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.
Alphavirus* ; Animals ; Cell Culture Techniques ; Diagnosis ; Disease Outbreaks ; DNA ; Genome ; Glycoproteins ; Horses ; Livestock* ; Methods ; Polymerase Chain Reaction* ; Reverse Transcription* ; RNA ; RNA, Viral ; Sensitivity and Specificity ; Swine

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.
Alphavirus* ; Antibodies* ; Enzyme-Linked Immunosorbent Assay* ; Horses* ; Humans ; Korea ; Mass Screening ; Sensitivity and Specificity ; Swine ; Togaviridae ; Vertebrates

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.
Alphavirus ; Animal Diseases ; Animals* ; Circovirus ; Communicable Diseases ; Diarrhea ; Encephalitis Virus, Venezuelan Equine ; Encephalomyelitis, Equine ; Immunity, Cellular ; Porcine epidemic diarrhea virus ; RNA ; Vaccines ; Vaccines, Synthetic ; Vaccines, Virus-Like Particle ; Venezuela

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.
Alphavirus* ; Amino Acids ; Animals ; Asian Continental Ancestry Group ; Humans ; Korea* ; Microscopy, Electron ; Molecular Epidemiology ; Nucleotides ; Sequence Analysis ; Trees ; Virulence

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.
Alphavirus ; drug effects ; genetics ; metabolism ; Animals ; Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; High-Throughput Screening Assays ; methods ; Luciferases ; genetics ; metabolism

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.
Alphavirus* ; Culicidae ; Edema ; Female ; Gyeonggi-do ; Horses* ; Humans ; Incidence ; Jeollabuk-do ; Korea* ; Lower Extremity ; Male

OBJECTIVETo investigate the distribution patterns of mosquito and mosquito-borne viruses in Dehong prefecture, Yunnan province, China.

METHODSMosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July, 2007 and 2010. Mosquito were cell cultured for viral isolation, and positive isolates were identified using RT-PCR and sequence analysis.

RESULTSA total of 43 634 mosquito comprised of 29 species representing six genera were collected. Culex tritaeniorhynchus 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. tritaeniorhynchus, identified as genotype I Japanese encephalitis virus (JEV). One strain was identified from Cx. tritaeniorhynchus, as Getah virus (GETV). Two strains isolated from Cx. tritaeniorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus (CppDNV).

CONCLUSIONCx. tritaeniorhynchus had been the major species of mosquito and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. Genotype I JEV, GETV and CppDNV were the vectors causing transmission of mosquito-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.

Alphavirus ; isolation & purification ; Animals ; Arboviruses ; classification ; isolation & purification ; China ; Culicidae ; virology ; Disease Vectors ; classification ; Encephalitis Virus, Japanese ; isolation & purification

In this review, we mainly focus on zoonotic encephalitides caused by arthropod-borne viruses (arboviruses) of the families Flaviviridae (genus Flavivirus) and Togaviridae (genus Alphavirus) that are important in both humans and domestic animals. Specifically, we will focus on alphaviruses (Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus) and flaviviruses (Japanese encephalitis virus and West Nile virus). Most of these viruses were originally found in tropical regions such as Africa and South America or in some regions in Asia. However, they have dispersed widely and currently cause diseases around the world. Global warming, increasing urbanization and population size in tropical regions, faster transportation and rapid spread of arthropod vectors contribute in continuous spreading of arboviruses into new geographic areas causing reemerging or resurging diseases. Most of the reemerging arboviruses also have emerged as zoonotic disease agents and created major public health issues and disease epidemics.
Africa ; Alphavirus* ; Animals, Domestic ; Arboviruses* ; Arthropod Vectors ; Asia ; Encephalitis ; Encephalitis Virus, Venezuelan Equine ; Encephalitis Virus, Western Equine ; Encephalitis Viruses ; Encephalomyelitis, Equine ; Epidemiology* ; Flaviviridae ; Flavivirus* ; Global Warming ; Humans ; Population Density ; Public Health ; South America ; Togaviridae ; Transportation ; Urbanization ; Zoonoses

Based on a pair of specific primers, a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold I which carried the His tag, this recombinant plasmid was then determined by enzyme digestion, PCR and DNA sequencing. This recombinant plasmid pCold-Cap was transformed into E. coli Rosetta 2, and PGETV Cap fusion protein was expressed through IPTG induction. The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0. Immol/L IPTG under 15"C for 24h, the expression quantity was 40. 2%. The product had a molecular mass about 32. 3kD as expected. The target protein was separated in gel slices and used to immunize Balb/c mice. The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained. The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.
Alphavirus ; genetics ; immunology ; metabolism ; Alphavirus Infections ; immunology ; veterinary ; virology ; Animals ; Antibodies, Viral ; blood ; immunology ; Blotting, Western ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; DNA Primers ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Swine ; Swine Diseases ; immunology ; virology ; Zoonoses

Traditional approach of inactivated or live-attenuated vaccine immunization has resulted in impressive success in the reduction and control of infectious disease outbreaks. However, many pathogens remain less amenable to deal with the traditional vaccine strategies, and more appropriate vaccine strategy is in need. Recent discoveries that led to increased understanding of viral molecular biology and genetics has rendered the used of viruses as vaccine platforms and as potential anti-cancer agents. Due to their ability to effectively induce both humoral and cell-mediated immune responses, viral vectors are deemed as an attractive alternative to the traditional platforms to deliver vaccine antigens as well as to specifically target and kill tumor cells. With potential targets ranging from cancers to a vast number of infectious diseases, the benefits resulting from successful application of viral vectors to prevent and treat human diseases can be immense.
Adenoviridae ; Alphavirus ; Communicable Diseases ; Disease Outbreaks ; Genetic Vectors ; Humans ; Immunization ; Molecular Biology ; Poxviridae ; Vaccines