Serological Evidence of Influenza A Viruses in Frugivorous Bats from Africa

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes – H17N10 and H18N11 – in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5μl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.     

New World Bats Harbor Diverse Influenza A Viruses

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.     

Molecular epidemiology of influenza A/H3N2 viruses circulating in Uganda

The increasing availability of complete influenza virus genomes is deepening our understanding of influenza evolutionary dynamics and facilitating the selection of vaccine strains. However, only one complete African influenza virus sequence is available in the public domain. Here we present a complete genome analysis of 59 influenza A/H3N2 viruses isolated from humans in Uganda during the 2008 and 2009 season. Isolates were recovered from hospital-based sentinel surveillance for influenza-like illnesses and their whole genome sequenced. The viruses circulating during these two seasons clearly differed from each other phylogenetically. They showed a slow evolution away from the 2009/10 recommended vaccine strain (A/Brisbane/10/07), instead clustering with the 2010/11 recommended vaccine strain (A/Perth/16/09) in the A/Victoria/208/09 clade, as observed in other global regions. All of the isolates carried the adamantane resistance marker S31N in the M2 gene and carried several markers of enhanced transmission; as expected, none carried any marker of neuraminidase inhibitor resistance. The hemagglutinin gene of the 2009 isolates differed from that of the 2008 isolates in antigenic sites A, B, D, and to a lesser extent, C and E indicating evidence of an early phylogenetic shift from the 2008 to 2009 viruses. The internal genes of the 2009 isolates were similar to those of one 2008 isolate, A/Uganda/MUWRP-050/2008. Another 2008 isolate had a truncated PB1-F2 protein. Whole genome sequencing can enhance surveillance of future seasonal changes in the viral genome which is crucial to ensure that selected vaccine strains are protective against the strains circulating in Eastern Africa. This data provides an important baseline for this surveillance. Overall the influenza virus activity in Uganda appears to mirror that observed in other regions of the southern hemisphere.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Avian Influenza A (H7N9) Virus in a Wild Land Bird in Central China,Late 2015

正Dear Editor,In early 2013,the first case of a human infection with avian influenza A(H7N9)virus was reported in the Yangtze River Delta region of China(Liu et al.2013).Since early2013,a total of 1533 laboratory-confirmed human infections with avian H7N9 viruses have been reported to the     

Origin of the 1918 pandemic H1N1 influenza A virus as studied by codon usage patterns and phylogenetic analysis

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.     

Origin and molecular characterization of the human-infecting H6N1 influenza virus in Taiwan

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain’s pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.     

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.     

Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.     

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn''t attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.     

Pathogenesis of avian influenza A (H5N1) viruses in ferrets

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.     

Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.     

Lethal H5N1 influenza viruses escape host anti-viral cytokine responses

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor alpha. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.     

H9N2 influenza virus in China: a cause of concern

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.     

Isolation of influenza viruses in different species of bird in Canada in 1978

As part of the international program on the ecology of influenza virus in animals sponsored by W.H.O., 357 influenza A viruses isolated from 2 293 cloacal samples collected from ducks and other bird species in Eastern Canada during the 1978 season were characterized antigenically. Seven hemagglutinin (Hsw 1, H2, H3, Hav2, Hav4, Hav6, Hav7) and six neuraminidase subtypes (N1, N2, Neq2, Nav1, Nav5, Nav6) in 18 different combinations were found. A comparison with viruses isolated during previous seasons indicates that subtypes do change from year-to-year and from place-to-place. Isolation of few viruses from passerine birds requires additional studies to determine if these species are truly infected with influenza virus in nature. This large reservoir of influenza A viruses circulating at the same time in ducks may well be involved in the appearance of new viruses in other species, including humans.     

Sentinel Surveillance of Influenza-Like Illness in Two Hospitals in Maracay,Venezuela: 2006–2010

Background

Limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical South American countries such as Venezuela. The objective of the present study was to examine the epidemiology of influenza-like illness (ILI) in two hospitals in Maracay, Venezuela.

Methodology/Principal Findings

We performed a prospective surveillance study of persons with ILI who presented for care at two hospitals in Maracay, Venezuela, from October 2006 to December 2010. A respiratory specimen and clinical information were obtained from each participant. Viral isolation and identification with immunofluorescent antibodies and molecular methods were employed to detect respiratory viruses such as adenovirus, influenza A and B, parainfluenza, and respiratory sincytial virus, among others. There were 916 participants in the study (median age: 17 years; range: 1 month – 86 years). Viruses were identified in 143 (15.6%) subjects, and one participant was found to have a co-infection with more than one virus. Influenza viruses, including pandemic H1N1 2009, were the most frequently detected pathogens, accounting for 67.4% (97/144) of the viruses detected. Adenovirus (15/144), parainfluenza virus (13/144), and respiratory syncytial virus (11/144) were also important causes of ILI in this study. Pandemic H1N1 2009 virus became the most commonly isolated influenza virus during its initial appearance in 2009. Two waves of the pandemic were observed: the first which peaked in August 2009 and the second - higher than the preceding - that peaked in October 2009. In 2010, influenza A/H3N2 re-emerged as the most predominant respiratory virus detected.

Conclusions/Significance

Influenza viruses were the most commonly detected viral organisms among patients with acute febrile respiratory illnesses presenting at two hospitals in Maracay, Venezuela. Pandemic H1N1 2009 influenza virus did not completely replace other circulating influenza viruses during its initial appearance in 2009. Seasonal influenza A/H3N2 was the most common influenza virus in the post-pandemic phase.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

High genetic and antigenic similarity between a swine H3N2 influenza A virus and a prior human influenza vaccine virus: A possible immune pressure-driven cross-species transmission

In late April of 2009, a global outbreak of human influenza was reported. The causative agent is a highly unusual reassortant H1N1 influenza virus carrying genetic segments derived from swine, human and avian influenza viruses. In this study, we compared the HA, NA and other gene segments of a swine H3N2 influenza A virus, A/Swine/Guangdong/z5/2003, which was isolated from pigs in 2003 in Guangdong Province, China, to the predominant human and swine H3N2 viruses. We found that the similarity of gene segments of A/Swine/Guangdong/z5/2003 was closer to Moscow/99-like human H3N2 virus than Europe swine H3N2 viruses during 1999-2002. These results suggest that A/Swine/Guangdong/z5/2003 may be porcine in origin, possibly being driven by human immune pressure induced by either natural H3N2 virus infection or use of A/Moscow/10/99 (H3N2)-based human influenza vaccine. The results further confirm that swine may play a dual role as a “shelter” for hosting influenza virus from humans or birds and as a “mixing vessel” for generating reassortant influenza viruses, such as the one causing current influenza pandemic.