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Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

ABSTRACT: Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/μl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against the putative novel genotypes. RT-qPCR assays targeting BTV segment 9 and 10 detected both strains (SPvvvv/02 and SPvvvv/03) whereas a BTV segment 1 RT-qPCR assay was unable to detect either BTV strain. The work presented here expands upon the current repertoire of RT-qPCR assays for BTV genotype determination.

SUBMITTER: King S

PROVIDER: S-EPMC8543067 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

King Simon S Flannery John J Batten Carrie C Rajko-Nenow Paulina P

Journal of virological methods 20210915

Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/μl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against ...[more]

PMID: 34536487

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Project description:Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.

Project description:IntroductionPorcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease that causes reproductive disorders in sows and respiratory problems in pigs of different ages. It first appeared in the late 20th century in the United States and Europe before spreading globally, leading to significant economic losses in the swine industry. Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has a high rate of genetic recombination, resulting in considerable genetic diversity within the virus. The lack of cross-protection between different lineages often leads to unsuccessful vaccination attempts.MethodsTo accurately distinguish PRRSV lineages and develop effective vaccination strategies for pigs, we have developed a fluorescence quantitative PCR (qPCR) method by designing specific primers and SYBR green dye. This method allows for the simultaneous identification of different PRRSV genotypes.ResultsOur experimental results show that these methods have good specificity and do not react with other common viral pathogens in pigs. This method also demonstrates good sensitivity, with the ability to detect low levels of the virus. The detection limits of these assay were 102 copies/μL for PRRSV-1 (European-type PRRS) and 101 copies/μL for PRRSV-2 (American-type PRRSV), HP-PRRSV (Highly Pathogenic PRRSV), and NL-PRRSV (NADC30-like PRRSV), respectively. Furthermore, the reproducibility of this method is commendable, with intra- and inter-assay coefficients of variation remaining below 3%. In the subsequent study, a total of 316 clinical samples of porcine with respiratory and reproductive failure symptoms were collected from 14 cities in Guangdong. The results showed that among these samples, 22.78% (72 out of 316) tested positive for PRRSV-2, 15.51% (49 out of 316) tested positive for HP-PRRSV, and 0.95% (3 out of 316) tested positive for NL-PRRSV. However, PRRSV-1 was not detected in any of the samples.DiscussionOur method provides a quick way to identify PRRSV genotypes in pig herds in Guangdong, which has certain significance for developing effective vaccination strategies against PRRS.

Dataset Information

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

Publications

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine.

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