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CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

ABSTRACT: Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 10³ pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

SUBMITTER: Yao R

PROVIDER: S-EPMC8219879 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBovine viral diarrhea virus (BVDV) is an important global viral pathogen of cattle and other ruminants. To survey the infection rate and genetic diversity of BVDV in western China, a total of 1234 serum samples from 17 herds of dairy cattle, beef cattle and yak in 4 provinces were collected in 2019.ResultsAll the 1234 serum samples were screened individually for BVDV by RT-PCR. Our results demonstrated that the average positive rate of BVDV was 7.2% (89/1234) in animals and 82.4% (14/17) in herds. Thirteen BVDV strains were isolated from RT-PCR positive clinical samples and they were all NCP biotype. BVDV-1a and 1c subgenotypes were identified from 22 selected virus isolates in 14 BVDV-positive herds. These results confirmed that BVDV-1a and BVDV-1c were circulating in western China, similar to the BVDV epidemics in cattle in other regions of China.ConclusionsThis study provides data for monitoring and vaccination strategies of BVDV in western China.

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Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick.

Project description:Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5-AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5-AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10-AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RT-qPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming.

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Analysis of codon usage in bovine viral diarrhea virus.

Project description:Bovine viral diarrhea virus (BVDV) is a widespread virus in beef and dairy herds. BVDV has been grouped into two genotypes, genotype 1 and genotype 2. In this study, the relative synonymous codon usage (RSCU) values, effective number of codon (ENC) values and nucleotide content were investigated, and a comparative analysis of codon usage patterns for open reading frames (ORFs) of 22 BVDV genomes, including 14 of genotype 1 and 8 of genotype 2, was carried out. A high A+U content and low codon bias were found in BVDV genomes. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage between the two genotypes, and a geographic factor exists only in genotype-1 of BVDV. The RSCU data have a negative correlation with general average hydrophobicity (GRAVY), aromaticity and nucleotide content. Furthermore, the overall abundance of C and U has no effect on the synonymous codon usage patterns. In contrast, the A and G content showed a significant correlation with the nucleotide content at the third position. In addition, the codon usage patterns of BVDV are similar to those of 22 conserved genes of Bos taurus. Taken together, the genetic characteristics of BVDV possibly result from interactions between natural selection and mutation pressure.

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Project description:Several studies suggest that synergisms between Mycoplasma bovis and other microorganisms might exacerbate disease outcome of bovine mycoplasmosis. Screening several bovine cell types to assess their potential use as in vitro infection models for M. bovis, it was observed that a widely used cell line of bovine macrophages (Bomac cells) is in fact persistently infected with bovine viral diarrhea virus (BVDV). The cell line was first cured of this virus allowing comparative studies between both cell lines. Subsequently, uptake and co-culture of two M. bovis strains of different clonal complexes with Bomac cells contaminated with BVDV and in BVDV-free Bomac cells were assessed. Additionally, cell viability, cytotoxicity and induction of apoptosis after infection with M. bovis were evaluated. No differences in the levels of uptake and growth in co-culture were observed between the two Bomac cell types and both M. bovis strains. Cytotoxicity was increased after infection of BVDV-free cells with one of the two strains, while apoptotic cell death was slightly induced by this strain in both cell lines. Overall, the presence or absence of BVDV in Bomac cells did not grossly change the parameters tested upon infection with M. bovis. Nevertheless, this cell model is very useful when studying viral co-infections with bacteria and could also be used for multiple co-infections. Considering the broad contamination of cell cultures with BVDV, careful screening for this virus should routinely be performed as its presence might be relevant depending on the molecular mechanisms being investigated.

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