Dataset Information

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

ABSTRACT: Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.

SUBMITTER: Wang C

PROVIDER: S-EPMC5249048 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

Wang Chenxi C Wang Qian Q Hu Junyi J Sun Honglei H Pu Juan J Liu Jinhua J Sun Yipeng Y

PloS one 20170120 1

Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and ...[more]

PMID: 28107507

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Project description:Since its first isolation in around 2007, the avian-origin H3N2 canine influenza virus (CIV) has become established and continues to circulate in dog populations. This virus serves as a useful model for deciphering the complex evolutionary process of interspecies transmission of influenza A virus (IAV) from one species to its subsequent circulation in another mammalian host. The present investigation is a comprehensive effort to identify and characterize genetic changes that accumulated in the avian-origin H3N2 CIV during its circulation in the dog. We revealed that H3N2 CIV experiences greater selection pressure with extremely high global non-synonymous to synonymous substitution ratios per codon (dN/dS ratio) for each gene compared to the avian reservoir viruses. A total of 54 amino acid substitutions were observed to have accumulated and become fixed in the H3N2 CIV population based on our comprehensive codon-based frequency diagram analysis. Of these substitutions, 11 sites also display high prevalence in H3N8 CIV, indicating that convergent evolution has occurred on different lineages of CIV. Notably, six substitutions, including HA-G146S, M1-V15I, NS1-E227K, PA-C241Y, PB2-K251R, and PB2-G590S, have been reported to play imperative roles in facilitating the transmission and spillover of IAVs across species barriers. Most of these substitutions were found to have become fixed in around 2015, which might have been a favorable factor that facilitating the spread of these CIV lineages from South Asia to North America and subsequent further circulation in these areas. We also detected 12 sites in six viral genes with evidence for positive selection by comparing the rates of non-synonymous and synonymous substitutions at each site. Besides, our study reports trends of enhanced ongoing adaptation of H3N2 CIV to their respective host cellular systems, based on the codon adaptation index analysis, which points toward increasing fitness for efficient viral replication. In addition, a reduction in the abundance of the CpG motif, as evident from an analysis of relative dinucleotide abundance, may contribute to the successful evasion of host immune recognition. The present study provides key insights into the adaptive changes that have accumulated in the avian-origin H3N2 viral genomes during its establishment and circulation into dog populations.

Project description:BACKGROUND:Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube. METHODS:Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay. RESULTS:The limitation of detection (LOD) of the multiplex assay was 5.4 × 10-2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10-2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10-2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples. CONCLUSIONS:These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

Project description:Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro, and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs.IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness.

Dataset Information

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

Publications

A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses.

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