Correlation between levels of apoptosis,levels of infection and haemagglutinin receptor binding interaction of various subtypes of influenza virus: does the viral neuraminidase have a role in these associations

Previously, we have shown that an H3N2 influenza virus (clone 7a) induced more apoptosis in MDCK cells than an H1N1 (A/Fiji) influenza virus and that the virion neuraminidase (NA) played a role in the induction of apoptosis. In this study we have examined a further 6 N2 (H3/H2) and 3 N1 (Hsw/H1) viruses and confirmed that the N2 viruses induce more apoptosis in MDCK cells than the N1 viruses. Furthermore, the level of apoptosis, the level of cell infection and the NA activity of the virus preparations paralleled each other for all the viruses. The levels of infection depended upon the degree of interaction of the viral haemagglutinin (HA) with its receptors: while all the viruses utilised NeuAc alpha-2,6 Gal containing receptors, the H3/H2 viruses showed a greater interaction than the Hsw/H1 viruses. Removal of sialic acid from virions by treatment with bacterial NA enhanced infection and apoptosis but the effect was much greater for the A/Fiji virus than for the clone 7a virus. Thus, while the relative interaction of the HAs for their receptors is the major factor influencing infectivity and apoptosis, the viral NA possibly plays an indirect role by removing sialic acid from the HA, thereby increasing its receptor binding.     

Genetic evolution of swine influenza A (H3N2) viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.     

Human influenza a viral genes responsible for the restriction of its replication in duck intestine

Although influenza A viruses are occasionally transmitted from one animal species to another, their host range tends to be restricted. Currently circulating human influenza A viruses are thought to have originated from avian viruses, yet none of these strains replicate in duck intestine, a major site of avian virus replication. Although the hemagglutinin (HA) and neuraminidase (NA) genes are known to restrict human virus replication in ducks, the contribution of the other viral genes remains unknown. To determine the genetic basis for host range restriction of the replication of human influenza A virus in duck intestine, we first established a reverse genetics system for generating A/Memphis/8/88 (H3N2) (Mem/88) and A/mallard/New York/6750/78 (H2N2) (Mal/NY) viruses from cloned cDNAs. Using this system, we then attempted to generate reassortant viruses with various combinations of candidate genes. We were able to generate single-gene reassortants, which possessed PB2, NP, M, or NS from Mem/88, with the remainder from Mal/NY. Despite unsuccessful production of other single-gene reassortants from Mem/88, we did generate reassortant viruses comprised of both the HA and the NA, all three polymerase genes (PB2, PB1, and PA), or all polymerase genes and the NP gene from Mem/88, with the rest derived from Mal/NY. Among these reassortants, only those possessing the M or NS gene from Mem/88 and the remainder from Mal/NY replicated in duck intestine. These results indicate incompatibility between the genes of avian and human influenza A viruses and indicate that all genes other than the M and NS restrict replication of human influenza A virus in duck intestine.     

Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role

Summary.  Previously, we have shown that apoptosis induced by influenza virus was inhibited by an anti-neuraminidase compound [4-guanidino-2, 3-dehydro-N-acetylneuraminic acid (GG167; Relenza; Zanamivir)], which does not enter cells, and acts at the attachment/entry phase of virus replication. Furthermore, a virulent virus, clone 7a, induced greater levels of apoptosis than the attenuated A/Fiji and had greater neuraminidase (NA) activity. To confirm more directly that NA induces apoptosis, the NA of clone 7a and A/Fiji was expressed fused to the Herpes simplex virus tegument coat protein VP22, transfected into HeLa cells and the level of apoptosis determined. VP22 translocates between cells via the medium thus allowing expressed proteins to transfer to a larger number of cells than those originally transfected. Clone 7a NA fused to VP22 induced a significant level of apoptosis whereas A/Fiji NA/VP22 did not, confirming that NA activity is an important determinant of apoptosis acting during fusion protein translocation between cells. Furthermore, the induction of apoptosis was abrogated by antibody to transforming growth factor-β, which is activated by NA. This approach also showed that VP22/NS1 proteins of both clone 7a and A/Fiji induced apoptosis when expressed alone but inhibited double stranded RNA-induced apoptosis suggesting that this protein may have a dual mode of action. Also, the M1 and M2 proteins of both viruses induced apoptosis but their NP proteins did not. Received June 27, 2001; accepted December 10, 2001     

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.     

Genetic characterisation of an influenza A virus of unusual subtype (H1N7) isolated from pigs in England

Summary.  An H1N7 influenza A virus, isolated from pigs in England in 1992, was examined genetically to determine the characteristics and probable origin of the eight gene segments. Six of the RNA segments encoding PB2, PB1, PA, HA, NP and NS were related most closely to those of human viruses, whilst two of the RNA segments (NA and M) were related most closely to those of equine viruses. The HA gene was most similar to that of A/USSR/90/77 (H1N1) but amino acid differences suggested independent genetic drift. In contrast, there were relatively few changes in the NA and M genes compared to those of A/equine/Prague/1/56 (H7N7). Accepted November 12, 1996 Received November 12, 1996     

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.     

Phylogenetic analysis of an H1N2 influenza A virus isolated from a pig in Korea

Summary. An influenza H1N2 virus was isolated from a pig during an severe outbreak of respiratory disease in a Korean herd. The neuraminidase (NA) and PB1 genes of the H1N2 isolate were of human origin, while the hemagglutinin (HA), matrix (M), nucleoprotein (NP), and non-structural (NS) genes were of swine origin and PA and PB2 gene were of avain origin. Phylogenetic results indicate that the Korean H1N2 isolate was closely related to H1N2 viruses isolated recently from pigs in the United States.     

Emergence of a new swine H3N2 and pandemic (H1N1) 2009 influenza A virus reassortant in two Canadian animal populations, mink and swine

A swine H3N2 (swH3N2) and pandemic (H1N1) 2009 (pH1N1) influenza A virus reassortant (swH3N2/pH1N1) was detected in Canadian swine at the end of 2010. Simultaneously, a similar virus was also detected in Canadian mink based on partial viral genome sequencing. The origin of the new swH3N2/pH1N1 viral genes was related to the North American swH3N2 triple-reassortant cluster IV (for hemagglutinin [HA] and neuraminidase [NA] genes) and to pH1N1 for all the other genes (M, NP, NS, PB1, PB2, and PA). Data indicate that the swH3N2/pH1N1 virus can be found in several pigs that are housed at different locations.     

Characterization of An Avian Influenza Virus of Subtype H7N2 Isolated from Chickens in Northern China

An H7N2 avian influenza virus was isolated from chickens during routine surveillance in northern China in 2002. To understand the origin of this virus, we completely sequenced its genome. The PB1, PA, HA, and M genes of this virus were highly homologous with those of the wild bird virus A/Africa starling/Eng-Q/983/79 (H7N1). The NP and NS genes were closely related to those of two other wild bird viruses isolated 30 years ago. The closest relatives of the PB2 and NA genes of the virus were those of the A/swine/Germany/2/81 (H1NI) and A/Leningrad/134/57 (H2N2), respectively. Animal inoculation tests showed that the virus cannot replicate efficiently in chickens. However, after intranasal inoculation, the virus induced 20% weight loss and replicated well in the lungs of mice. The virus was also recovered from the hearts and brains of the mice. These results suggest that the influenza virus isolated in chickens in northern China in 2002 originated in wild birds and may pose a threat for both avian species and mammalian hosts.     

Characterization of a new avian-like influenza A virus from horses in China.

In March 1989 a severe outbreak of respiratory disease occurred in horses in the Jilin and Heilongjiang provinces of Northeast China that caused up to 20% mortality in some herds. An influenza virus of the H3N8 subtype was isolated from the infected animals and was antigenically and molecularly distinguishable from the equine 2 (H3N8) viruses currently circulating in the world. The reference strain A/Equine/Jilin/1/89 (H3N8) was most closely related to avian H3N8 influenza viruses. Sequence comparisons of the entire hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and NS genes along with partial sequences of the three polymerase (PB1, PB2, PA) genes suggest that six of the eight gene segments (PA, HA, NP, NA, M, NS) are closely related to avian influenza viruses. Since direct sequence analysis can only provide a crude measure of relationship, phylogenetic analysis was done on the sequence information. Phylogenetic analyses of the entire HA, NP, M, and NS genes and of partial sequences of PB1, PB2, and PA indicated that these genes are of recent avian origin. The NP gene segment is closely related to the gene segment found in the newly described H14 subtype isolated from ducks in the USSR. The A/Equine/Jilin/1/89 (H3N8) influenza virus failed to replicate in ducks, but did replicate and cause disease in mice on initial inoculation and on subsequent passaging caused 100% mortality. In ferrets, the virus caused severe influenza symptoms. A second outbreak of influenza in horses in Northeast China occurred in April 1990 in the Heilongjiang province with 48% morbidity and no mortality. The viruses isolated from this outbreak were antigenically indistinguishable from those in the 1989 outbreak and it is probable that the reduced mortality was due to the immune status of of the horses in the region. No influenza was detected in horses in Northern China in the spring, summer, or fall of 1991 and no influenza has been detected in horses in adjacent areas. Our analysis suggests that this new equine influenza virus in horses in Northeast China is the latest influenza virus in mammals to emerge from the avian gene pool in nature and that it may have spread to horses without reassortment. The appearance of this new equine virus in China emphasizes the potential for whole avian influenza viruses to successfully enter mammalian hosts and serves as a model and a warning for the appearance of new pandemic influenza viruses in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Virulence of H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene

More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263–277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID₅₀ or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD₅₀ in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively_. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.     

First isolation of an H1N1 avian influenza virus from wild terrestrial non-migratory birds in Argentina

A type A avian influenza (AI) virus was isolated from dead or severely ill red-winged tinamous (Rhynchotus rufescens) found in a hunting ground in April 2008 in Argentina. The subtype of A/red-winged tinamou/Argentina/MP1/2008 was determined as H1N1 by sequence analysis. The cleavage site of the viral hemagglutinin corresponded to a low pathogenic influenza virus, although the clinical presentation and pathological studies suggest that the virus was pathogenic for red-winged tinamous. Phylogenetic analysis of the viral genome suggested that while the hemagglutinin and neuraminidase genes were related to AIV from North America, the internal genes were most closely related to other South American isolates. These findings support the postulated South American phylogenetic lineage for AIV PB2, PB1, PA, M and NS genes, and suggest that the evolutionary pathways of HA and NA genes involve exchanges between the Northern and Southern hemispheres.     

Detection and characterization of new influenza B virus variants in 2002

One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.     

Mutations in the genes coding for hemagglutinin and neuraminidase in cold-adapted variants of the influenza virus A/Leningrad/134/57 (H2N2)

An analysis of ts-mutations in the genomes of native and cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus based on the use of fowl plague virus ts mutants was carried out. The recombination test was done by the conventional method in chick embryo fibroblast culture (genes PB2, PB1, PA, NP, NA, M and NS) or cell systems permissive for reproduction of human influenza virus (gene HA). The cold-adapted strain A/Len/17 used for preparation of live influenza vaccine (LIV) for adults was shown to have 4 ts mutations: three in "internal" genes (PB2, NP, and M) and one in gene 4 coding for hemagglutinin (HA). The more attenuated cold-adapted donor A/Len/47 for preparation of similar LIV for children acquired three additional ts mutations: two (PB1 and NS) in "internal" genes and one in gene 6 coding for neuraminidase (NA). The accumulation of ts mutations in the genome of cold-adapter strains was found to be accompanied by a decrease in their pneumotropicity for mice as well as their detectability in different organs of these animals.     

Isolation and phylogenetic analysis of H1N1 swine influenza virus isolated in Korea

A swine influenza H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in Korea. All genes of the H1N1 isolate, including hemagglutinin (HA), neuraminidase (NA), matrix (M), nucleoprotein (NP), non-structural (NS), PA, PB1 and PB2, were of swine origin. Also, all these genes showed a close phylogenic relationship with those of H1N1 viruses previously isolated from pigs in the United States. These results suggest that North American swine influenza virus has actually been transmitted to pigs in Korea.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

Genetic analysis of mouse-adapted influenza A virus identifies roles for the NA, PB1, and PB2 genes in virulence.

Adaptation of the prototype A/FM/1/47 H1N1 strain to mice resulted in selection of the A/FM/1/47-MA variant with increased virulence. Earlier analysis identified mutations in the HA and M1 genes that increase virulence in the mouse. Complete sequence analysis identified mutations in the PB1, PB2, HA, NA, and M1 genes. Reassortants were produced between the parental FM and FM-MA strains to obtain viruses that differ due to combinations of mutant genes. To assess the relationship between virulence and replication, the median lethal dose was determined for mice and growth properties were assessed in mouse lung, MDCK cells and chicken embryo. Not only were all five mutations shown to control virulence but also the replicative capacity in the mouse. The HA, NA and M1 mutations increased yield in all three hosts whereas in combination the PB1 and PB2 mutations were host restrictive changing the virus to a mouse specific strain. For the NA and M1 mutations the increase in growth in mouse lung was proportional to a 2-fold (log10) increase in virulence however the HA mutation increased virulence largely independent of increased growth indicating a change in pathological properties that damage the host. Thus mutations that affect virulence can be classified according to host-dependent and independent ability to increase growth as well as changes in pathological properties. Each of the PB1, PB2, NA, HA, and M1 genes acquired gain-of-function mutations for mouse infection that involve structural motifs that may serve as markers for virulence or targets for antiviral therapy.     

Genetic characterization of low pathogenic H5N1 and co-circulating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008

As part of a long-term wild bird monitoring programme, five different low pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards (subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating (same location, same time period) viruses were selected for full genome sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus (A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later in December 2008. All genes of these co-circulating viruses shared common ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses. The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A double infection with two different PA segments from H5N1 and from H5N3 could be observed for the H1N1 sample. The observed gene constellations resulted from multiple reassortment events between viruses circulating in wild birds in Eurasia. Several internal gene segments from these 2008 viruses and the N3 sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the growing sequence evidence of the dynamic nature of the avian influenza natural reservoir in Eurasia, and underline the importance of monitoring AIV in wild birds. Genetic information of potential hazard to commercial poultry continues to circulate in this reservoir, including H5 and H7 subtype viruses and genes related to previous AIV outbreaks.