUV s in Asia.     

Duck Tembusu virus detection and characterization from mosquitoes in duck farms,Thailand

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, belongs to the Flavivirus genus and Flaviviridae family. The transmission of DTUMV involves mosquito vectors; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. This study was conducted to examine DTMUV detection and characterization from mosquitoes in duck farms in central Thailand. Mosquitoes were collected from two duck farms in Sing Buri Province and two duck farms in Ang Thong Province from September 2015 to July 2016 using four CDC‐light traps. A total of 30,841 mosquitoes were collected and identified to seven species (Anopheles (An.) barbirostris, An. stephensi, Culex (Cx.) gelidus, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Mansonia (Ma.) annulifera and Ma. uniformis). The most common collected species from each duck farm and each collection time was Cx. tritaeniorhynchus. Mosquitoes were pooled according to species, location, and collection time and then examined for DTMUV by RT‐PCR. A total of 273 mosquito pools were examined, with only one pool of Cx. tritaeniorhynchus collected from Sing Buri Province in November 2015 testing positive for DTMUV. Phylogenetic analysis of the polyprotein genes demonstrated that a mosquito‐derived Thai DTMUV was grouped into subcluster 2.1 and most closely related to the 2013 Thai DTMUVs. Thus, this study indicated that Cx. tritaeniorhynchus may play a role as a vector in the transmission of DTMUV in Thailand. However, additional studies concerning the vector competence of this mosquito for DTMUV are needed.     

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010–2015

Duck hepatitis A virus (DHAV ) is the most common aetiologic agent of duck virus hepatitis (DVH ), causing substantial economic losses in the duck industry worldwide. In China, officially approved DHAV ‐1 live‐attenuated vaccines have been used widely to vaccinate breeder ducks since 2013. However, following the reports of DVH outbreaks, it has become necessary to assess the epidemiological situation of this virus in China. We conducted molecular epidemiological analyses of 32 DHAV field isolates while analysing the samples from ducks suspected of having hepatitis collected from commercial duck farms in China between May 2010 and December 2015. Considerable changes were observed in the epidemiology of DHAV ‐1 and DHAV ‐3 in China over time. A higher number of DHAV ‐1 strains were isolated during 2010–2012, coinciding with the widespread use of officially approved DHAV ‐1 live vaccine strains beginning in 2013. In contrast, a higher rate of DHAV ‐3 causing DHAV infections was observed between 2013 and 2015. Phylogenetic analyses based on the full‐length VP 1 gene were performed on these field isolates and using reference strains available in GenBank. DHAV ‐1 field isolates were evaluated in two groups: one group closely related to prototype strains and circulating in China between 2010 and 2012 and another group exhibiting genetic and serological differences from prototype strains. All DHAV ‐3 strains isolated in this study were grouped as monophyletic, which has become the predominant viral type, particularly in Shandong and Sichuan provinces, since 2013. In conclusion, these data provide updated information on the genetic and serological diversity of DHAV ‐1 and DHAV ‐3, and our findings may serve as a foundation for the prevention of, and vaccine development for, DHAV in China.     

Isolation and Characterization of a Novel Tembusu Virus Circulating in Muscovy Ducks in South China

Duck Tembusu virus (DTMUV) is an infectious pathogen that can cause epidemics in egg‐laying ducks. Here, we isolated and characterized a DTMUV, designated GDLH01, thought to be responsible for the noticeable egg drop in Muscovy duck flocks in South China since 2011. The genome sequence of GDLH01 shared 97–99% homology with other avian‐origin Tembusu viruses, and 99.5% homology with the mosquito‐borne strain SDMS recently reported in China. Phylogenetic analysis based on the nucleotide sequence of the entire open reading frame confirmed that the isolate was of avian origin and closely related to a mosquito‐borne strain. Our findings characterize a novel Tembusu virus circulating in Muscovy ducks in South China and emphasize the importance of reinforcing biosecurity measures and developing vaccines to prevent the spread of this viral pathogen.     

Isolation and Genetic Characterization of a Tembusu Virus Strain Isolated From Mosquitoes in Shandong,China

Tembusu virus (TMUV) is a flavivirus, presumed to be a mosquito‐borne flavivirus of the Ntaya virus subgroup. To date, however, there have been no reports indicating that mosquitoes are involved in the spread of TMUV. In this study, we report the first isolation of TMUV from Culex mosquitoes. We describe the isolation and characterization of a field strain of TMUV from mosquitoes collected in Shandong Province, China. The virus isolate, named TMUV‐SDMS, grows well in mosquito cell line C6/36, in Vero and duck embryo fibroblast (DEF) cell lines, and causes significant cytopathic effects in these cell cultures. The TMUV‐SDMS genome is a single‐stranded RNA, 10 989 nt in length, consisting of a single open reading frame encoding a polyprotein of 3410 amino acids, with 5′ and 3′ untranslated regions of 142 and 617 nt, respectively. Phylogenetic analysis of the E and NS5 genes revealed that the TMUV‐SDMS is closely related to the TMUV YY5 and BYD strains which cause severe egg‐drop in ducks. The 3′NTR of TMUV‐SDMS contains two pairs of tandem repeat CS and one non‐duplicate CS, which have sequence similarities to the same repeats in the YY5 and BYD strains. Our findings indicate that mosquitoes carrying the TMUV may play an important role in the spread of this virus and in disease outbreak.     

Molecular and pathological characterization of duck enteritis virus in Egypt

In winter 2016, a fatal disease outbreak suspected to be duck virus enteritis (DVE) stroke over a million ducklings in 10 white Pekin and Muscovy ducks flocks in Dakahlia and Gharbia Governorates, Egypt, causing heavy economic losses. The disease quickly killed 20%–60% of affected farms. The clinical signs were inappetence, ataxia, crowding in corners, partially closed eye lids and blue beaks. Post mortem examination revealed white necrotic foci in liver, mottled spleen and sometimes cecal core. A total of 10 intestines, livers and spleens samples were collected from diseased flocks. Each sample was pooled randomly from eight to ten ducklings. Polymerase chain reaction (PCR) and histopathological examination were utilized for DEV identification in collected samples. Nucleotides sequences of the amplified DNA polymerase gene were compared with the other DEVs available on GeneBank. Also, existence of co‐infection with Salmonella spp. was verified via PCR. DEV nucleic acid was detected by PCR in 8 of 10 collected samples (80%) with positive amplification of polymerase gene. Histopathological examination revealed eosinophilic and basophilic intranuclear inclusion bodies in enterocytes. In some infected enterocytes, intranuclear and intracytoplasmic inclusions were observed in the same cell. Respectively, eosinophilic intranuclear inclusion bodies found in hepatocytes and reticular cells of liver and spleen of diseased ducklings. Four of the 10 collected samples showed positive results for Salmonella spp. infection that may be involved in enhancing infection with DEV. The identified DEVs revealed close genetic relationship with DEVs detected previously in India and China indicating potential transmission of the virus from there that crucially needs further work for better understanding of virus origin. In conclusion, our study revealed infection of duckling farms with DEV and Salmonella that necessitate the implementation of restricted early preventive and control measures for both diseases to decrease the expected economic losses.     

Characterization of a Tembusu Virus Isolated from Naturally Infected House Sparrows (Passer domesticus) in Northern China

The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV‐SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species.     

Genetic and phylogenetic analysis of reemerged novel Seneca Valley virus strains in Guangdong province, 2017

From June to July 2017, six Seneca Valley virus (SVV ) strains were isolated from swine herds exhibiting SVV ‐associated porcine idiopathic vesicular disease (PIVD ) in Guangdong province, China. Complete genomic sequences of these six newly identified strains were genetically and phylogenetically analysed. The results revealed that these six SVV strains were genetically closely related to USA /GBI 29/2015 and notably distinct from all previous Chinese strains, indicating the reemergence of new SVV strains in Guangdong province.     

Analysis of the Complete Genome of Tembusu Virus,a Flavivirus Isolated from Ducks in China

During investigations into the outbreak of duck viral infection in 2010 in China, with a severe drop in egg production, a flavivirus was isolated from the affected ducks. It was characterized as a Tembusu virus (TMUV). In this study, we obtained a complete genome sequence of Tembusu virus using RT‐PCR and RACE techniques. TMUV genome is a singled‐stranded RNA, with 10 990 nucleotides in length, and contains a single open reading frame (3410 amino acids) encoding 11 viral proteins with 5′and 3′non‐translated regions (NTRs) of 142 and 618 nt, respectively. We characterized the open reading frame (ORF) with respect to gene sizes, cleavage sites and potential glycosylation sites. The different genomic regions of the virus were also compared with those of six other flaviviruses including Japanese encephalitis virus, West Nile virus (WNV), dengue‐2 virus, yellow fever virus, tick‐borne encephalitis virus (TBEV) and Bagaza virus. TMUV demonstrated the highest similarity to Bagaza virus. The result of entire ORF scanning shows that TMUV was close to Bagaza viruses in genetic relatedness. These data demonstrate that TMUV is a unique virus among the mosquito‐borne flaviviruses and also provide a useful reference for a critically important study to determine why TMUV is a serious pathogen for ducks.     

Genetic diversity and comparison of diagnostic tests for characterization of foot‐and‐mouth disease virus strains from Pakistan 2008–2012

We report the laboratory analysis of 125 clinical samples from suspected cases of foot‐and‐mouth disease (FMD ) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT ‐PCR , of which 88 were also found to contain infectious FMD virus (FMDV ) by virus isolation (VI ), with strong correlation between these tests (κ = 0.96). Samples that were VI ‐positive were serotyped by antigen detection ELISA (Ag‐ELISA ) and VP 1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n  = 13), O (n  = 36) and Asia‐1 (n  = 41), including three samples from which both serotypes Asia‐1 and O were detected. Serotype A viruses were classified within three different Iran‐05 sublineages: HER ‐10, FAR ‐11 and ESF ‐10. All serotype Asia‐1 were within Group VII (Sindh‐08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia‐2 within two different sublineages: ANT ‐10 and BAL ‐09. Using VP 1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag‐ELISA to correctly determine serotype was 74%, and serotype‐specific sensitivity was 8% for serotype A, 88% for Asia‐1 and 89% for O. Serotype‐specific specificity was 100% for serotype A, 93% for Asia‐1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag‐ELISA . This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV .     

Genetic evolution analysis of novel recombinant pseudorabies virus strain in Sichuan,China

Pseudorabies is a disease that seriously endangers the pig industry in China. Recently, we successfully isolated a pseudorabies virus from the brain tissue of piglets at a farm in Sichuan, China, and named it the FJ62 strain. In order to understand the molecular biological characteristics of the strain, primers were designed for glycoproteins gB, gC, gD and gE, which were amplified by a polymerase chain reaction (PCR) and sequenced. After comparing the sequence with the GenBank 22 pseudorabies virus reference strains and establishing the genetic evolutionary tree, it was found that the gB gene of pseudorabies virus was highly homologous (up to 100%) with the MY‐1 strain which is isolated from a wild boar in Japan ( AP018925 ) but that homology with other strains in China was low. The gC gene was in the same branch as most of the representative strains in China, with 99.5% homology. The gD gene is in the same branch as the domestic strain LA in China ( KU552118 ), and the homology was 99.9%. The gE gene was in the same branch as the domestic BJ/YT strain in China ( KC981239 ), with 99.9% homology. The results showed that the FJ62 strain of the pseudorabies virus isolated here may be a variant strain of FJ62 isolated from a domestic pig after natural recombination of pseudorabies virus genotype I from wild boar and genotype II from pigs in China. There have been no similar reports in Sichuan. The discovery of the recombinant virus strain provides a reference basis for the prevention and control of pseudorabies and a design strategy for a vaccine in Sichuan, China, in the future.     

Identification and characterization of lumpy skin disease virus isolated from cattle in the Republic of North Ossetia‐Alania in 2015

The first notifications of the unknown disease of cattle appeared in September–October 2015 in North Caucasus region of Russia (Republic of North Ossetia‐Alania). The clinical signs included watery discharge from eyes, apathy, loss of appetite, salivation, lameness and nodular skin lesions. Capripoxvirus genome was detected by real‐time PCR in the tissue samples of sick animals. The aetiological agent was isolated in the primary cell cultures of lamb testis and goat testis, as well as in the continuous MDBK cell culture. Further sequencing of the GPCR gene and phylogenetic analysis showed the close genetic relationship of isolated capripoxvirus with a group of lumpy skin disease virus. Koch's postulates were fulfilled by the experimental infection of four calves with a suspension of tissue samples from sick animals.     

Different dynamics of Usutu virus infections in Austria and Hungary, 2017–2018

Usutu virus (USUV), a mosquito‐borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass‐mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV‐related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT‐PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV‐associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future.     

Emergence of novel Seneca Valley virus strains in China, 2017

Since early 2017, several outbreaks of Seneca Valley virus (SVV) infection have re‐emerged in China. We report the identification of novel SVV KS15‐01‐like strains showing a notable distinction with previous Chinese SVV strains. The determined SVV strains are currently causing new outbreaks in two provinces in China where no SVV infection has been reported previously, implying the increased epidemic regions and potential threat to local pig breeds of SVV in China.     

Isolation and characterization of a variant duck orthoreovirus causing spleen necrosis in Peking ducks,China

Since December 2017, an infectious disease has caused economic hardship for duck farms and breeding ducks in many regions of China. This disease characterized by spleen necrosis and swelling, is due to a variant strain of duck orthoreovirus (DRV) (Duck/N‐DRV‐XT18/China/2018), which we isolated from the spleen of diseased ducks. After isolating the virus, we used next‐generation sequencing technology to determine the entire genomic of the virus. Our phylogenetic analysis of 10 genomic segments showed that the N‐DRV‐XT18 strain is closely related to orthoreovirus isolates derived from ducks and geese, with nucleotide sequence identities for 10 genomic fragments ranging between 49.8% and 99.3%. In contract, the nucleotide sequence of N‐DRV‐XT18 genomic fragments are only 38.6% to 78.8% similar to the chicken orthoreovirus isolate. Therefore, we determined that this pathogen, causing duck spleen necrosis, is a new variant of a duck orthoreovirus that is significantly different from any previously reported waterfowl‐derived othoreovirus.     

Detection and molecular characterization of Peste des Petits Ruminants virus from outbreaks in Burundi,December 2017–January 2018

In December 2017, Peste des Petits Ruminants (PPR) emerged in Burundi (East Africa) and rapidly spread to five provinces (Gitega, Kirundo, Mwaro, Muramvya and Karuzi) in the country, causing severe disease and killing more than 4,000 goats in the province of Gitega alone. An initial outbreak investigation was conducted in December 2017 by the Burundi Government Veterinary Services and samples were collected for laboratory confirmation. A competitive Enzyme Linked Immuno‐Sorbent Assay (cELISA: Chinese Patent No. ZL201210278970.9) supplied by the Lanzhou Veterinary Research Institute was used to test 112 sera and results showed around 37.5% positive samples. This high level of PPR positive sera in an animal population where PPR infection and vaccination had not been previously reported indicated the exposure of the animals to PPRV. Subsequently in January 2018, the laboratory tests conducted at the African Union‐Pan African Veterinary Vaccine Centre (AU‐PANVAC) laboratories following a joint investigative mission by the African Union‐Interafrican Bureau for Animal Resources (AU‐IBAR), AU‐PANVAC and the East African Community (EAC) confirmed the presence of PPR in Burundi. Samples tested by conventional RT‐PCR indicated the presence of the PPR virus (PPRV). Confirmatory isolation of the virus was also performed. Phylogenetic analysis revealed that the virus belongs to lineage III and shows a close relationship with PPRV isolates from Kenya in 2011 and Uganda in 2012. A possible explanation for the outbreaks of PPR in Burundi between December 2017 and February 2018 is presented.     

Foot‐and‐mouth disease outbreaks due to an exotic serotype Asia 1 virus in Myanmar in 2017

In January 2017, two villages located in Rakhine State of Myanmar reported clinical signs in cattle suggestive of foot‐and‐mouth disease virus (FMDV) infection. Laboratory analysis identified the outbreak virus as FMDV serotype Asia 1, which represented the first detection of this serotype in Myanmar since 2005 and in the region of South‐East Asia (SEA) since 2007. Genetic analysis revealed that the outbreak virus was different from historical viruses from Myanmar and was more closely related to viruses circulating in Bangladesh and India during 2012–2013, indicating that a novel viral introduction had occurred. The precise origin of the outbreaks was not clear, but frequent informal livestock trade with South Asia was reported. Responses to the outbreaks involved disinfection, quarantine and animal movement restrictions; no further outbreaks were detected under the present passive surveillance system. Detection of serotype Asia 1 highlights the complex and dynamic nature of FMDV in SEA. Active surveillance is needed to assess the extent and distribution of this exotic Asia 1 strain and continued vigilance to timely detect the occurrence of emerging and re‐emerging FMDV strains is essential.