A Multiplex Polymerase Chain Reaction for Differential Detection of Turkey Coronavirus from Chicken Infectious Bronchitis Virus and Bovine Coronavirus
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC7122580 | biostudies-literature | 2015 Sep
REPOSITORIES: biostudies-literature
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Project description:Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/μL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/μL, 1 copy/μL and 2.4 copies/μL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.
| S-EPMC10523346 | biostudies-literature
Project description:Phylogenetic analysis of complete genomes of the avian coronaviruses avian infectious bronchitis (AIBV) and turkey coronavirus (TCoV) supported the hypothesis that numerous recombination events have occurred between these viruses. Although the two groups of viruses differed markedly in the sequence of the spike protein, the gene (S) encoding this protein showed no evidence of positive selection or of an elevated mutation rate. Rather, the data suggested that recombination events have homogenized the portions of the genome other than the S gene between the two groups of viruses, while continuing to maintain the two distinct, anciently diverged versions of the S gene. The latter hypothesis was supported by a phylogeny of S proteins from representative coronaviruses, in which S proteins of AIBV and TCoV fell in the same clade.
| S-EPMC7086623 | biostudies-literature
Project description:Bovine coronavirus (BCoV) is an economically significant cause of enteric and respiratory diseases in cattle throughout the world. BCoV is a known cause of neonatal calf diarrhea, winter dysentery in adult cattle, and respiratory disorders in cattle of all ages. In this chapter, we describe a simple and efficient protocol for total nucleic acids extraction to be used in conventional RT-PCR assay. This is a technique used routinely in our virology laboratory to detect BCoV from stool and nasopharyngeal samples of cattle.
| S-EPMC7121124 | biostudies-literature
Project description:Identification of avian infectious bronchitis virus (IBV) genotypes is essential for controlling infectious bronchitis (IB) disease, because vaccines that differ from the circulating strains might not provide efficient cross-protection. In Egypt, IBV strain typing is a difficult process, due to the widespread distribution of four genotype lineages (GI-13, GI-23, GI-1, and GI-16), which may contribute to IBV vaccination failure. In this study, we developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (mRT-qPCR) assay that targets highly conserved areas of the S1 gene in order to detect classical (G1) and Egyptian variant II (G23) strains in allantoic fluids and clinical samples. The viral genotyping technique was assessed using commercially available vaccines as well as local strains, and 16 field isolates were tested to investigate its clinical applicability. The assay was found to be specific for the detection of classical and VAR II strains and did not detect the VAR I strain or other avian pathogens such as Newcastle disease virus, avian influenza virus (H9N2 and H5N8), or infectious bursal disease virus. The results also showed that 28 out of 41 samples tested positive for IBV utilizing rt-qRT-PCR targeting the N gene and that 26 out of the 28 positive samples were genotyped by mRT-qPCR targeting the S1 gene, whereas the remaining two samples that were not genotyped were VAR 1 (4/91) and VAR I (793/B). Interestingly, the testing could identify combined infections in one sample, indicating a mixed infection with both genotypes. The real-time RT-PCR assay could detect viral RNA at concentrations as low as 102 EID50 /ml for both classical and variant II. This assay is rapid, specific, and sensitive. It appears to be a valuable tool for regular disease monitoring that can be used to differentiate as well as identify viruses.
| S-EPMC9741560 | biostudies-literature
Project description:OBJECTIVE:To analyze the 3'-end structural protein-encoding region of turkey coronavirus (TCoV) isolates associated with outbreaks of acute enteritis in Indiana, North Carolina, or Minnesota. METHODS:Four isolates of TCoV were sequenced over the entire 3'-end structural protein-encoding region and compared phylogenetically along with the corresponding sequences of infectious bronchitis virus (IBV) strains. RESULTS:The sequence similarity between TCoV and IBV was lower than that among TCoV isolates or that among IBV strains. The variation of sequences between TCoV and IBV was mainly contributed by the S protein gene. The sequence similarity of S gene between TCoV and IBV was lower than that among TCoV isolates or that among IBV strains. The phylogenetic tree based on the S protein region was similar to that based on the entire 3'-end structural protein-encoding region with TCoV isolates and IBV strains grouped in two separate clusters. The phylogenetic tree based on other genes had a very different topology with TCoV isolates randomly forming groups with different IBV strains. CONCLUSIONS:These results suggested that TCoV probably shared the same origin with that of IBV and acquired sequences of S gene for turkey intestine tropism during the process of evolution in a separate environment.
| S-EPMC7179528 | biostudies-literature
Project description:Using viral metagenomics, the complete genome sequence of an infectious bronchitis virus (IBV) strain (named ahysx-1) from a fecal sample from a healthy chicken in Anhui province, China, was determined. The genome sequence of ahysx-1 was found to be very similar to that of IBV strain ck/CH/LLN/131040 (KX252787), except for the spike gene region, which is similar to that of a turkey coronavirus strain (EU022526), suggesting that ahysx-1 is a recombinant. Recombination analysis and phylogenetic analysis based on the genomic sequences of ahysx-1 and other related strains confirmed that ahysx-1 appears to be a recombinant resulting from a recombination event that occurred between a chicken coronavirus and a turkey coronavirus. Further studies need to be performed to determine whether this recombinant IBV strain is pathogenic and whether it is transmitted between chickens and turkeys.
| S-EPMC7087231 | biostudies-literature
Project description:Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997-0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05-3.0% (wt/wt) for the bovine and turkey targets, and 0.01-1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.
| S-EPMC5552122 | biostudies-literature
Project description:UnlabelledCoronaviruses from both the Alphacoronavirus and Betacoronavirus genera interfere with the type I interferon (IFN) response in various ways, ensuring the limited activation of the IFN response in most cell types. Of the gammacoronaviruses that mainly infect birds, little is known about the activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells, and a chicken cell line. In fact, Ifnβ expression is delayed with respect to the peak of viral replication and the accompanying accumulation of double-stranded RNA (dsRNA). In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the response at the transcriptional and translational levels. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, the signaling of nonself dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.ImportanceOur results demonstrate that IBV has evolved multiple strategies to avoid the activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as the pathogenesis of gammacoronaviruses.
| S-EPMC4300645 | biostudies-literature
Project description:BackgroundInfectious bronchitis virus (IBV), a prototype of the Coronaviridae family, is an economically important causative agent of infectious bronchitis in chickens and causes an acute and highly contagious upper respiratory tract infections that may lead to nephritis. However, the molecular antiviral mechanisms of chickens to IBV infection remain poorly understood. In this study, we conducted global gene expression profiling of chicken kidney tissue after nephropathogenic IBV infection to better understand the interactions between host and virus.ResultsIBV infection contributed to differential expression of 1777 genes, of which 876 were up-regulated and 901 down-regulated in the kidney compared to those of control chickens and 103 associated with immune and inflammatory responses may play important roles in the host defense response during IBV infection. Twelve of the altered immune-related genes were confirmed by real-time RT-PCR. Gene ontology category, KEGG pathway, and gene interaction networks (STRING analysis) were analyzed to identify relationships among differentially expressed genes involved in signal transduction, cell adhesion, immune responses, apoptosis regulation, positive regulation of the I-kappaB kinase/NF-kappaB cascade and response to cytokine stimulus. Most of these genes were related and formed a large network, in which IL6, STAT1, MYD88, IRF1 and NFKB2 were key genes.ConclusionsOur results provided comprehensive knowledge regarding the host transcriptional response to IBV infection in chicken kidney tissues, thereby providing insight into IBV pathogenesis, particularly the involvement of innate immune pathway genes associated with IBV infection.
| S-EPMC3870970 | biostudies-literature
Project description:We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.
| S-EPMC1906892 | biostudies-literature