Physicochemical properties of the RNA and proteins of an influenza virus H1N3 isolated from an ill child and antigenically analogous to A/whale/TO/19/76

A comparative analysis of RNA and proteins of influenza A/Baku/799/82, A/whale/TO/19/76, and A/PR8/34 viruses was carried out. The viruses were shown to be similar in their polypeptide composition and oligopeptide maps of the heavy (HA1) and light (HA2) chains of hemagglutinin; in their migration properties of RNA fragments in polyacrylamide gel the A/Baku/799/82 and A/whale/TO/19/76 viruses were similar but not identical. Marked differences in the electrophoretic mobility in gel of RNA fragments coding for P proteins, HA, NP, and NA polypeptides were demonstrated. All these fragments of A/whale/TO/19/76 virus had higher electrophoretic mobility in gel. RNA fragments coding for M and NS proteins had a similar electrophoretic mobility. The A/Baku/799/82 and A/whale/TO/19/76 viruses differed considerably in migration properties of the RNA fragment coding for neuraminidase from the epidemic A/PR8/34 virus. In the latter, this fragment had a higher electrophoretic mobility in gel. Experiments of RNA-RNA hybridization demonstrated a high degree of homology of the primary structure of all RNA fragments of A/Baku/799/82 and A/whale/TO/19/76 viruses.     

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.     

Preparation of influenza B virus recombinant strains

The study of antigenic and biologic properties of influenza B epidemic viruses isolated in 1979 and 1983 and laboratory strain B/Lee/40 has revealed some differences in their biologic properties. The most marked changes have been found in the haemagglutinin (HA) and neuraminidase (NA) indicating that influenza B viruses underwent dramatic antigenic drifts during the period in question. The strains obtained by genetic recombination have inherited surface antigens of epidemic influenza B/Singapore/222/79 and B/USSR/100/83 viruses and preserved the HA thermolability inherent to these viruses. They have, however, acquired the marker of reproduction in chick embryos and the immunogenicity from the donor strain B/Lee/40. These recombinants can be, therefore, recommended as vaccine strain candidates.     

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.     

Comparative studies on influenza B virus. I. Biological properties and polypeptide composition of strains isolated in different years

Influenza B viruses isolated in 1940, 1950 and 1970 belonged to antigenically remote subgroups according to the antigenic composition of haemagglutinin (HA). Antigenically related strains isolated in 1972-1976 differed by their reproduction capability, by their sensitivity to inhibitors in mammalian sera and by affinity to specific antibodies in human sera. Certain correlation between these properties was observed by strains of the Hong Kong variant. Polyacrylamide gel electrophoresis (PAGE) showed that the viruses under study differed in the mobility of the major HA chain (HA1). Molecular weights (MW) of HA1 of antigenically similar influenza B viruses isolated in 1972-1976 revealing different biological properties varied from 52.5 K to 58 K. Neuraminidase (NA) of influenza B/Leningrad/369/75 virus purified by electrophoresis in acetate cellulose consisted of 2 polypeptides with apparent MW of 62 K and 57 K. All viruses examined showed similar mobility of the NA polypeptide MW 57 K; the mobility of the former polypeptide was similar to that of nucleoprotein (NP).     

Increased sensitivity and reduced specificity of hemagglutination inhibition tests with ether-treated influenza B/Singapore/222/79

Hemagglutination inhibition (HI) tests against whole virus (WV) influenza B/Singapore/222/79 antigen detected prevaccination serum antibody in only 15 (20%) of 50 predominantly elderly volunteers and fourfold or greater titer rises in only three (6%) after they received 1981-1982 trivalent influenza vaccine containing antigens of this virus. HI titers against ether-treated (ET) B/Singapore/222/79 were about eightfold higher than those against WV antigen and were comparable to microneutralization titers against this virus. The ET HI detected prevaccination antibody in 84%, a postvaccination titer rise in 32%, and a final titer of 80 or higher in 66%. Among 51 additional persons with known or presumed influenza B virus infections early in 1982, ET B/Singapore/222/79 was also more sensitive than WV for serodiagnosis (69 versus 49%), but eight persons with both WV and ET B/Singapore/222/79 HI responses also had an HI titer rise to WV A/Brazil/11/78 (H1N1) antigen. Conversely, among 14 college students with febrile, culture-proven influenza A (H1N1) infections early in 1982, 6 (43%) developed HI titer rises to ET B/Singapore/222/79 with no other serological evidence of influenza B virus infection. Moreover, young adult volunteers with mild experimental influenza A (H1N1) infections also exhibited a 17% (3 of 18) incidence of ET B/Singapore/222/79 HI titer rises, versus none in matched, uninfected volunteers. These data indicate that ET B/Singapore/222/79 virus has increased sensitivity but reduced specificity compared to WV as an HI antigen and that caution is needed in interpretation of a single HI test for serodiagnosis, whether with WV or ET antigen.     

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.     

Reassortment of American and Eurasian genes in an influenza A virus isolated from a great black-backed gull (Larus marinus), a species demonstrated to move between these regions

The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.     

Comparison of the 35S-methionine oligopeptide maps of influenza A virus NP proteins

Tryptic mapping of radioactive methionine-labeled NP proteins of 15 species of human influenzae A viruses and 11 animal viruses was performed. On the basis of similarities and differences of peptide maps, NP proteins were divided into 4 groups designated A, B, C, and D. Group A included viruses A/WS/33 and A/PR/8/34; Group B viruses H1N1 (apart from those isolated after 1977 and WSN virus), H2N2, H3N2, and 8 species of animal influenza viruses, Group C 4 species of H1N1 viruses isolated in 1977-1979 (A/USSR/90/77, A/USSR/086/79, A/USSR/093/79, A/Brazil/79); Group D three species of animal influenza viruses (A/swine/Iowa/30, A/horse/Praha/56, A/duck/England/56).     

Isolation of the influenza B virus in MDCK cells

The frequency of influenza B virus isolation from clinical specimens is much higher when a continuous line of dog kidney cells, MDCK, is employed, and not the developing chick embryos. Among 9 influenza B virus strains isolated during the influenza epidemic of 1983-1984 winter, 8 strains were isolated in MDCK cells and only 1 in chick embryos. The influenza B virus isolates were similar to influenza B/Singapore/222/79 virus differing from it in HI titres 2-16-fold.     

Genetic analysis of an influenza B virus isolated from a patient with encephalopathy in Japan

An influenza B virus, B/Saga/S172/99 (SAG99), was isolated from the nasopharynx of a patient with encephalopathy/encephalitis in Japan in 1999. To clarify the molecular characteristics of this virus, detailed analysis of the gene segments coding for the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M) and non-structural protein (NS) was undertaken. All five genes of SAG99 showed high nucleotide and predicted amino acid similarities with those of recent non-encephalopathic strains isolated in the same epidemic season. Subsequent phylogenetic analysis revealed that all five gene segments of SAG99 analyzed in the present study were most similar to those of the recent Yamagata/16/88-like viruses. The hemagglutinin and neuraminidase proteins of SAG99 were each distinguished from those of recent epidemic strains by one characteristic amino acid substitution. These substitutions were not found in the previously reported encephalopathy/encephalitis-derived influenza B viruses, and we could not find any common characteristic amino acid changes in SAG99 and these viruses. Similarly, among the internal proteins studied, only the M2 protein of SAG99 was found to contain a single novel amino acid change when compared with other recent isolates. Thus, it was apparent that SAG99 contained very few amino acid differences when compared with other epidemic viruses. The association of recent B/Yamagata/16/88-like viruses with encephalitis/encephalopathy observed in the present study and previously suggest that these viruses may have a higher potential for causing neurological complications in certain individuals.     

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)     

Antigenic and molecular analysis of influenza A(H3N2) virus strains isolated in 1985 in open and closed communities of northern Germany.

Antigenic and molecular analyses of influenza A(H3N2) virus strains isolated in 1985 during outbreaks in open and closed communities of North Germany were carried out. The data obtained have shown that 11 strains isolated in a closed orphanage were antigenically similar to each other. The electrophoretic mobilities of either HA, NP, M1 and NS1 polypeptides or of double stranded RNA segments were indistinguishable. Analysis of viruses isolated at the same time from open communities has revealed that they contained at least three groups of strains differing in homology of 3-5 RNA segments. These data support the idea that an outbreak of influenza in a community is caused by single virus strain, from which their slightly different variants of the virus arise during circulation among sensitive persons.     

Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice

In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host.     

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.     

Structural polypeptides of antigenically distinct strains of influenza B virus

Analyses of the polypeptide composition of influenza B viruses by 13 per cent SDS-polyacrylamide gel electrophoresis are reported. The viruses contained polypeptides of eight species ranging in molecular weight from 27,000 to 78,000. Four of them were glocypeptides and were selectively removed from the surface of the virion by Bromelain treatment. One of the blycopeptides was identified as viral neuraminidase. Three antigenically distinct strains of influenza virus, B/Lee/40, B/Massachusetts/1/71 and B/Hong Kong/5/72, showed an essentially identical electrophoretic picture, although strain-to-strain difference was observed in the migration rate of HA1 and HA2 polypeptides.     

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.     

Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses

Sequence analyses and comparison of the genes coding for the nonstructural (NS) and hemagglutinin (HA) proteins of different influenza B viruses isolated between 1940 and 1987 reveal that the number of substitutions is not always proportional to the time between isolates. Examination of 14 influenza B virus NS gene and 10 HA gene sequences by the maximum parsimony method suggested that--as with influenza C viruses--there are multiple evolutionary lineages which can coexist for considerable periods of time. Comparison of the sequence divergence among genes of viruses belonging to type A, B, and C virus suggests that, in man, influenza B viruses evolve slower than A viruses and faster than C viruses. We propose an evolutionary model for influenza B viruses that is intermediate between the pattern for human influenza A viruses and that for influenza C viruses.     

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