RESUMO
Getah virus (GETV) mainly causes disease in livestock and may pose an epidemic risk due to its expanding host range and the potential of long-distance dispersal through animal trade. Here, we used metagenomic next-generation sequencing (mNGS) to identify GETV as the pathogen responsible for reemerging swine disease in China and subsequently estimated key epidemiological parameters using phylodynamic and spatially-explicit phylogeographic approaches. The GETV isolates were able to replicate in a variety of cell lines, including human cells, and showed high pathogenicity in a mouse model, suggesting the potential for more mammal hosts. We obtained 16 complete genomes and 79 E2 gene sequences from viral strains collected in China from 2016 to 2021 through large-scale surveillance among livestock, pets, and mosquitoes. Our phylogenetic analysis revealed that three major GETV lineages are responsible for the current epidemic in livestock in China. We identified three potential positively selected sites and mutations of interest in E2, which may impact the transmissibility and pathogenicity of the virus. Phylodynamic inference of the GETV demographic dynamics identified an association between livestock meat consumption and the evolution of viral genetic diversity. Finally, phylogeographic reconstruction of GETV dispersal indicated that the sampled lineages have preferentially circulated within areas associated with relatively higher mean annual temperature and pig population density. Our results highlight the importance of continuous surveillance of GETV among livestock in southern Chinese regions associated with relatively high temperatures. IMPORTANCE Although livestock is known to be the primary reservoir of Getah virus (GETV) in Asian countries, where identification is largely based on serology, the evolutionary history and spatial epidemiology of GETV in these regions remain largely unknown. Through our sequencing efforts, we provided robust support for lineage delineation of GETV and identified three major lineages that are responsible for the current epidemic in livestock in China. We further analyzed genomic and epidemiological data to reconstruct the recent demographic and dispersal history of GETV in domestic animals in China and to explore the impact of environmental factors on its genetic diversity and its diffusion. Notably, except for livestock meat consumption, other pig-related factors such as the evolution of live pig transport and pork production do not show a significant association with the evolution of viral genetic diversity, pointing out that further studies should investigate the potential contribution of other host species to the GETV outbreak. Our analysis of GETV demonstrates the need for wider animal species surveillance and provides a baseline for future studies of the molecular epidemiology and early warning of emerging arboviruses in China.
Assuntos
Arbovírus , Genoma Viral , Filogenia , Animais , Humanos , Camundongos , Arbovírus/genética , China/epidemiologia , Genômica , Gado/virologiaRESUMO
Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: ⢠We established a rapid antibody detection method that can monitor GETV infection ⢠We developed colloidal gold test strips with high sensitivity and specificity ⢠The development of colloidal gold test strips will aid in the field serologic detection of GETV.
Assuntos
Alphavirus , Anticorpos Antivirais , Coloide de Ouro , Sensibilidade e Especificidade , Animais , Coloide de Ouro/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Alphavirus/imunologia , Suínos , Cromatografia de Afinidade/métodos , Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/imunologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Fitas Reagentes , China , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
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.
Assuntos
Infecções por Alphavirus , Alphavirus , Substituição de Aminoácidos , Proteínas do Envelope Viral , Alphavirus/genética , Alphavirus/patogenicidade , Infecções por Alphavirus/virologia , Animais , Sítios de Ligação/genética , Células Cultivadas , Modelos Animais de Doenças , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Alphavirus is a type of arbovirus that can infect both humans and animals. The amino acid sequence of the 6K protein, being one of the structural proteins of the alphavirus, is not conserved. Deletion of this protein will result in varying effects on different alphaviruses. Our study focuses on the function of the Getah virus (GETV) 6K protein in infected cells and mice. We successfully constructed infectious clone plasmids and created resulting viruses (rGETV and rGETV-Δ6K). Our comprehensive microscopic analysis revealed that the 6 K protein mainly stays in the endoplasmic reticulum. In addition, rGETV-Δ6K has lower thermal stability and sensitivity to temperature than GETV. Although the deletion of the 6K protein does not reduce virion production in ST cells, it affects the release of virions from host cells by inhibiting the process of E2 protein transportation to the plasma membrane. Subsequent in vivo testing demonstrated that neonatal mice infected with rGETV-Δ6K had a lower virus content, less significant pathological changes in tissue slices, and milder disease than those infected with the wild-type virus. Our results indicate that the 6K protein effectively reduces the viral titer by influencing the release of viral particles. Furthermore, the 6K protein play a role in the clinical manifestation of GETV disease.
Assuntos
Alphavirus , Humanos , Animais , Camundongos , Alphavirus/metabolismo , Virulência , Proteínas Virais/metabolismo , Replicação Viral , Sequência de AminoácidosRESUMO
Getah virus (GETV) was becoming more serious and posing a potential threat to animal safety and public health. Currently, there is limited comprehension regarding the pathogenesis and immune evasion mechanisms employed by GETV. Our study reveals that GETV infection exhibits the capacity for interferon antagonism. Specifically, the nonstructural protein nsP2 of GETV plays a crucial role in evading the host immune response. GETV nsP2 effectively inhibits the induction of IFN-ß by blocking the phosphorylation and nuclear translocation of IRF3. Additionally, GETV nsP2 hinders the phosphorylation of STAT1 and its nuclear accumulation, leading to significantly impaired JAK-STAT signaling. Furthermore, the amino acids K648 and R649, situated in the C-terminal region of GETV nsP2, play a crucial role in facilitating nuclear localization. Not only do they affect the interference of nsP2 with the innate immune response, but they also exert an influence on the pathogenicity of GETV in mice. In summary, our study reveals novel mechanisms by which GETV evades the immune system, thereby offering a foundation for comprehending the pathogenic nature of GETV. Video Abstract.
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Alphavirus , Interferons , Animais , Camundongos , Linhagem Celular , Imunidade Inata , Evasão da Resposta ImuneRESUMO
A new Getah virus (GETV) strain, B254, was isolated from Culex fuscocephalus mosquitoes captured at Mount Ophir, Malaysia, in 2012. Phylogenetic analyses revealed that GETV B254 is distinct from the old Malaysia GETV MM2021 strain but closely related to group IV GETV from Russia (LEIV16275Mag), China (YN12031), and Thailand (GETV/SW/Thailand/2017).
Assuntos
Alphavirus , Culex , Culicidae , Animais , Malásia/epidemiologia , FilogeniaRESUMO
Getah virus (GETV), a mosquito-borne virus belonging to the Alphavirus genus of family Togaviridae, has become increasingly problematic, which poses a huge threat to the safety of animals and public health. In order to detect GETV quickly and accurately, we have developed a SYBR Green I real-time quantitative reverse transcription PCR (RT-qPCR) assay for GETV with the detection limit of 66 copies/µL, excellent correlation coefficient (R2) of 0.9975, and amplification efficiency (E) of 98.90%, the target selected was the non-structural protein 3 of GETV. The sensitivity of it was higher than that of ordinary RT-PCR by 1000 folds, and the inter-assay and intra-assay CV values were all less than 0.99%. The newly developed RT-qPCR assay exhibited good sensitivity and reproducibility, which will provide technical support for the reliable and specific rapid diagnosis, and quantitative analysis of GETV infection.
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Alphavirus , Culicidae , Alphavirus/genética , Animais , Benzotiazóis , Diaminas , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transcrição Reversa , Sensibilidade e Especificidade , SuínosRESUMO
In the present study, Getah virus (GETV) isolate, GETV-V1, was isolated from a commercial PRRSV attenuated live vaccine (MLV), which has been widely used to immunize pigs against porcine reproductive and respiratory syndrome virus (PRRSV). Further analysis demonstrated that nine batches of the PRRSV MLV vaccine (three batches per year from 2017 to 2019) from the same manufacturer were all positive for GETV. Genomic analyses indicated that the GETV-V1 isolate shared the highest sequence identity with the GETV strain, 16-I-674, which was isolated from horses in Japan. The phylogenetic analysis based on the genomic sequences showed that the GETV-V1 strain was clustered with the Japanese GETV strains. Taken together, this is the first report of GETV contamination in live swine vaccines in China. Our findings demonstrate that immunization with commercial live vaccines might be a potential novel route of GETV transmission in swine. This highlights the need for more extensive monitoring of commercial live vaccines.
Assuntos
Alphavirus/classificação , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vacinas Virais/análise , Alphavirus/genética , Alphavirus/isolamento & purificação , Animais , Linhagem Celular , China , Contaminação de Medicamentos , Cavalos , Japão , Filogenia , Filogeografia , SuínosRESUMO
We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/patogenicidade , Doenças dos Animais/epidemiologia , Doenças dos Animais/virologia , Raposas/virologia , Alphavirus/classificação , Alphavirus/genética , Alphavirus/ultraestrutura , Doenças dos Animais/história , Doenças dos Animais/transmissão , Animais , China/epidemiologia , História do Século XXI , Filogenia , Vigilância em Saúde Pública , RNA Viral , Análise de Sequência de DNA , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologiaRESUMO
BACKGROUND: Getah virus (GETV) is a neglected mosquito-borne Alphavirus that causes pyrexia, body rash, and leg oedema in horses and foetal death and reproductive disorders in pigs. Infected animals may play a critical role in the amplification and circulation of the virus. The present study aimed to investigate GETV infection in clinically infected cattle and vector mosquito species in northeastern China. RESULTS: Serum samples were collected from beef cattle that presented sudden onset of fever in forest grazing areas, and metagenomic sequencing was conducted, revealing 29 contigs from ten serum samples matching the GETV genome. Quantitative RT-PCR (RT-qPCR) was performed with GETV RNA from 48 beef cattle serum samples, showing that the overall prevalence of GETV in the beef cattle samples was 6.25% (3/48). Serological investigation indicated that GETV neutralizing antibodies were detected in 83.3% (40/48, 95% CI 67-100) of samples from the study region. The GETV JL1808 strain was isolated from clinically infected cattle showing fever. Sequence comparisons showed high identity with the HuN1 strain, a highly pathogenic swine epidemic isolate obtained in Hunan province in 2017, at the nucleotide level (99.5%) and at the deduced amino acid level (99.7-99.9%). The phylogenetic analysis of JL1808 clustered in Group III, and also revealed a close genetic relationship with the HuN1 strain. Additionally, about 12,000 mosquitoes were trapped in this region. The presence of GETV infection was detected in mosquitoes, suggesting that the minimum infection rate (MIR) was 1.50, with MIRs of 1.67 in Culex pseudovishnui, 1.60 in Culex tritaeniorhynchus, and 1.21 in Anopheles sinensis. CONCLUSIONS: To the best of our knowledge, this is the first report of GETV infection in cattle. These results demonstrated that a highly pathogenic, mosquito-borne swine GETV can infect and circulate in cattle, implying that it is necessary to conduct surveillance of GETV infection in animals in northeastern China.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Doenças dos Bovinos/virologia , Alphavirus/classificação , Infecções por Alphavirus/virologia , Animais , Bovinos , China , Culicidae/virologia , Vetores de Doenças , Filogenia , RNA Viral/isolamento & purificaçãoRESUMO
BACKGROUND: We studied a recent epizootic of Getah virus infection among pigs in the southern part of Ibaraki Prefecture and the northern part of Chiba Prefecture, Japan, focusing on its possible association with outbreaks in racehorses in 2014 and 2015. The genomic sequence of a Getah virus strain from an infected pig was analyzed to evaluate the degree of identity with the strains from horses. RESULTS: Sera were collected from pigs from September to December 2012 to 2015 in south Ibaraki (380 pigs in 29 batches), and from September to December 2010 to 2015 in north Chiba (538 pigs in 104 batches). They were examined by using a virus-neutralizing test for Getah virus. Seropositivity rates in 2012-2013 in south Ibaraki and 2010-2012 in north Chiba ranged from 0% to 1.6%. In south Ibaraki, seropositivity rates in 2014 (28.8%) and 2015 (65.0%) were significantly higher than those in the previous years (P < 0.01); 4/5 batches had positive sera in 2014 and 7/7 in 2015. In north Chiba, seropositivity rates in 2013 (14.1%), 2014 (17.8%), and 2015 (48.0%) were significantly higher than those in the previous years (P < 0.01); 6/27 batches had positive sera in 2013, 3/9 in 2014, and 5/5 in 2015. Complete genome analysis revealed that the virus isolated from an infected pig had 99.89% to 99.94% nucleotide identity to the strains isolated from horses during the outbreaks in 2014 and 2015. CONCLUSIONS: Serological surveillance of Getah virus in pigs revealed that the virus was circulating in south Ibaraki and north Chiba in 2014 and 2015; this was concomitant with the outbreaks in racehorses. The Getah virus strain isolated from a pig was closely related to the ones from horses during the 2014 and 2015 outbreaks. To our knowledge, this is the first convincing case of simultaneous circulation of Getah virus both among pigs and horses in specific areas.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Doenças dos Cavalos/virologia , Doenças dos Suínos/virologia , Alphavirus/classificação , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/transmissão , Animais , Chlorocebus aethiops , DNA Viral , Surtos de Doenças/veterinária , Genoma Viral , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/transmissão , Cavalos , Japão/epidemiologia , Filogenia , RNA Viral/sangue , Análise de Sequência de DNA , Especificidade da Espécie , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Células VeroRESUMO
In this study, we isolated a virus strain (YN12031) from specimens of Armigeres subalbatus collected in the China-Laos border. BHK-21 cells infected with YN12031 exhibited an evident cytopathic effect (CPE) 32 h post-infection. The virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers. Molecular genetic analysis revealed that YN12031 was closely related to alpha viruses such as Chikungunya virus and Sindbis virus, and located in the same clade as MM2021, the prototype of Getahvirus (GETV) isolated in Malaysia in 1955. Phylogenetic analysis of the E2 and capsid genes further revealed that YN12031 was located in the same clade as the Russian isolate LEIV/16275/Mag. Analysis of the homology of nucleotides and amino acids in the coding area and E2 gene demonstrated that the YN12031 isolated from the China-Laos border (tropical region) was related closest to the LEIV/16275/Mag isolate obtained in Russia (North frigid zone area) among other isolates studied. These results suggest that GETV can adapt to different geographical environments to propagate and evolve. Thus, strengthening the detection and monitoring of GETV and its related diseases is very crucial.
Assuntos
Alphavirus/genética , Alphavirus/isolamento & purificação , Culicidae/virologia , Animais , Linhagem Celular , China , Cricetinae , Laos , FilogeniaRESUMO
BACKGROUND: As we reported previously, Getah virus infection occurred in horses at the Miho training center of the Japan Racing Association in 2014. This was the first outbreak after a 31-year absence in Japan. Here, we report a recurrent outbreak of Getah virus infection in 2015, sequential to the 2014 one at the same site, and we summarize its epizootiological aspects to estimate the risk of further outbreaks in upcoming years. RESULTS: The outbreak occurred from mid-August to late October 2015, affecting 30 racehorses with a prevalence of 1.5% of the whole population (1992 horses). Twenty-seven (90.0%) of the 30 affected horses were 2-year-olds, and the prevalence in 2-year-olds (27/613 [4.4%]) was significantly higher than that in horses aged 3 years or older (3/1379 [0.2%], P < 0.01). Therefore, the horses newly introduced from other areas at this age were susceptible, whereas most horses aged 3 years or older, which had experienced the previous outbreak in 2014, were resistant. Among the 2-year-olds, the prevalence in horses that had been vaccinated once (10/45 [22.2%]) was significantly higher than that in horses vaccinated twice or more (17/568 [3.0 %], P < 0.01). Horse anti-sera raised against an isolate in 2014 neutralized both the homologous strain and a 2015 isolate at almost the same titers (256 to 512), suggesting that these viruses were antigenically similar. Among horses entering the training center from private surrounding farms in 2015, the seropositivity rate to Getah virus increased gradually (11.8% in August, 21.7% in September, and 34.9% in October). Thus, increased virus exposure due to the regional epizootic probably allowed the virus to spread in the center, similarly to the outbreak in 2014. CONCLUSIONS: The 2015 outbreak was caused by a virus which was antigenically close to the 2014 isolate, affecting mostly 2-year-old susceptible horses under epizootiological circumstances similar to those in 2014. The existence of 2-year-olds introduced from regions free from Getah virus could continue to pose a potential risk of additional outbreaks in upcoming years. Vaccination on private farms and breeding farms would help to minimize the risk of outbreaks.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus , Surtos de Doenças/veterinária , Doenças dos Cavalos/epidemiologia , Alphavirus/isolamento & purificação , Infecções por Alphavirus/epidemiologia , Animais , Chlorocebus aethiops , Estudos Transversais , Suscetibilidade a Doenças/veterinária , Doenças dos Cavalos/virologia , Cavalos , Fatores de Risco , Células Vero , Vacinas Virais/administração & dosagemRESUMO
An outbreak of Getah virus infection occurred among racehorses in Japan during September and October 2014. Of 49 febrile horses tested by reverse transcription PCR, 25 were positive for Getah virus. Viruses detected in 2014 were phylogenetically different from the virus isolated in Japan in 1978.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Alphavirus/classificação , Alphavirus/genética , Animais , Surtos de Doenças , Genes Virais , História do Século XXI , Doenças dos Cavalos/história , Cavalos , Japão/epidemiologia , FilogeniaRESUMO
In recent years, the epidemiological profile of Getah virus (GETV) has become increasingly serious, posing a huge threat to animal and public health in China. GETV can cause multi-species infection, including horses, pigs, rats, cattle, kangaroos, reptiles and birds. However, there were few reports on the efficiency of the virus entering the host via routes of different systems. In the present study, a GETV strain (SC201807) was obtained from a piglet's blood in 2018 in Sichuan, China. First, we established a quantitative real-time polymerase chain reaction (qRT-PCR) SYBR assay specific to GETV. Then, we evaluated the infection efficiency of different routes using mouse animal model. 108 male mice were randomly divided into four groups as follows: intramuscular, intraoral and intranasal infection routes, and negative control. All mice in the experimental group were inoculated with 4 × 102.85 TCID50 GETV virus. Tissue tropism experiments show that GETV has a wide range of tissue distribution, and intramuscular infection is the first to infect all tissues of the body, and suggest that oral infection may be a new GETV transmission route. Histopathological examination results showed that intramuscular injection of GETV mainly caused different degrees of pathological damage to the tissues, and could rapidly induce a large amount of inflammatory regulatory factors such as IL-6 and TNF-α. Our data may help us to evaluate the risk of transmission of Porcine Getah virus and provide an experimental basis for the prevention and control of Porcine Getah virus.
RESUMO
Getah virus (GETV) is an emerging mosquito-borne alphavirus that can infect horses, pigs and other animals. Given the public health threat posed by GETV, research on its pathogenesis, diagnosis and prevention is urgently needed. In the current study, prokaryotic expression systems were used to express the capsid protein of GETV. This protein was then used to immunize BALB/c mice in order to generate monoclonal antibodies (mAbs). Subsequently, hybridoma cells secreting a mAb (2B11-4) against the capsid protein were obtained using the hybridoma technique. A B cell linear epitope, 18-PAYRPWR-24, located at the capsid protein's N-terminal region was identified using western blotting analysis with the produced mAb, 2B11-4. Sequence alignment indicated that this epitope was highly conserved in group III (GIII) strains of GETV, but varied among the other genotypes. Western blotting showed that mAb 2B11-4 could discriminate Group III GETVs from other genotypes. This study describes the preparation of a mAb against the GETV capsid protein and the identification of the specific localization of B-cell epitopes, and will contribute towards a better understanding of the biological importance of the GETV capsid protein. It will also pave the way for developing immunological detection methods and genotype diagnosis for GETVs.
Assuntos
Alphavirus , Culicidae , Camundongos , Animais , Suínos , Cavalos , Alphavirus/genética , Proteínas do Capsídeo/genética , Anticorpos Monoclonais , Epitopos de Linfócito B/genéticaRESUMO
Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.
RESUMO
The prevalence and impact of Getah virus (GETV) are significant concerns in China. GETV can infect a wide range of animals, including horses, pigs, sheep, cattle, birds, and humans, resulting in substantial losses in the livestock and agricultural industries. GETV infection can cause the development of ulcers and inflammation in the mouth and gums of horses, which result in pain and discomfort and lead to symptoms such as reduced appetite, drooling, and difficulty chewing. As a result, there is a pressing need for efficient and rapid disease diagnosis methods. However, the currently available diagnostic methods have limitations in terms of operational time, equipment, and the experience of the individuals using them. In this study, a rapid, specific, and sensitive detection method was developed using a colloidal gold-based immunochromatographic strip (ICS) for the detection of antibodies against GETV in horses. To prepare the ICS, the antigen domain of the E2 glycoprotein of GETV was expressed using the Escherichia coli expression system after analysis with DNAstar v7.1 software. The nitrocellulose membrane was coated with rE2 protein or SPA to form the test line and control line, respectively. After optimizing the reaction conditions, the sensitivity, specificity, and repeatability of the strip were verified. The results showed that the test strip had a detection limit of up to 1:320 dilutions for GETV-positive serum, with no cross-reactivity observed with other equine-susceptible pathogens such as equine arteritis virus (EAV), equine herpesvirus-1 (EHV-I), equine infectious anemia virus (EIAV), equine influenza virus (EIV), African horse sickness virus (AHSV), and Japanese encephalitis virus (JEV). Furthermore, the ICS exhibited a concordance rate of 94.0% when testing 182 clinical serum samples compared to the virus neutralization test. Overall, this ICS diagnosis method will be an effective tool for the rapid detection of GETV in the field.
RESUMO
Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3' end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests.
Assuntos
Antivirais , Genes Reporter , Proteínas de Fluorescência Verde , Testes de Neutralização , Animais , Antivirais/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Replicação Viral/efeitos dos fármacos , Alphavirus/genética , Alphavirus/efeitos dos fármacos , Suínos , CricetinaeRESUMO
Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.