Development and application of an indirect ELISA for detecting equine IgG antibodies against Getah virus with recombinant E2 domain protein.

Getah virus (GETV) disease is a mosquito-borne infectious disease that causes fever, aseptic meningitis, and abortion in a variety of animals. Currently, the epidemic trend of GETV disease increases seriously worldwide, especially in China, posing a potential threat to animal safety and public health. However, there are few reports about the epidemiological investigation of GETV disease in China as well as a lack of commercial diagnostic kit for GETV antibody. Therefore, the establishment of a rapid, sensitive and suitable GETV antibody detection method for large-scale samples is an urgent request to fully understand the prevalence of GETV disease. Here, a recombinant plasmid pET22b-GETV-E2d that contained the domain of GETV-E2 (E2d) fused to His-tag was constructed to express recombinant protein E2d (rE2d) in Escherichia coli. The rE2d was mainly expressed in inclusion bodies. And it was purified successfully by nickel affinity column so that it could be used to develop an indirect ELISA (rE2d-ELISA). After optimizing reaction conditions of rE2d-ELISA, the cut-off value was determined as 0.396 with 100 equine sera tested by virus neutralization test (VNT). Furthermore, rE2d-ELISA method showed the positive rate of IgG antibodies against GETV was 54.3% based on testing 646 clinical serum samples obtained in Xinjiang whereas the overall coincidence rate between rE2d-ELISA and VNT was 94.0%, with 98.2% sensitivity and 92.6% specificity. The findings suggest that the developed IgG ELISA employing recombinant E2d promises was an efficient and low-cost type of antibody detection method for horse, which will benefit for prevention of GETV outbreaks in horses.

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.

Synergistic pathogenicity by coinfection and sequential infection with JXA1-like HP-PRRSV and PCV2d in PCV2 antibody-positive post-weaned pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus (PCV) are two important pathogens, which caused respiratory disease in pigs. PRRSV and PCV2 had caused great economic losses to the pig industry. Pigs coinfection with PCV2 and PRRSV were common in the clinic, PCV2 antibodies can be detected in most of the pigs. PCV2d and HP-PRRSV(JXA1-like) were two major viruses circulating in the pigs in China. In this study, HP-PRRSV (JXA1-like) and PCV2d were used to coinfect and (or) sequential infect 5-week-old weaned PCV2-antibody positive pigs and the clinical indications, pathological, virus load, and specific antibodies of the challenged post-weaned piglets were evaluated. Thirty 5-week-old post-weaned pigs were divided into six groups infected with PBS, PCV2, PRRSV, PCV2-PRRSV, PRRSV-PCV2, and Co-PRRSV-PCV2 according to the PCV2 specific antibodies. Pigs infected with PRRSV can experience diarrhea, increased body temperature, weight loss, and even death. The pigs in the PRRSV infected group and PRRSV-PCV2 infected group showed severe clinical symptoms, high mortality, and low average daily gain. The main pathological changes were widening of the lung interstitium, lung adhesion, and so on. The PRRSV-PCV2 infected group showed high levels of TNF-α and IL-2. In conclusion, PRRSV and PRRSV-PCV2 sequential infected pigs showed most pathogenic signs, and PCV2-PRRSV sequential infected pigs showed less pathogenicity than pigs of PCV2 and PRRSV coinfection and PRRSV monoinfection from day 10-14, partially suppressing the cytokine storm produced by PRRSV.

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.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Origin, genetic diversity, adaptive evolution and transmission dynamics of Getah virus.

As a member of the Alphavirus, Getah virus (GETV) was becoming more serious and posing a serious threat to animal safety and public health. However, the circulation, distribution and evolution of GETV is not well understood. Hence, we integrated a variety of bioinformatic methodologies, from genomic alterations to systematic analysis, phylogeography, selection, adaptive analysis, prediction of protein modification, structural biology and molecular dynamics simulations to understand the characteristics of GETV. The results of phylogeography and molecular evolution show that due to the lack of vaccine, GETV is rapidly expanding its host range and geographical distribution at a high evolutionary rate. We also predicted the important modification sites, and identified the adaptive and active selection sites. Finally, the analysis of spatial structure and function showed that six adaptive sites may be related to the structural stability, receptor binding ability, immunogenicity and immune evasion of the virus, respectively. The data from this study have important implications for the understanding of ongoing GETV outbreaks worldwide and will guide future efforts to develop effective preventive and control measures against GETV. In particular, biosafety measures should be strengthened immediately to prevent GETV from becoming a pandemic, especially in China, South Korea and Japan.