Human H3N2 Viruses Research Articles

Response to Comment on “Seroevidence for H5N1 Influenza Infections in Humans: Meta-Analysis”

We address points made in the comment by Van Kerkhove et al . and explain why human H5N1 virus infections are much more common than was previously thought.

Mammalian Innate Resistance to Highly Pathogenic Avian Influenza H5N1 Virus Infection Is Mediated through Reduced Proinflammation and Infectious Virus Release

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

Evolution of Novel Reassortant A/H3N2 Influenza Viruses in North American Swine and Humans, 2009–2011

Novel H3N2 influenza viruses (H3N2v) containing seven genome segments from swine lineage triple-reassortant H3N2 viruses and a 2009 pandemic H1N1 (H1N1pdm09) matrix protein segment (pM) were isolated from 12 humans in the United States between August and December 2011. To understand the evolution of these novel H3N2 viruses in swine and humans, we undertook a phylogenetic analysis of 674 M sequences and 388 HA and NA sequences from influenza viruses isolated from North American swine during 2009-2011, as well as HA, NA, and M sequences from eight H3N2v viruses isolated from humans. We identified 34 swine influenza viruses (termed rH3N2p) with the same combination of H3, N2, and pM segments as the H3N2v viruses isolated from humans. Notably, these rH3N2p viruses were generated in swine via reassortment events between H3N2 viruses and the pM segment approximately 4 to 10 times since 2009. The pM segment has also reassorted with multiple distinct lineages of H1 virus, especially H1δ viruses. Importantly, the N2 segment of all H3N2v viruses isolated from humans is derived from a genetically distinct N2 lineage that has circulated in swine since being acquired by reassortment with seasonal human H3N2 viruses in 2001-2002, rather than from the N2 that is associated with the 1998 H3N2 swine lineage. The identification of this N2 variant may have implications for influenza vaccine design and the potential pandemic threat of H3N2v to human age groups with differing levels of prior exposure and immunity.

Serologic evidence of human influenza virus infections in swine populations, Cambodia

Background This study was conducted from 2006 to 2010 and investigated the seroprevalence of influenza A viruses in Cambodian pigs, including human H1N1, H3N2, 2009 pandemic H1N1 (A(H1N1)pdm09), and highly pathogenic avian H5N1 influenza A viruses. Methods A total of 1147 sera obtained from pigs in Cambodia were tested by haemagglutination inhibition (HI) assays for antibody to human influenza A viruses along with both HI and microneutralization (MN) tests to assess immunological responses to H5N1 virus. The results were compared by year, age, and province. Results Antibodies against a human influenza A virus were detected in 14·9% of samples. A(H1N1)pdm09 virus were dominant over the study period (23·1%), followed by those to human H1N1 (17·3%) and H3N2 subtypes (9·9%). No pigs were serologically positive for avian H5 influenza viruses. The seroprevalence of human H1N1 and H3N2 influenza viruses peaked in 2008, while that of A(H1N1)pdm09 reached a peak in 2010. No significant differences in seroprevalence to human influenza subtypes were observed in different age groups. Conclusions Cambodian pigs were exposed to human strains of influenza A viruses either prior to or during this study. The implications of these high prevalence rates imply human‐to‐swine influenza virus transmission in Cambodia. Although pigs are mostly raised in small non‐commercial farms, our preliminary results provide evidence of sustained human influenza virus circulation in pig populations in Cambodia.

Identification of Reassortant Pandemic H1N1 Influenza Virus in Korean Pigs

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.

Bird flu in mammals

An engineered influenza virus based on a haemagglutinin protein from H5N1 avian influenza, with just four mutations, can be transmitted between ferrets, emphasizing the potential for a human pandemic to emerge from birds. See Letter p.420 Whether avian H5N1 viruses can gain the ability to transmit between humans was uncertain. The viral haemagglutinin protein (HA) mediates virus binding to host-specific cellular receptors, but previous studies have shown that alterations in HA that enable binding to human-type receptors are not sufficient to enable respiratory droplet transmission of H5N1 viruses in ferrets, the best animal model for human-to-human transmission. Imai et al. show that only four mutations in H5N1 HA are required to enable ferret-to-ferret transmission of a reassortant virus containing H5 HA, with the remaining genes from human pandemic H1N1 influenza virus. It is probable that further adaptations in other avian virus genes would be required to mediate transmission of wholly avian H5N1 in mammals, but human H1N1 and H5N1 viruses are genetically compatible and the emergence of H5-HA-containing viruses might be expected to cause a pandemic because humans lack immunity to H5 viruses. Knowledge of the mutations involved in adapting H5 HA to mammalian transmission could help with surveillance and monitoring of H5N1 viruses adapting towards pandemic potential.

Infection of Pulmonary Vascular Endothelial Cells by H5N1 Avian Influenza Virus and its Role in Pathogenesis

Evaluation of: Zeng H, Pappas C, Belser JA et al. Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection. J. Virol. 86(2), 667–678 (2012). The H5N1 highly pathogenic avian influenza (HPAI) virus is still a major threat for pandemic influenza. The severity of the infection, with mortality reaching up to 60%, is of particular concern, and the detailed mechanism of this high pathogenicity is not fully understood. While alveolar epithelial cells are considered to be the main target cells in humans, Zeng et al. reported that human pulmonary vascular endothelial cells were more susceptible to the H5N1 HPAI virus than to seasonal, pandemic or low pathogenic avian influenza viruses. Infection induced the endothelial cells to produce a number of proinflammatory cytokines, which raised the possibility of a new virulence mechanism that may link to the systemic dissemination of the infection in humans. However, there are still many unanswered questions regarding the determinants of the higher infectivity of H5N1 HPAI in this cell type and the role of the infection of this cell type in the pathogenesis of H5N1 HPAI.

Identification of Dual Receptor-binding Specific Strains of Human H5N1 Viruses in China

ObjectiveBoth the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China. MethodsThe binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers. ResultsDual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses. ConclusionThe findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

Identification of three H1N1 influenza virus groups with natural recombinant genes circulating from 1918 to 2009

In this study, we identify a recombinant pb1 gene, a recombinant MP segment and a recombinant PA segment. The pb1 gene is recombined from two Eurasia swine H1N1 influenza virus lineages. It belongs to a H1N1 swine clade circulating in Europe and Asia from 1999 to 2009. The mosaic MP segment descends from H7 avian and H1N1 human virus lineages and pertains to a large human H1N1 virus family circulating in Asia, Europe and America from 1918 to 2007. The recombinant PA segment originated from two swine H1N1 lineages is found in a swine H1N1 group prevailing in Asia and Europe from 1999 to 2003. These results collectively falsify the hypothesis that influenza virus do not evolve by homologous recombination. Since recombination not only leads to virus genome diversity but also can alter its host adaptation and pathogenecity; the genetic mechanism should not be neglected in influenza virus surveillance.

Prediction of Biological Functions on Glycosylation Site Migrations in Human Influenza H1N1 Viruses

Protein glycosylation alteration is typically employed by various viruses for escaping immune pressures from their hosts. Our previous work had shown that not only the increase of glycosylation sites (glycosites) numbers, but also glycosite migration might be involved in the evolution of human seasonal influenza H1N1 viruses. More importantly, glycosite migration was likely a more effectively alteration way for the host adaption of human influenza H1N1 viruses. In this study, we provided more bioinformatics and statistic evidences for further predicting the significant biological functions of glycosite migration in the host adaptation of human influenza H1N1 viruses, by employing homology modeling and in silico protein glycosylation of representative HA and NA proteins as well as amino acid variability analysis at antigenic sites of HA and NA. The results showed that glycosite migrations in human influenza viruses have at least five possible functions: to more effectively mask the antigenic sites, to more effectively protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. The information here can provide some constructive suggestions for the function research related to protein glycosylation of influenza viruses, although these predictions still need to be supported by experimental data.

The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.

PB1-F2 Modulates Early Host Responses but Does Not Affect the Pathogenesis of H1N1 Seasonal Influenza Virus

In the context of infections with highly pathogenic influenza A viruses, the PB1-F2 protein contributes to virulence and enhances lung inflammation. In contrast, its role in the pathogenesis of seasonal influenza viral strains is less clear, especially in the H1N1 subtype, where strains can have a full-length 87- to 90-amino-acid protein, a truncated 57-amino-acid version, or lack the protein altogether. Toward this, we introduced the full-length 1918 PB1-F2, or prevented PB1-F2 expression, in H1N1 A/USSR/90/77, a seasonal strain that naturally expresses a truncated PB1-F2. All viruses replicated with similar efficiency in ferret or macaque ex vivo lung cultures and elicited similar cytokine mRNA profiles. In contrast, the virus expressing the 1918 PB1-F2 protein caused a delay of proinflammatory responses in ferret blood-derived macrophages, while the PB1-F2 knockout virus resulted in a more rapid response. A similar but less pronounced delay in innate immune activation was also observed in the nasal wash cells of ferrets infected with the 1918 PB1-F2-expressing virus. However, the three viruses did not differ in their virulence or clinical course in ferrets, supporting speculations that PB1-F2 is of limited importance for the pathogenesis of primary viral infection with human seasonal H1N1 viruses.

Evolution and adaptation of hemagglutinin gene of human H5N1 influenza virus

The H5N1 HPAI virus has brought heavy loss to poultry industry. Although, there exists limited human-to-human transmission, it poses potential serious risks to public health. HA is responsible for receptor-binding and membrane-fusion and contains the host receptor-binding sites and major epitopes for neutralizing antibodies. To investigate molecular adaption of HPAI H5N1 viruses, we performed a phylogenetic analysis of HA sequences with 240 HPAI virus strains isolated from human. The topology of the tree reveals overall clustering of strains in four major clusters based on geographic location, and shows antigenic diversity of HA of human H5N1 isolates co-circulating in Asia, Africa, and Europe. The four clusters possess distinct features within the cleavage site and glycosylation sites, respectively. We identified six sites apparently evolving under positive selection, five of which persist in the population. Three positively selected sites are found to be located either within or flanking the receptor-binding sites, suggesting that selection at these sites may increase the affinity to human-type receptor. Furthermore, some sites are also associated with glycosylation and antigenic changes. In addition, two sites are found to be selected differentially in the two clusters. The analyses provide us deep insight into the adaptive evolution of human H5N1 viruses, show us several candidate mutations that could cause a pandemic, and suggest that efficiency measures should be taken to deal with potential risks.

N-Linked Glycosylation in the Hemagglutinin of Influenza A Viruses

Since the 1918 influenza A virus (IAV) pandemic, H1N1 viruses have circulated in human populations. The hemagglutinin (HA) of IAV determines viral antigenicity and often undergoes N-linked glycosylation (NLG) at several sites. Interestingly, structural analysis of the 1918 and 2009 H1N1 pandemic viruses revealed antigenic similarities attributable to the conserved epitopes and the NLG statuses of their HA proteins. NLG of the globular head of HA is known to modulate the antigenicity, fusion activity, virulence, receptor-binding specificity, and immune evasion of IAV. In addition, the HA of IAV often retains additional mutations. These supplemental mutations compensate for the attenuation of viral properties resulting from the introduced NLG. In human H1N1 viruses, the number and location of NLG sites has been regulated in accordance with the antigenic variability of the NLG-targeted antibody-binding site. The relationship between the NLG and the antigenic variance in HA appears to be stably controlled in the viral context.

Molecular Features of Highly Pathogenic Avian and Human H5N1 Influenza A Viruses in Asia

The highly pathogenic avian H5N1 influenza virus could infect humans with high mortality rate, even though it has not yet become efficiently transmissible among humans. This proteomic study investigated the molecular basis of interspecies transmission and host range of this lethal virus in Asia, due to its potential pandemic threat. Although there are host markers located in previous research between general avian and human influenza viruses, the novelty of our work was to uncover host markers between highly pathogenic avian and human H5N1 viruses in Asia. Many host markers we found were not present in the previous general markers, thus expanding the current repertoire of host markers with these strain-specific host markers. Ranked by their order of importance, the top 10 host markers discovered in this report were PB2_627, HA_325, NS1_205, PB2_524, HA_86, NA_201, NP_373, NS1_7, HA_156, NA_74, confirming our current knowledge that PB2_627 is the most critical site for distinguishing avian and human H5N1. We also identified several naturally-occurred mutations in the HA protein that might shift the receptor binding preference of Asian avian H5N1, since early detection of mutations that might lead to emergence of a new pandemic virus is of prime importance. Finally, we analyzed the distinctive interaction patterns within and between proteins of avian and human H5N1 in Asia at protein level and individual residue level. From multiple viewpoints, our findings reinforced the experimental observation that multiple genes of Asian avian H5N1 are involved in its gradual adaptation to human hosts.

Computational analysis of adaptive antigenic mutations of the human influenza hemagglutinin for vaccine strain selection

The adaptive evolution of the hemagglutinin (HA) segment of the human influenza H1N1 and H3N2 viruses is not well understood due to antigenic mutations under selection pressures. This paper identified significant antigenic selection sites and discussed the structural relevance of these mutations. We discovered four positively selected sites for H1N1 (position 206 for the 1A lineage - 2009 swine influenza, and positions 144, 190, and 192 for the 1B lineage), and eight positively selected sites for H3N2 with the loop regions near residues 157 and 225 as the latest antigenic mutations, which may be targeted for vaccine development.

In silico biology of H1N1: molecular modelling of novel receptors and docking studies of inhibitors to reveal new insight in flu treatment.

Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. The new influenza H1N1 viral stain has emerged by the genetic combination of genes from human, pig, and bird's H1N1 virus. The influenza virus is roughly spherical and is enveloped by a lipid membrane. There are two glycoproteins in this lipid membrane; namely, hemagglutinin (HA) which helps in attachment of the viral strain on the host cell surface and neuraminidase (NA) that is responsible for initiation of viral infection. We have developed homology models of both Hemagglutinin and Neuraminidase receptors from H1N1 strains in eastern India. The docking studies of B-Sialic acid and O-Sialic acid in the optimized and energy-minimized homology models show important H-bonding interactions with ALA142, ASP230, GLN231, GLU232, and THR141. This information can be used for structure-based and pharmacophore-based new drug design. We have also calculated ADME properties (Human Oral Absorption (HOA) and % HOA) for Oseltamivir which have been subject of debate for long.

Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses

The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

Human Pulmonary Microvascular Endothelial Cells Support Productive Replication of Highly Pathogenic Avian Influenza Viruses: Possible Involvement in the Pathogenesis of Human H5N1 Virus Infection

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

Genomic characterization of H1N2 swine influenza viruses in Italy

Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of whole genome sequences of 26 strains isolated from 1998 to 2010. Phylogenetic analysis of HA and NA genes showed differences between the older (1998–2003) and the more recent strains (2003–2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Two other reassortant H1N2 strains have been detected: A/sw/It/22530/02 has the HA gene that is closely related to H1N1 viruses; A/sw/It/58769/10 is an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs. Amino acid analysis revealed interesting features: a deletion of two amino acids (146–147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009 to 2010, which is said to have contributed to the increased virulence. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses.