Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health

Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.     

An early ‘classical’ swine H1N1 influenza virus shows similar pathogenicity to the 1918 pandemic virus in ferrets and mice

The 1918 pandemic influenza virus has demonstrated significant pathogenicity in animal models and is the progenitor of ‘classical’ swine and modern seasonal human H1N1 lineages. Here we characterize the pathogenicity of an early ‘classical’ swine H1N1 influenza A virus isolated in 1931 compared to the pathogenicity of the 1918 pandemic virus and a seasonal H1N1 virus in mice and ferrets. A/Swine/Iowa/31 (Sw31) and the 1918 influenza viruses were uniformly lethal in mice at low doses and produced severe lung pathology. In ferrets, Sw31 and 1918 influenza viruses caused severe clinical disease and lung pathology with necrotizing bronchiolitis and alveolitis. The modern H1N1 virus caused little disease in either animal model. These findings revealed that in these models the virulence factors of the 1918 influenza virus are likely preserved in the Sw31 virus and suggest that early swine viruses may be a good surrogate model to study 1918 virulence and pathogenesis.     

Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets

A 2009 H1N1 influenza virus pandemic, which had its origin in swine, caused severe illness and mortality in humans. Inflammatory responses may be responsible for pathogenesis caused by infection with influenza viruses. To better understand the pathogenic mechanism, clinical signs and inflammatory responses in ferrets infected with the pandemic H1N1 were compared with those caused by seasonal H1N1 influenza virus. Ferrets infected with the 2009 pandemic H1N1 virus displayed higher body temperatures, greater reduction in body weight, and higher viral titers in the tracheae and lungs. Levels of inflammatory cytokines, including interleukin-6, interferon-alpha, and tumor necrosis factor-alpha, were higher in the lungs of ferrets infected with the 2009 pandemic H1N1. The data support the idea that increased pathogenesis caused by the 2009 pandemic H1N1 influenza virus may have been partially mediated by a higher induction of pro-inflammatory cytokines in the lungs of affected humans or animals.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.     

Nucleotide sequence analysis of the HA1 coding portion of the haemagglutinin gene of swine H1N1 influenza viruses.

The nucleotide and deduced amino acid sequences coding for the HA1 portion of the haemagglutinin (HA) genes of three swine influenza viruses were determined and compared with published HA sequence data for human H1N1 influenza viruses. Sequence differences between the classic swine influenza HAs sw37 (A/swine/29/37) and NJ76 (A/New Jersey/11/76) were randomly distributed in the molecule without being confined to antigenic sites. In contrast, sequence differences between the HAs of sw37 and the antigenically atypical strains sw38 (A/swine/Northern Ireland/38) and sw39 (A/swine/Cambridge/39) were clustered in hypervariable regions, similar to the pattern of changes that was present between sw37 and the human strains PR834 (A/PR/8/34) and WSN33 (A/WSN/33). Sequence homologies of the European swine influenza strains (sw38, sw39) were higher with the HAs of the human strains (PR834, WSN33) than with the classic swine influenza HAs (sw37, NJ76). Phylogenetic analysis showed that the HA genes of these two European swine influenza strains emerged from a different evolutionary lineage of H1 HAs than the HAs of classic swine influenza strains.     

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus and potential application to serodiagnosis

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were generated after fusing spleen cells from a mouse immunized with rHA1 protein derived from influenza strain A/California/06/09 H1N1 with a mouse myeloma cell line. Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4, and 9E12 were reactive in Western blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 inhibited haemagglutination of turkey red blood cells with recombinant NIBRG‐121 virus derived from A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with NIBRG‐121 was localized to the membrane/cytoplasm for four of the reactive Mabs. The differing reactivity of the Mabs in Western blots, immunostaining, EIA, and haemagglutination inhibition assay suggest that at least four of the five Mabs recognize different epitopes on HA1 of the pandemic influenza A/H1N1 2009 virus. Ferret antisera to pandemic influenza A/H1N1 2009 (A/England/195/09 and A/California/07/09 strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine (CELTURA®, Novartis Vaccines, Germany), inhibit binding of 1F5‐HRP to biotinylated rHA1 derived from A/California/06/09 in a competitive EIA, suggesting that the epitope recognized by this Mab also evokes an antibody response in infected ferrets and vaccinated humans. J. Med. Virol. 83:559–567, 2011. © 2011 Wiley‐Liss, Inc.     

Characterization of an influenza A virus in Mexican swine that is related to the A/H1N1/2009 pandemic clade

In the spring of 2009, swine-origin influenza H1N1pdm09 viruses caused the first influenza pandemic of this century. We characterized the influenza viruses that circulated early during the outbreak in Mexico, including one newly sequenced swine H1N1pdm09 virus and three newly sequenced human H1N1pdm09 viruses that circulated in the outbreak of respiratory disease in La Gloria, Veracruz. Phylogenetic analysis revealed that the swine isolate (A/swine/Mexico/4/2009) collected in April 2009 is positioned in a branch that is basal to the rest of the H1N1pdm09 clade in two (NP and PA) of the eight single-gene trees. In addition, the concatenated HA-NA and the complete whole-genome trees also showed a basal position for A/swine/Mexico/4/2009. Furthermore, this swine virus was found to share molecular traits with non-H1N1pdm09 H1N1 viral lineages. These results suggest that this isolate could potentially be the first one detected from a sister lineage closely related to the H1N1pdm09 viruses.     

A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice

Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naïve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses.     

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 first isolation of swine H1N1 influenza viruses from pigs in Thailand

Summary Two influenza A viruses were isolated from pigs in Thailand in January 1988 during the early febrile stage of an influenza-like illness. The isolates contained hemagglutinin and neuraminidase antigens related to those of swine H1N1 influenza virus. This result based on the virus isolation is compatible with the epizootiological evidence that, unlike the human influenza with peak activity in summer (May–July), swine influenza virus is prevalent in the winter season (November–January) in Thailand. The proportion of sera with hemagglutination-inhibiting antibody was higher to A/NJ/8/76 than to A/sw/Iowa/15/30. Likewise, hemagglutination-inhibition tests with monoclonal antibodies indicated that hemagglutinin antigen of the isolates was very similar to that of A/NJ/8/76 virus. In agreement with the serological survey and antigenic characteristic, genetic relatedness between the isolates from Thailand and A/NJ/8/76 virus was also demonstrated by the oligonucleotide mapping of RNA, suggesting that they may be of the same origin.     

Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi, India

Genetic analysis of pandemic 2009 influenza A (H1N1; H1N1pdm09) virus was undertaken to understand virus evolution during 2009 and 2010 in India. Surveillance of influenza viruses from July 2009 to December 2010 revealed major peaks of circulating H1N1pdm09 viruses in August–September and December–January 2009 and then in August–September 2010. To understand the diversity of the H1N1pdm09 virus, selected specimens (n = 23) from 2009 or 2010 were characterized by nucleotide sequence determination of the HA1 subunit of the HA gene. Phylogenetic analysis revealed that 22 clustered with clade 7 viruses characterized by S203T mutations, whereas one virus from 2010 fell within clade 6. None of the viruses from either 2009 or 2010 formed a monophyletic group, suggesting a continuum of independent introduction of circulating viral strains. Amino acid analysis revealed minor amino acid changes in the antigenic or receptor‐binding domains. Importantly, we observed mutations that were also present in 1918 pandemic virus, which includes S183P in 4 and S185T mutation in 3 of 13 viruses analyzed from 2010, while none of the 2009 viruses carried these mutations. Whether antibody‐mediated pressure is imposing such changes remains to be determined. Continued genetic surveillance is warranted to monitor pathogenicity as the virus evolves to acquire new features. J. Med. Virol. 84:386–393, 2012. © 2011 Wiley Periodicals, Inc.     

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.     

Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs

Swine influenza, apart from its importance in animal health, may also be of public health significance. Although the first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure, this virus was shown to be related genetically to swine influenza viruses (SIV) and easily transmissible to pigs. In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences on the risk of human infection by increasing the reservoir of this virus and the risk of possible emergence of new reassortant viruses with increased virulence for pigs and/or humans. Sensitive tools to monitor and detect rapidly such an infection are therefore mandatory. In this study, five commercial real-time RT-PCR assays developed by manufacturers LSI and Adiagène were assessed and validated, (i) for rapid detection of influenza A viruses, including pH1N1/09, in pig and (ii) for the differentiation of pH1N1/09 in that species. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene. All five kits are ready-to-use, one-step duplex RT-PCR and contain an internal positive control (IPC), appropriate for porcine biological samples, for assessing RNA extraction efficiency and the presence of PCR inhibitors. They have been used successfully by veterinary laboratories and shown to be powerful tools for the diagnosis and epidemiological surveillance of influenza virus infections in pigs.     

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.     

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.     

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.     

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.     

Determination of the antigenic composition of the hemagglutinins of influenza viruses H1N1

A comparative immunological analysis of the antigenic composition of hemagglutinins (HA) of influenza A viruses isolated from man and animals was done by the method of specific adsorption of immunoglobulin fractions recovered from sera of rabbits and rats immunized with influenza H1N1 viruses isolated in 1930-1948. A single determinant which remains antigenically stable from 1930 up to the present time was found in HA of human influenza H1N1 viruses (1933-1957, 1977-1981), swine virus A/Swine/Iowa/30, and whale virus A/whale/Pacific Ocean/8/76. Animal influenza viruses A/swine/Iowa/30 and A/whale/Pacific Ocean/8/76 have in their HA one more determinant contributing to the similarity of the swine virus with human A/PR8/34 virus, and the whale virus with human A/W.Smith/33 virus.