Validation and diagnostic application of NS and HA gene-specific real-time reverse transcription-PCR assays for detection of 2009 pandemic influenza A (H1N1) viruses in clinical specimens.
ABSTRACT: Real-time reverse transcription-PCR assays specific for the nonstructural (NS) and hemagglutinin (HA) genes of the 2009 pandemic influenza A (H1N1) virus were developed and evaluated with clinical samples from infected patients. The tests are characterized by high sensitivity and specificity and performed well throughout the first year of the 2009 pandemic.
Project description:During May 2009-April 2010, we analyzed 692 samples of pandemic (H1N1) 2009 virus from patients in Mexico. We detected the H275Y substitution of the neuraminidase gene in a specimen from an infant with pandemic (H1N1) 2009 who was treated with oseltamivir. This virus was susceptible to zanamivir and resistant to adamantanes and oseltamivir.
Project description:In June-July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission.
Project description:BACKGROUND: The 2009 influenza A (H1N1) pandemic has required decision-makers to act in the face of substantial uncertainties. Simulation models can be used to project the effectiveness of mitigation strategies, but the choice of the best scenario may change depending on model assumptions and uncertainties. METHODS: We developed a simulation model of a pandemic (H1N1) 2009 outbreak in a structured population using demographic data from a medium-sized city in Ontario and epidemiologic influenza pandemic data. We projected the attack rate under different combinations of vaccination, school closure and antiviral drug strategies (with corresponding "trigger" conditions). To assess the impact of epidemiologic and program uncertainty, we used "combinatorial uncertainty analysis." This permitted us to identify the general features of public health response programs that resulted in the lowest attack rates. RESULTS: Delays in vaccination of 30 days or more reduced the effectiveness of vaccination in lowering the attack rate. However, pre-existing immunity in 15% or more of the population kept the attack rates low, even if the whole population was not vaccinated or vaccination was delayed. School closure was effective in reducing the attack rate, especially if applied early in the outbreak, but this is not necessary if vaccine is available early or if pre-existing immunity is strong. INTERPRETATION: Early action, especially rapid vaccine deployment, is disproportionately effective in reducing the attack rate. This finding is particularly important given the early appearance of pandemic (H1N1) 2009 in many schools in September 2009.
Project description:Influenza A pandemic (H1N1) 2009 virus continues to rapidly spread worldwide. In 2009, pandemic (H1N1) 2009 infection in a domestic cat from Iowa was diagnosed by a novel PCR assay that distinguishes between Eurasian and North American pandemic (H1N1) 2009 virus matrix genes. Human-to-cat transmission is presumed.
Project description:To demonstrate that pandemic (H1N1) 2009 virus may cause respiratory disease in cats, we intratracheally infected cats. Diffuse alveolar damage developed. Seroconversion of sentinel cats indicated cat-to-cat virus transmission. Unlike in cats infected with highly pathogenic avian influenza virus (H5N1), extrarespiratory lesions did not develop in cats infected with pandemic (H1N1) 2009 virus.
Project description:BACKGROUND:Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans. METHODS:We established a real-time reverse-transcription (RT)-PCR-based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green-based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds. RESULTS:All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1-6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus. CONCLUSIONS:Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.
Project description:Adaptive mutations that have contributed to the emergence of influenza A pandemic (H1N1) 2009 virus, which can replicate and transmit among humans, remain unknown. We conducted a large-scale scanning of influenza protein sequences and identified amino acid-conserving positions that are specific to host species, called signatures. Of 47 signatures that separate avian viruses from human viruses by their nonglycoproteins, 8 were human-like in the pandemic (H1N1) 2009 virus. Close examination of their amino acid residues in the recent ancestral swine viruses of pandemic (H1N1) 2009 virus showed that 7 had already transitioned to human-like residues and only PA 356 retained an avianlike K; in pandemic (H1N1) 2009 virus, this residue changed into a human-like R. Signatures that separate swine viruses from human viruses were also present. Continuous monitoring of these signatures in nonhuman species will help with influenza surveillance and with evaluation of the likelihood of further adaptation to humans.
Project description:In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host.
Project description:Recent case reports describe multidrug-resistant influenza A pandemic (H1N1) 2009 virus infection in immunocompromised patients exposed to neuraminidase inhibitors because of an I223R neuraminidase mutation. We report a case of multidrug-resistant pandemic (H1N1) 2009 bearing the I223R mutation in an ambulatory child with no previous exposure to neuraminidase inhibitors.
Project description:Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.