Transcriptome screening identifies TIPARP as an antiviral host factor against the Getah virus.

IMPORTANCE: Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses.

Evolutionary characterization and pathogenicity of Getah virus from pigs in Guangdong Province of China.

Getah virus (GETV) is an emerging zoonotic virus that can infect humans and many mammals through mosquitoes. In this study, a novel pathogenic GETV strain, GDQY2022, was isolated from a pig farm in Guangdong Province, China. Sequence comparisons and phylogenetic analysis showed that GDQY2022 belongs to group III (GIII) and was most closely related to strain HeN202009-2, with 99.78% nucleotide sequence identity. Histopathological examination revealed significant pathological changes, such as widened alveolar septum in the lungs with mild congestion and hemorrhage. Differences in viral load between tissues were assessed by real-time RT-PCR, and significantly higher levels of GETV were found in abdominal lymph nodes and lungs of subclinically and clinically affected pigs (P < 0.01). This study provides valuable data for understanding the risk of GETV infection in the pig industry and a reliable basis for studying the pathogenic mechanisms and diagnostic surveillance of GETV.

Establishment of a reverse transcription real-time quantitative PCR method for Getah virus detection and its application for epidemiological investigation in Shandong, China.

Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus Alphavirus of the family Togaviridae. Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV Nsp1 gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and Cq value with a correlation coefficient (R 2) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects.

Getah Virus (Alphavirus): An Emerging, Spreading Zoonotic Virus.

Getah virus (GETV) is a zoonotic virus transmitted by mosquitoes, belonging to the Togaviridae family, Alphavirus genus. It was first isolated from mosquitoes in Malaysia in 1955, being widespread in island countries in the South Pacific region. Since the beginning of the 21st century, GETV expanded its range and geographical distribution from low-latitude tropical regions to 60° north latitude, being isolated from 17 different species of mosquitoes belonging to five genera of Culicidae (Culex, Anopheles, Armigeres, Aedes and Mansonia), as well as from midges in Eurasia. Molecular genetic evolution analysis revealed large molecular differences between the mosquitoes currently circulating Eurasia and those in the South Pacific in 1950s. The number of disease outbreaks caused by GETV in animals is increasing alongside the types of animals infected, from horses and pigs to cattle, blue foxes and red pandas. The disease burden is severely underestimated, and the economic cost to livestock production remains unknown. Herein, we review GETV temporal and spatial distribution, molecular genetic evolution, transmission and data on disease outbreaks. This work provides a reference for public health workers engaged in GETV research and zoonotic disease prevention and control.

Molecular and serological surveillance of Getah virus in the Xinjiang Uygur Autonomous Region, China, 2017-2020.

The Getah virus (GETV), a mosquito-borne RNA virus, is widely distributed in Oceania and Asia. GETV is not the only pathogenic to horses, pigs, cattle, foxes and boars, but it can also cause fever in humans. Since its first reported case in Chinese mainland in 2017, the number of GETV-affected provinces has increased to seventeen till now. Therefore, we performed an epidemiologic investigation of GETV in the Xinjiang region, located in northwestern China, during the period of 2017-2020. ELISA was used to analyze 3299 serum samples collected from thoroughbred horse, local horse, sheep, goat, cattle, and pigs, with thoroughbred horse (74.8%), local horse (67.3%), goat (11.7%), sheep (10.0%), cattle (25.1%) and pigs (51.1%) being positive for anti-GETV antibodies. Interestingly, the neutralizing antibody titer in horses was much higher than in other species. Four samples from horses and pigs were positive for GETV according to RT-PCR. Furthermore, from the serum of a local horse, we isolated GETV which was designated as strain XJ-2019-07, and determined its complete genome sequence. From the phylogenetic relationships, it belongs to the Group III lineage. This is the first evidence of GETV associated to domestic animals in Xinjiang. Overall, GETV is prevalent in Xinjiang and probably has been for several years. Since no vaccine against GETV is available in China, detection and monitoring strategies should be improved in horses and pigs, especially imported and farmed, in order to prevent economic losses.

Construction and characterization of a full-length infectious clone of Getah virus in vivo.

Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK-21 â€‹cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 3'UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 â€‹cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

Attenuation of Getah Virus by a Single Amino Acid Substitution at Residue 253 of the E2 Protein that Might Be Part of a New Heparan Sulfate Binding Site on Alphaviruses.

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo. Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the three-dimensional structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

Isolation and characterization of Getah virus from pigs in Guangdong province of China.

Getah virus (GETV) is a mosquito-borne virus that is widely distributed in Asian countries including China, in which the first case of equine GETV infection was reported in Guangdong province in August 2018. In this study, GETVs were detected in two classical swine fever virus-positive samples collected from swine herds in Foshan city, Guangdong province, 2018. Infection of porcine PK-15 cells produced rapid cytopathic effects (CPEs), including shrinking, rounding and detaching, and peak titre of 109.3 TCID50 /ml occurred at 24 hr post-infection. Electron microscopy and ultra-thin sectioning revealed spherical GETV particles of 70 nm diameter with an isometric interior and are found to be lining the outer membrane of infected cells. Whole-genome analysis showed that the two GETV isolates are identical to each other and cluster with Group III strains of GETV, sharing 96.1%-99.7% nucleotide sequence identity with all available reference strains. The most closely relative of the obtained GETV isolates was porcine strain HNJZ-S2 from Henan province (99.7%), with 98.6% sequence identity shared with equine GETV strain GZ201808 first identified in Guangdong province, indicating different sources for porcine and equine GETV infections in this region. No evidence of GETV infection was found in 497 archived porcine samples collected between 1990 and 2018 in Guangdong province.

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.