Rapid detection of the pandemic 2009 H1N1 virus M gene by real-time and gel-based RT-PCR assays.
ABSTRACT: Since the first pandemic 2009 H1N1 (pH1N1) virus was isolated from humans, it has also been detected in other mammalian (pigs, cats, dogs, ferrets) and avian (turkey) species, most likely because of cross-species transmission from humans. The pH1N1 contains six genes derived from swine influenza viruses (SIVs) currently circulating in North America of human- (PB1), avian- (PB2, PA), and swine- (HA, NP, and NS) origin and two genes (NA and M) derived from Eurasian SIVs. The novel genetic composition of pH1N1 necessitates development of novel molecular and serological assays to differentiate the pH1N1 virus from circulating human, swine, turkey, canine, and feline influenza viruses.To detect and discriminate the pH1N1 from currently circulating SIVs in North America, we developed and evaluated a TaqMan probe-based real-time and a gel-based RT-PCR assay, both targeting the pH1N1 matrix gene.The real-time and gel-based RT-PCR assays were able to specifically detect the pH1N1 M gene and differentiate it from SIVs circulating in North America, including the classical and novel human-like H1N1 influenza virus as well as H1, H2, and H3 subtype triple reassortant SIVs. Both assays were highly sensitive and specific for the pH1N1 virus.The newly developed pH1N1-specific real-time and gel-based RT-PCR assays can be used to detect and differentiate the pH1N1 virus from currently circulating SIVs in North America. We suggest a combinational diagnostic approach where the real-time RT-PCR is used for high-throughput detection of influenza positive or suspect samples and the gel-based RT-PCR for confirmation and sequencing of the M-gene.
Project description:The emergence in humans of pandemic (H1N1) 2009 (pH1N1) with similarities to swine influenza viruses (SIVs) caused much concern for both human and animal health as potential for interspecies transmission was initially unknown.The goal of this study was to develop a real-time RT-PCR test for the detection and differentiation of 2009 pH1N1 and endemic influenza A viruses in North America.Matrix (M) gene sequences from U.S. human pH1N1 cases and U.S. SIVs were aligned to determine a suitable region for an assay target. Primers were selected to amplify all influenza A. Two probes were designed to differentiate pH1N1 (EA matrix) from endemic (NA matrix) SIVs. The assay was validated using the first U.S. pH1N1 strain, 10 human pH1N1-positive specimens and nine U.S. SIV isolates, then evaluated on 165 specimens of swine and other animal origin submitted to the Iowa State University Veterinary Diagnostic Laboratory. Results were compared to other influenza A PCR assays. Sequences from additional pH1N1 strains and contemporary H1N1 SIVs were used to assess robustness of the selected primers and probes for the intended purpose.The new assay's results from clinical specimens concurred with confirmatory PCR testing. The additional probe designed from sequence analysis improved detection of the NA matrix subtype when added to the reaction mixture.This assay detects and differentiates pH1N1 and US influenza A viruses in various sample matrices and species. Good bioinformatics support is critical when designing RT-PCR assays and monitoring their performance.
Project description:While much is known about the prevalence of influenza viruses in North America and Eurasia, their prevalence in birds and mammals in South America is largely unknown. To fill this knowledge gap and provide a baseline for future ecology and epidemiology studies, we conducted 2 years of influenza surveillance in the eastern plains (Los Llanos) region of Colombia. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) identified influenza viruses in wild birds, domestic poultry, swine and horses. Prevalence ranged from 2.6% to 13.4% across species. Swine showed the highest prevalence and were infected primarily with 2009 pandemic H1N1 (pH1N1) viruses genetically related to those in humans. In addition, we isolated H5N2 viruses from two resident species of whistling ducks (genus Dendrocygna) that differed completely from previous South American isolates, instead genetically resembling North American wild bird viruses. Both strains caused low pathogenicity in chickens and mammals. The prevalence and subtype diversity of influenza viruses isolated from diverse species within a small area of Colombia highlights the need for enhanced surveillance throughout South America, including monitoring of the potential transmissibility of low-pathogenic H5N2 viruses from wild birds to domestic poultry and the emergence of reassortant viruses in domestic swine.
Project description:The 2009 pandemic H1N1 virus (pH1N1) was derived through reassortment of North American triple reassortant and Eurasian avian-like swine influenza viruses (SIVs). To date, when, how and where the pH1N1 arose is not understood. To investigate viral reassortment, we coinfected cell cultures and a group of pigs with or without preexisting immunity with a Eurasian H1N1 virus, A/Swine/Spain/53207/2004 (SP04), and a North American triple reassortant H1N1 virus, A/Swine/Kansas/77778/2007 (KS07). The infected pigs were cohoused with one or two groups of contact animals to investigate viral transmission. In coinfected MDCK or PK15 continuous cell lines with KS07 and SP04 viruses, more than 20 different reassortant viruses were found. In pigs without or with preexisting immunity (immunized with commercial inactivated swine influenza vaccines) and coinfected with both viruses, six or seven reassortant viruses, as well as the parental viruses, were identified in bronchoalveolar lavage fluid samples from the lungs. Interestingly, only one or two viruses transmitted to and were detected in contact animals. No reassortant containing a gene constellation similar to that of pH1N1 virus was found in either coinfected cells or pigs, indicating that the reassortment event that resulted in the generation of this virus is a rare event that likely involved specific viral strains and/or a favorable, not-yet-understood environment. IMPORTANCE The 2009 pandemic-like H1N1 virus could not be reproduced either in cell cultures or in pigs coinfected with North American triple reassortant H1N1 and Eurasian H1N1 swine influenza viruses. This finding suggests that the generation of the 2009 pandemic H1N1 virus by reassortment was a rare event that likely involved specific viral strains and unknown factors. Different reassortant viruses were detected in coinfected pigs with and without preexisting immunity, indicating that host immunity plays a relevant role in driving viral reassortment of influenza A virus.
Project description:Surveillance of swine influenza viruses (SIVs) in 31 pig farms in northern and southern parts of Vietnam was conducted. Six H3N2 influenza A viruses were isolated from a pig farm in southern Vietnam. They were novel genetic reassortants between a triple-reassortant SIV and a human seasonal H3N2 virus. Their hemagglutinin and neuraminidase genes were derived from a human virus circulating around 2004-2006 and the remaining genes from a triple-reassortant SIV that originated in North America. This is the first report describing the isolation of a novel triple-reassortant SIV in Vietnam.