Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009. © 2009 Wiley‐Liss, Inc.     

Contemporary seasonal influenza A (H1N1) virus infection primes for a more robust response to split inactivated pandemic influenza A (H1N1) Virus vaccination in ferrets

Human influenza pandemics occur when influenza viruses to which the population has little or no immunity emerge and acquire the ability to achieve human-to-human transmission. In April 2009, cases of a novel H1N1 influenza virus in children in the southwestern United States were reported. It was retrospectively shown that these cases represented the spread of this virus from an ongoing outbreak in Mexico. The emergence of the pandemic led to a number of national vaccination programs. Surprisingly, early human clinical trial data have shown that a single dose of nonadjuvanted pandemic influenza A (H1N1) 2009 monovalent inactivated vaccine (pMIV) has led to a seroprotective response in a majority of individuals, despite earlier studies showing a lack of cross-reactivity between seasonal and pandemic H1N1 viruses. Here we show that previous exposure to a contemporary seasonal H1N1 influenza virus and to a lesser degree a seasonal influenza virus trivalent inactivated vaccine is able to prime for a higher antibody response after a subsequent dose of pMIV in ferrets. The more protective response was partially dependent on the presence of CD8(+) cells. Two doses of pMIV were also able to induce a detectable antibody response that provided protection from subsequent challenge. These data show that previous infection with seasonal H1N1 influenza viruses likely explains the requirement for only a single dose of pMIV in adults and that vaccination campaigns with the current pandemic influenza vaccines should reduce viral burden and disease severity in humans.     

Induction of inflammatory cytokines and toll-like receptors in human normal respiratory epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, and highly pathogenic H5N1 influenza virus

Respiratory epithelial cells are one of main targets for infections caused by influenza viruses. Recently, the induction of proinflammatory cytokines and toll-like receptors (TLRs) in normal human bronchial/tracheal epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, or highly pathogenic H5N1 influenza virus were studied to understand the pathogenesis and early immune responses. The cells were productively infected with the viruses. Among the inflammatory cytokines tested, interleukin (IL)-8 was predominantly induced in virus-infected cells. Among the chemokines tested, interferon-γ-inducible protein-10 (IP-10) and growth-related oncogene-α (GRO-α) were predominantly induced in virus-infected cells. TLR-5 was predominantly induced in cells infected with seasonal H1N1, pandemic H1N1, or H5N1 influenza virus, and TLR-3 was predominantly induced in cells infected with seasonal H3N2 influenza virus. Taken together, the results suggest that IL-8, IP-10, and GRO-α are predominantly induced in respiratory epithelial cells infected with influenza A viruses, and that TLR-5 and TLR-3 are involved in the stimulation of virus-infected respiratory epithelial cells.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.     

The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009)

Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.     

A novel H1N1 virus causes the first pandemic of the 21st century

A novel H1N1 virus of swine origin (H1N1v ) is currently spreading in humans, giving rise to the first pandemic in 40 years. The disease is of moderate severity but has notable differences from seasonal influenza. In contrast to seasonal influenza, those over 60 years are relatively spared, a likely consequence of the presence of H1N1v cross‐neutralizing antibody in this age group. Most patients appear to have mild influenza‐like illness and many of the complications leading to hospitalization and mortality occur in those with underlying disease conditions or pregnancy. Studies in animal models suggest that the novel H1N1v pandemic virus causes a more severe illness and appears to have a greater predilection for the alveolar epithelium than seasonal influenza viruses. As there are as yet little data on the pathogenesis and immunology of H1N1v infection in humans, we have reviewed relevant data from past pandemics, from seasonal influenza and avian influenza H5N1 to highlight key issues pertaining to pathogenesis and immunology.     

Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States

The evolution of classical swine influenza viruses receptor specificity preceding the emergence of the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical swine influenza viruses from the α, β, and γ antigenic clusters isolated between 1945 and 2009 revealed a binding profile very similar to that of 2009 pandemic H1N1 viruses, with selectivity for α2-6-linked sialosides and very limited binding to α2-3 sialosides. Despite considerable genetic divergence, the ‘human-like’ H1N1 viruses circulating in swine retained strong binding preference for α2-6 sialylated glycans. Interspecies transmission of H1N1 influenza viruses from swine to humans or from humans to swine has not driven selection of viruses with distinct novel receptor binding specificities. Classical swine and human seasonal H1N1 influenza viruses have conserved specificity for similar α2-6-sialoside receptors in spite of long term circulation in separate hosts, suggesting that humans and swine impose analogous selection pressures on the evolution of receptor binding function.     

Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice

A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and a virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6 months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.     

Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.     

Identification of oseltamivir resistance among pandemic and seasonal influenza A (H1N1) viruses by an His275Tyr genotyping assay using the cycling probe method

Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

Preexisting Antibody Response against 2009 Pandemic Influenza H1N1 Viruses in the Taiwanese Population

A novel pandemic influenza H1N1 (pH1N1) virus spread rapidly across the world in 2009. Due to the important role of antibody-mediated immunity in protection against influenza infection, we used an enzyme-linked immunosorbent assay-based microneutralization test to investigate cross-reactive neutralizing antibodies against the 2009 pH1N1 virus in 229 stored sera from donors born between 1917 and 2008 in Taiwan. The peak of cumulative geometric mean titers occurred in donors more than 90 years old and declined sharply with decreasing age. Sixteen of 27 subjects (59%) more than 80 years old had cross-reactive antibody titers of 160 or more against the 2009 pH1N1 virus, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) from 6 months to 9 years old had an antibody titer of 40. We further tested the antibody responses in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed that only the 2 sera from children with antibody responses to the 2009 pH1N1 virus had high titers of neutralizing antibody against recent seasonal influenza virus strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years old or older, and the elderly subjects who may already have been exposed to the 1918 virus had high titers of neutralizing antibody to the 2009 pH1N1 virus. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce a cross-reactive antibody response to the 2009 pH1N1 virus.Influenza A viruses have caused several pandemics during the past century and continue to cause epidemics around the world yearly. Pandemics are typically caused by the introduction of a virus with a hemagglutinin (HA) subtype that is new to human populations (14). In 2009, a novel pandemic influenza H1N1 (pH1N1) virus of swine origin spread rapidly and has caused variable disease globally via interhuman transmission (2, 3).The 2009 pH1N1 virus contains a unique combination of gene segments from both the North American and Eurasian swine lineages and is antigenically distinct from any known seasonal human influenza virus (14). Since H1N1 influenza A viruses have been circulating in human populations for decades, much of the world has encountered these viruses repeatedly, either through infection or through vaccination. Under the threat of a pandemic outbreak, however, a major concern is whether preexisting immunity can provide some protection from the novel 2009 pH1N1 virus.Recent reports from the United States suggested that 33% of individuals over the age of 60 years had neutralization antibodies to the novel 2009 pH1N1 virus, probably due to previous exposure to antigenically similar H1N1 viruses (1, 7). In Japan, however, appreciable neutralization antibodies against the 2009 pH1N1 virus were found only in individuals more than 90 years old (9). The differences in geographical location and vaccination programs against influenza in 1976 may account for the different age distributions of neutralization antibodies in the two countries. In the early 1900s, Taiwan had had a close relationship with Japan historically and geographically. The prevalence of influenza in Taiwan may be quite similar to that in Japan. In recent years, however, sequence analysis of epidemic influenza virus strains revealed that the Taiwanese strains usually circulate in Taiwan prior to their circulation in many other countries, including Japan. (16). The differences between the studies from United States and Japan, and the unique epidemic situation in Taiwan, highlight the need for us to assess the level of preexisting immunity in the Taiwanese population.In this study, we measured the titers of neutralizing antibodies against the 2009 pH1N1 virus in sera obtained from previous influenza infection or vaccination of different age groups. In addition, we also assessed the antibodies against the local seasonal H1N1 strains isolated in Taiwan in 2006 and 2008 (A/Taiwan/N86/06, A/Taiwan/N94/08, and A/Taiwan/N510/08) to evaluate whether there is a cross-reactive antibody response between recent local strains and the 2009 pH1N1 virus.     

Comparison of pro-inflammatory cytokine expression and cellular signal transduction in human macrophages infected with different influenza A viruses

Influenza A virus infection of macrophages and virus-induced pro-inflammatory gene expression are regarded to contribute to severity of influenza A virus-caused diseases. Although some data are available on cytokine production by influenza A virus-infected macrophages, systematic comparisons of the virus types are currently considered to be of high relevance in humans (pandemic H1N1/2009, seasonal H1N1, seasonal H3N2, highly pathogenic avian influenza H5N1) on pro-inflammatory potential, and relevant underlying cellular signalling events are missing. Here, we show that the infection of human monocyte-derived macrophages with pandemic H1N1/2009 (A/HH/01/2009), seasonal H1N1/1999 (A/New Caledonia/20/99), seasonal H3N2/2004 (A/California/7/2004) or highly pathogenic H5N1/2004 (A/Thailand/1(Kan-1)/04) results in similar infection rates. However, the investigated H1N1 strains caused delayed and decreased apoptosis in comparison with H3N2/2004 or H5N1/2004. Moreover, human macrophage infection with H3N2/2004 or H5N1/2004 but not with H1N1 viruses was associated with pronounced pro-inflammatory cytokine production and activation of relevant mitogen-activated protein kinase pathways as indicated by phosphorylation of p38, JNK and ERK 1/2. These findings are in line with clinical observations indicating enhanced disease severity in H3N2- or H5N1-infected patients compared to individuals infected with pandemic H1N1/2009 or seasonal H1N1.     

Differential host determinants contribute to the pathogenesis of 2009 pandemic H1N1 and human H5N1 influenza A viruses in experimental mouse models

Influenza viruses are responsible for high morbidities in humans and may, eventually, cause pandemics. Herein, we compared the pathogenesis and host innate immune responses of a seasonal H1N1, two 2009 pandemic H1N1, and a human H5N1 influenza virus in experimental BALB/c and C57BL/6J mouse models. We found that both 2009 pandemic H1N1 isolates studied (A/Hamburg/05/09 and A/Hamburg/NY1580/09) were low pathogenic in BALB/c mice [log mouse lethal dose 50 (MLD(50)) >6 plaque-forming units (PFU)] but displayed remarkable differences in virulence in C57BL/6J mice. A/Hamburg/NY1580/09 was more virulent (logMLD(50) = 3.5 PFU) than A/Hamburg/05/09 (logMLD(50) = 5.2 PFU) in C57BL/6J mice. In contrast, the H5N1 influenza virus was more virulent in BALB/c mice (logMLD(50) = 0.3 PFU) than in C57BL/6J mice (logMLD(50) = 1.8 PFU). Seasonal H1N1 influenza revealed marginal pathogenicity in BALB/c or C57BL/6J mice (logMLD(50) >6 PFU). Enhanced susceptibility of C57BL/6J mice to pandemic H1N1 correlated with a depressed cytokine response. In contrast, enhanced H5N1 virulence in BALB/c mice correlated with an elevated proinflammatory cytokine response. These findings highlight that host determinants responsible for the pathogenesis of 2009 pandemic H1N1 influenza viruses are different from those contributing to H5N1 pathogenesis. Our results show, for the first time to our knowledge, that the C57BL/6J mouse strain is more appropriate for the evaluation and identification of intrinsic pathogenicity markers of 2009 pandemic H1N1 influenza viruses that are "masked" in BALB/c mice.     

2009甲型H1N1疫苗株与普通季节性H1N1疫苗株在呼吸道细胞中感染效率的比较

 摘要：目的 探讨2009甲型H1N1疫苗株与季节性H1N1疫苗株在人呼吸道细胞中感染效率的差异。方法 用间接免疫荧光法检测H1N1病毒感染A549、CNE-2Z、H1650、Hep-2和MRC-5细胞的效率;用间接免疫荧光法检测抑制因子对H1N1病毒侵入细胞的影响。结果 （1）2009甲型H1N1疫苗株感染人呼吸道细胞的效率：H1650>A549>CNE-2Z>MRC-5>Hep-2;季节性H1N1疫苗株感染人呼吸道细胞的效率A549>CNE-2Z>H1650>MRC-5>Hep-2;（2）2009甲型H1N1疫苗株和季节性H1N1疫苗株均依赖于内吞作用进入细胞。结论 在A549细胞中,季节性H1N1疫苗株的感染效率明显高于甲型H1N1疫苗株;在H1650细胞中,甲型H1N1疫苗株的感染效率明显高于季节性H1N1疫苗株。