Full length genome sequencing and phylogenetic analysis of two strains of Getah virus / 中国热带医学

@#Abstract: Objective　The main aim of the study is to sequence the complete genome of two Getah virus strains (GS11-155 and HNDZ1712-1) isolated in Gansu Province and Hainan Province in 2011 and 2017 respectively and analyze the molecular and genetic evolution of the two strains compared with M1, which was first isolated in 1964 in Hainan Province, China. Methods　Genome of two newly isolated Getah viruses were sequenced by virus gene amplification technique, and the genomic database of Getah viruses was established. The molecular characteristics and genetic evolution of the viruses were analyzed by bioinformatics software. Results　The genome length of two new isolated Getah virus strains (GS11-155 and HNDZ1712-1) was 11 690 nt and 11 621 nt, respectively. Both strains had the structural characteristics of Alphavirus genome. Although the nucleotide sequence lengths of structural genes, non-structural genes and non-coding junction regions of the two strains were identical, the nucleotide sequence lengths of the 5' and 3' non-coding regions of the viral genomes were a few different. The 3'UTR repeats elements in the genomes of the two virus strains did not change. It was 97.7% and 98.1% different of nucleotide and amino acid homology between both strains of Getah virus, HNDZ1712-1 isolated in 2017 and M1 isolated in 1964 in Hainan Province. Interesting, Gansu 2011 cluster and Hainan 2017 cluster were emerged leading by both strains GS11-155 and HNDZ1712-1 respectively, those two clusters totally independent with M1 virus isolated from Hainan in 1964 in whole genome phylogenetic analysis first. Conclusions　Although the HNDZ1712-1 was also isolated from mosquito samples in Hainan Province, it was in a completely different evolutionary branch from the M1 isolated from Hainan Island in 1964, and was closely related to the strain isolated from Gansu Province (GS11-155) thousands of kilometers away. It is suggested that the two new strains of Getah virus are different from the Getah virus isolated in 1964.

Isolation and identification of Getah virus from Culicoides in Yunnan / 中华实验和临床病毒学杂志

Objective@#To discuss the taxonomy and molecular characteristics of one virus strain (SZC30) isolated from Culicoides in Yunnan.@*Methods@#Culicoides were collected with light trap method in the Wulong Village of Shizong County of Yunnan Province in July, 2013. BHK-21 and C6/36 cells were used for virus isolation. The positive isolates were inoculated into brain of one-day suckling mice. Alphavirus and Getah virus specific primers were used to amplify the genome of the virus isolation by RT-PCR. The products of RT-PCR were sequenced. Clustal X1.83, DNAStar, Mega5.1 were used for bioinformatics analysis.@*Results@#Totally 3 500 culicoides were collected and divided into 41 batches for virus isolation. One isolate (SZC30) produced cytopathic effect (CPE) on BHK-21 and C6/36 cells; the result of RT-PCR with Alphavirus and Getah virus NS1 specific primer were positive; the sequence analysis of NS1 gene suggested that SZC30 and two Getah virus strains (YN0540, SC1210) from China were in the same evolutionary branching, the nucleotide homology were 97%-100%, and the amino acid was 97%-100%.@*Conclusions@#SZC30 isolated in Yunnan province was identified as Getah virus.

First isolation and identification of Getah virus SC1210 in Sichuan / 中华实验和临床病毒学杂志

Objective@#To study the genome molecular characteristics of Getah virus (SC1210) which isolated in Sichuan province in 2012.@*Methods@#Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the isolate and the genome was sequenced by the second Ion Torrent PGM. Computer softwares, including Mega Align and Mega 6, were used to analyze the nucleotide and deduced amino acid sequence, and draw phylogenetic trees.@*Results@#SC1210 was identified as Getah virus. The full genome sequence was 11 690nt, the nucleotide and amino acid homology of the full sequence with other strains were 99.2%-99.7% and 96.5%-99.4%.The capsid protein of SC1210 consisting of 804 nucleotides, encoding 268 amino acids and the full-length of E2 protein, had 1 266 nucleotides, encoding 422 amino acids. The nucleotide homology of the capsid protein and the E2 protein with other strains were 94.9%-99.2% and 94.6%-99.6%, and the amino acid were 97%-99.6% and 97.1%-99.5%. The 3′ UTR of the virus included 402 nucleotides and there were three repeat sequence elements and 19 nucleotides conservation sequence.@*Conclusions@#The first GETV isolate SC1210 in Sichuan province has a closer relationship with Yunnan strain YN040 and a far genetic relationship with MM2021.

Establishment of reverse transcription polymerase chain reaction for detection of Getah virus infection in livestock

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of 10(2.0) TCID₅₀/mL. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

Phylogenctics of Getah virus and its migration / 中国人兽共患病学报

In order to investigate the molecular evolution and spatio-temporal migration of Getah viruses (GETV) isolated around the world,the nucleotide and deduced amino acid sequence of GETVs were analyzed and phylogenetic trees were constructed by using informatics software including ClustalX1.83,MegaAlign,GeneDOC and Mega6.0.The Bayesian Stochastic Search Variable Selection (BSSVS) program in the BEAST v 1.8.1 software package was used to analyze the spatial dynamics of the Getah virus.Results showed that the full-length of Getah virus E2 gene consists of 1 266 nueleotides,encoding 422 amino acids.And the homology of nucleotide and amino acid were 94.5％ 100％ and 96.4％ 100％ respectively.The molecular evolution analysis revealed that there were no species and geographical distribution difference existing among GETV host animals (e.g.horses and pigs) and vectors (e.g.mosquitoes).Bioinformatics analysis showed that GETV originated in Malaysia,then it was spread to Japan,China,South Korea,Mongolia,Russia,etc.GETV E2 gene was relatively stable since GETV was first isolated in 1955.The differences of species and geographical distribution did not exist among GETV host animals and vectors,and the virus has spread from tropical regions to Eurasian continent.Thus,strengthening the detection and monitoring of GETV and its infections in humans and livestock is critical.

Development and Evaluation of Indirect ELISA for Detection of Antibodies to Getah Virus in Horse Serum

Getah virus (GETV) is a member of the genus Alphavirus in the family Togaviridae. GETV infection can occur in a wide range of vertebrate species, and the virus has been known for a pathogen of horses and pigs. To rapidly and accurately diagnose GETV infection of a racehorse, an indirect ELISA (I-ELISA) was developed in the present study for detection of antibodies to GETV in serum samples. To evaluate the developed I-ELISA, a total of 240 serum samples from Thoroughbred racehorses raised in Korea were screened in parallel by a serum neutralization (SN) test. The developed I-ELISA exhibited an efficacy comparable to that of the SN test in terms of a high diagnostic sensitivity (86.3%) and specificity (94.5%) at a cut-off absorbance value of 0.25. In addition, our results showed that the developed I-ELISA had a significant correlation with the SN test (r = 0.91; p < 0.05). Taken together, our findings suggest that the I-ELISA developed in this study is a valuable diagnostic tool for the screening of horses suspected to be infected with GETV.

Sero-surveillance of Getah Virus among Thoroughbred Horses in Korea

Getah virus (GETV), which is transmitted by mosquitoes, causes lower limb edema and stiffness in horses. In this study, we investigated the sero-surveillance of GETV among Thoroughbred racehorses in Korea during 2013 and 2014. A total of 1,182 equine serum samples collected from Thoroughbred racehorses in four provinces (Gyeongnam, Gyeonggi, Jeonbuk and Jeju provinces) were analyzed using virus neutralization (VN) tests. An antibody titer of > or = 1:2 was considered positive. Overall, the seropositivity rate for GETV was found to be 12.4% (146/1,182) among the racehorses; the annual seropositivity rates were 12.4% and 12.2% in 2013 and 2014, respectively. The seropositivity rates in April and September in 2013 turned out to be 8.6% and 15.2%, respectively. The regional distribution of seropositivity ranged from 5.0% to 22.3% in 2013 and from 0.0% to 15.0% in 2014, respectively. Gyeongnam province had the highest seropositivity rate than other provinces. By analyzing the distribution of VN titers according to horse age, we found that the highest GETV seropositivity rate was in horses over 6 years of age (22.4% and 28.1%, 2013 and 2014, respectively), and that the incidence of GETV was higher in geldings (17.6% and 18.6%, 2013 and 2014, respectively) than in males and females. These results indicate that Thoroughbred horses raised in Korea were bitten by mosquitoes harboring GETV.

Recharacterization of Morphological and Genetic Feature of Getah Virus Isolated from South Korea

Three QIAG93 strains, QIAG9301, QIAG9302 and QIAG9303 that have been identified as Getah virus (GETV) are analyzed in this study. The morphological features of three virus isolates were observed by using electron microscopy, suggesting that the QIAG9301, QIAG9302 and QIAG9303 isolate can be classified as tentative member of Alphavirus species in the Semliki Forest complex. The full length of the structural polyprotein gene of each QIAG93 isolate (QIAG9301, QIAG9302 and QIAG9303) was determined that are identical in size, comprising 3759 nucleotides that encoded 1253 amino acids. The sequence analysis of the structural polyprotein gene, including the C, E3, E1, 6K and E2 domain, showed that each QIAG93 isolate shares >98.9% sequence identity. The phylogenetic analysis and evolutionary distance (ED) estimation based on the structural polyprotein gene sequence showed that the QIAG9301 isolate is closely related to GETV South Korea strain (99.9% sequence identity and ED value 0.001) and Chinese GETV YN0540 strain (99.3% sequence identity ED value 0.007) than other Alphavirus species analyzed in this study. Both QIAG9032 and QIAG9303 isolate exhibited genetically close relationship with Mongolian GETV LEIV17741MPR strain (at least 99.3% sequence identity and mean ED value 0.0065). Therefore, our findings will be valuable for molecular epidemiological analyses of GETV in Korea and contribute to a further study on pathogenicity of three QIAG93 isolates in animals.

Investigation on mosquitoes and mosquito-borne viruses in Dehong prefecture,Yunnan province,2007 and 2010 / 中华流行病学杂志

Objective To investigate the distribution patterns of mosquitoes and mosquito-borne viruses in Dehong prefecture,Yunnan province,China. Methods Mosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July,2007 and 2010. Mosquitoes were cell cultured for viral isolation,and positive isolates were identified using RT-PCR and sequence analysis. Results A total of 43 634 mosquitoes comprised of 29 species representing six genera were collected. Culex tritaeriorhynchus and Anopheles sinensis comprised 78.69% and 14.77% of the total. Six strains of viruses were isolated from the mosquito pools. RT-PCR and phylogenetic analysis revealed three strains from Cx. tritaeriorhynchus,identified as genotypeⅠJapanese encephalitis virus(JEV). One strain was identified from Cx. tritaeriorhynchus,as Getah virus (GETV). Two strains isolated from Cx. tritaeriorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus(CppDNV). Conclusion Cx. tritaeriorhynchus had been the major species of mosquitoes and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. GenotypeⅠJEV,GETV and CppDNV were the vectors causing transmission of mosquitoe-borne diseases in this area. Data from phylogenetic analysis showed that these newly discovered isolates seemed to have had close relationship with those viruses previously circulating in Yunnan and other provinces of China.

Investigation on mosquitoes and mosquito-borne viruses in Dehong prefecture,Yunnan province,2007 and 2010 / 中华流行病学杂志

Objective To investigate the distribution patterns of mosquitoes and mosquito-borne viruses in Dehong prefecture,Yunnan province,China. Methods Mosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July,2007 and 2010. Mosquitoes were cell cultured for viral isolation,and positive isolates were identified using RT-PCR and sequence analysis. Results A total of 43 634 mosquitoes comprised of 29 species representing six genera were collected. Culex tritaeriorhynchus and Anopheles sinensis comprised 78.69% and 14.77% of the total. Six strains of viruses were isolated from the mosquito pools. RT-PCR and phylogenetic analysis revealed three strains from Cx. tritaeriorhynchus,identified as genotypeⅠJapanese encephalitis virus(JEV). One strain was identified from Cx. tritaeriorhynchus,as Getah virus (GETV). Two strains isolated from Cx. tritaeriorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus(CppDNV). Conclusion Cx. tritaeriorhynchus had been the major species of mosquitoes and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. GenotypeⅠJEV,GETV and CppDNV were the vectors causing transmission of mosquitoe-borne diseases in this area. Data from phylogenetic analysis showed that these newly discovered isolates seemed to have had close relationship with those viruses previously circulating in Yunnan and other provinces of China.

Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay / 中国病毒学·英文版

Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation.We developed a cost-effective,rapid,and highly sensitive one-step triplex RT-PCR enzyme hybridizationassay for simultaneous detections of Japanese Encephallitis virus (JEV,Flaviviridae)Getah virus (GETV,Togaviridae),and Tahyna virus (TAHV,Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction.The analytical sensitivity of this assay was 1 PFU/mL for JEV,10PFU/mL for GETV,and 10 PFU/mL for TAHV.This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods.When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid(CSF)samples that were JEV-positive by normal RT-PCR assay,all samples were strongly positive for JEV,but negative for GETV and TAHV,demonstrating a good sensitivity,specificity,and performance at CSF specimen detection.

Partial genome molecular characteristics of Getah virus newly isolated in China / 中华微生物学和免疫学杂志

Objective To study the genome molecular characteristics of Getah virus(DY0824)which isolated in Shandong province,2008 by molecular biology methods.Methods Reverse transcriptasepolymerase chain reaction(RT-PCR)was used to amplify the structural gene and 3'UTR fragments then the RT-PCR products were inserted into PGEM-T easy to be sequenced.Computer software was used to analyze the nucleotide and deduced amino acid sequence,and draw phylogenetic trees,including Clustal X1.83 and MegaAlign and Mega4.Results The capsid protein of DY0824 consists of 804 nucleotides,encoding 268 amino acids and the full-length of E2 protein is 1266 nucleotides,encoding 422 amino acids.The nucleotide homology of the capsid protein and the E2 protein with other strains were 95.4%-99.9%and 94.8%-99.5%,and the amino acid were 97.4%-100%and 97.6%-100%.The 3'UTR of the virus include 401 nucleotides and there are three repeat sequence elements.Conclusion Compared with the prototype virus,the Getah virus isolated in Shandong province had 7 amino acid differences in capsid protein genes and 10 amino acid differences in E protein genes.The 3'UTR region had multi-nucleotide changes.

Isolation and identification of arboviruses from mosquito pools in some regions of Liaoning province,China / 中华流行病学杂志

Objective To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province.Methods Mosquitoes were collected from Shenyang,Yingkou,Panjin,Jinzhou and Dandong cities of Liaoning province in 2006.Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells.The new isolates were identified using serological and molecular biological methods.Results 5410 mosquitoes were collected from the five cities in total.Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BI-IK-21 cell.Three isolates (LN0684,LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus.Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates.The identity of nucleotide sequence was between 91.2% and 94.7%,compared with other Banna virus strains.The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine).The identity of nucleotide sequence was 99.2%,when comparing with the strain of South Korea and was 95% to 99% with the strains fi'om Russia,mainland of China and Taiwan region.Conehmion Eight virus isolates,including three Banna virus,one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province.Banna virus and Getah virus were reported for the first time in Liaoning province,while Getah virus showed the highest nucleotide homology with the South Korea strains.