Studies on the origin of pandemic influenza. IV. Selection and transmission of "new" influenza viruses in vivo

The emergence of recombinant influenza viruses as dominant strains after recombinant in vivo was studied in an avian system. Turkeys possessing low levels of antibodies to the hemagglutinin of turkey influenza virus and to the neuraminidase of fowl-plague virus were mixedly infected with fowl-plague virus and turkey influenza viruses. Recombinant influenza viruses possessing the hemagglutinin of FPV and the neuraminidase antigen of turkey influenza virus became the dominant viruses present and killed the turkeys. These recombinant influenza viruses spread under natural conditions of transmission to contact turkeys and caused a miniepidemic of disease. The recombinant viruses were genetically stable but differed in transmissibility. Some failed to transmit to contact birds while others were highly transmissible and highly virulent.These studies suggest a mechanism of recombination, selection, and transmission consistent with the emergence of new pandemic strains of influenza viruses.     

Experimental assessment of the pathogenicity of eight avian influenza A viruses of H5 subtype for chickens, turkeys, ducks and quail

Clinical signs, death, virus excretion and immune response were measured in 2-week-old chickens, turkeys, quail and ducks infected by intramuscular, intranasal and contact routes with eight influenza viruses of H5 subtype. Six of the viruses: A/chicken/Scotland/59 (H5N1), ck/Scot; A/tern/South Africa/61 (H5N3), tern/SA; A/turkey/Ontario/ 7732/66 (H5N9); ty/Ont; A/chicken/Pennsylvania/1370/83 (H5N2); Pa/1370; A/turkey/Ireland/83 (H5N8); ty/Ireland, and A/duck/Ireland/ 113/84 (HSN8); dk/Ireland, were highly pathogenic for chickens and turkeys. Two viruses, A/chicken/Pennsylvania/1/83 (H5N2), Pa/1 and A/turkey/Italy/ZA/80 (H5N2), ty/Italy, were of low pathogenicity. Ck/Scot was more pathogenic for chickens than turkeys while ty/Ont was more pathogenic for turkeys than chickens. Other viruses showed little difference in their pathogenicity for these two hosts. No clinical signs or deaths were seen in any of the infected ducks. Only two viruses, dk/Ireland and ty/Ireland, produced consistent serological responses in ducks, although intramuscular infection with tern/SA and ty/Italy resulted in some ducks with positive HI titres. These four were the only viruses reisolated from ducks. Quail showed some resistance to viruses which were highly pathogenic for chickens and turkeys, most notably to ck/Scot and ty/Ont and to a lesser extent tern/SA and Pa/1370. Transmission of virus from intranasally infected birds to birds placed in contact varied considerably with both host and infecting virus and the various combinations of these.     

Characterisation of influenza A viruses isolated from turkeys in England during March-May 1979

During the early spring of 1979 turkeys on at least twelve sites in England became infected with influenza A viruses. On five of these sites no virus was isolated but birds were shown to have antibodies to Havl (four sites) and Hav2 antigenic subtypes of influenza A viruses. The eight viruses isolated were typed: A/turkey/England/192-328/79 (Havl Nav2/3), A/turkey/England/192-329/79 (Hav1 N2), A/turkey/England/199/79 (Hav1 Neq1), A/turkey/ England/214/79 (Hav1 Neq1), A/turkey/England/250/79 (Hsw1 N1), A/turkey/England/262/79 (Hav1 Nav2/3), A/turkey/England/272/79 (Havl Neq1), A/turkey/England/384/79 (Hav2 Nav4). Pathogenicity index tests in 6-week-old chickens agreed with the clinical signs seen in turkeys in the field. Three of the isolates: 199, 214 and 272 were of extremely high virulence, 384 showed intermediate virulence, while the other isolates were of low virulence.     

The prevalence of influenza viruses in swine and the antigenic and genetic relatedness of influenza viruses from man and swine.

Serological and virological surveillance of swine during 1976-77 showed that Hsw1N1 influenza viruses were prevalent throughout the swine population of the U.S., particularly in the northern states. A low incidence of H3N2 virus infections was detected serologically in pigs and confirmed by the isolation of a virus antigenically similar to A/Vic/3/75 from one herd. Both the hemagglutinin and neuraminidase antigens of the human New Jersey isolate, A/NJ/8/76, were indistinguishable from those of selected Hsw1N1 influenza viruses isolated from pigs from 1970 to 1977 and from man in 1976; these antigenically similar viruses were serologically separable from earlier swine viruses. The RNAs from Hsw1N1 viruses were separated by polyacrylamide-gel electrophoresis and the RNA migration patterns among viruses from both species were noticeably different. The only viruses with identical RNA migration patterns were human and swine isolates from the same farm in Wisconsin.     

Characterization of recent H5 subtype avian influenza viruses from US poultry.

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.     

Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza A viruses.

Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza. A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6. The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 degrees C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals.     

Ultrastructural changes induced by influenza viruses in permissive and nonpermissive cells

Human diploid fibroblast (LEP) cells and hamster embryo fibroblast (HEF) cells were infected with four influenza viruses, viz., A/NWS, A/WS, and their recombinants r₁₂ and r₁₄. NWS virus could be propagated in both cell systems and the recombinants could be propagated in HEF cells only, while WS virus could not be passed in either type of cells. After 24 hr, or earlier, all of the cells in both types of culture infected with any of the four viruses produced hemagglutinin and a great majority of them also produced ribonucleo-protein antigen. Electron microscopic examination revealed all of the ultrastructural changes typical of influenza virus replication in HEF and LEP cells when these were infected with NWS virus and in HEF cells when these were infected with WS virus or either of the recombinants. In LEP cells infected with the latter three viruses, only a low percentage (less than 5%) of cells exhibited such alterations; a great majority were free of any detectable change. These results indicate that cells can be infected with influenza virus and synthesize some virus-specific products without being morphologically altered.     

Correlation of pathogenicity and gene constellation of influenza A viruses. III. Non-pathogenic recombinants derived from highly pathogenic parent strains

We have demonstrated by recombination of two highly pathogenic avian influenza viruses [A/FPV/Rostock (Hav1N1) x A/turkey/England/63 (Hav1Nav3)] that recombinants can be isolated which are pathogenic as well as non-pathogenic for chickens. They carried the glycoproteins of either parent strains, and all are produced in infectious form in chick embryo cells. Genetic analysis revealed that the non-pathogenic recombinants possess a mixed RNA polymerase complex, consisting of pol 1, pol 2, ptra and NP gene products, while, with one exception, the pathogenic recombinants have the genes coding for the polymerase activity from one or other parent virus. The biological properties of the recombinant viruses did not correlate with their pathogenicity for chickens.     

Occurrence of Hsw1N1 subtype influenza A viruses in wild ducks in Europe

Summary Two identical strains of influenza A viruses antigenically related to swine influenza (Hsw1N1) have been isolated from adult mallard ducks(Anas platyrhynchos) in Southern Germany. They were designated A/Duck/Bavaria/1/77 and A/Duck/Bavaria/2/77. Serologic tests revealed a close antigenic relationship to the strain A/Duck/Alberta/35/76.Experimental infections of piglets with strain A/Duck/Bavaria/1/77 demonstrated the susceptibility of swine to this virus strain. The virus was isolated from nasal swabs of infected piglets up to 8 days p. inf. and from contact animals up to 9 days. No seroconversion was detected during an observation period of 30 days.     

The NS1 gene of H5N1 influenza viruses circumvents the 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 anti-viral 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 and provide insight into the virulence of 1918 Spanish influenza.     

Identification and characterization of a highly virulent triple reassortant H1N1 swine influenza virus in the United States

A highly virulent H1N1 influenza A virus, A/Swine/Kansas/77778/2007 (KS07), which caused approximately 10% mortality in finishing pigs, was isolated from herds in the Midwestern United States. Molecular and phylogenic analysis revealed this swine isolate was a triple reassortant virus, similar to an H1N1 virus that infected humans and pigs at an Ohio county fair in August 2007. A pig challenge model was developed to evaluate the pathogenicity and transmission capacity of the KS07 virus. The results confirmed that the KS07 virus is highly virulent in pigs and easily transmitted to sentinel animals. The KS07 virus failed to cross-react with a panel of H1-specific swine sera. Interestingly, the KS07 virus shed for a prolonged period up to 7 days in infected pigs, indicating that this virus can spread efficiently between animals. The highly virulent H1N1 swine influenza virus is further evidence of reassortment among avian, human and swine influenza viruses and justifies the need for continued surveillance of influenza viruses in swine.     

Electrophoretic migration rate differences of polypeptides of human influenza A viruses: Partial analysis of the genome of influenza vaccine recombinant viruses

Summary Electrophoretic migration rate differences were detected in high resolution SDS polyacrylamide gels for nucleoprotein (NP), matrix protein (M), non structural protein (NS1), haemagglutinin (HA) and, less regularly, for the polymerase polypeptides P1, P2 and P3 induced by different influenza A viruses. The technique allowed parental assignation of the corresponding genes in certain recombinant viruses including A/PR/8/34 (H0N1)—A/HK/117/77 (H1N1), A/Okuda/57 (H2N2)—A/HK/119/77 (H1N1) and A/Leningrad/76 (H3N2)—A/Leningrad/46 (H0N1) recombinants, thus considerably extending the technique which had been applied previously to A/PR/8/34—A/HK/68 (H3N2) only. Agreement in gene assignment between three recombinants of the former group and 11 of 17 recombinants in the A/Okuda/57—A/HK/119/77 group was noted when the data obtained using the polypeptide method was correlated with a direct genetic analysis by others using RNA:RNA hybridisation techniques. The polypeptide method appears to have wide application for the initial rapid analysis of influenza A virus recombinants obtained using parents of different influenza subtypes although complete analysis of the total genome requires the use of RNA hybridisation techniques. Two additional virus induced proteins are described, a phosphorylated form of NS 1 and a non structual polypeptide with a molecular weight of 16 K daltons.With 6 Figures     

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

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

Genetic reassortment of influenza A viruses in the intestinal tract of ducks

Genetic reassortment between influenza A viruses was shown to occur in the intestinal tract of ducks during mixed infection; this phenomenon was examined both in naturally and laboratory-infected ducks. Studies on cloacal samples from Canadian feral ducks demonstrated that 7% of these samples contained two or more antigenically distinguishable influenza viruses, indicating that mixed infections occur rather frequently in nature. The RNAs of multiple viruses isolated from one cloacal sample were separated by polyacrylamide gel electrophoresis; the RNA migration patterns showed that this sample contained a mixture of viruses, including antigenically identical viruses with heterogeneous RNAs. To determine if genetic reassortment may be responsible for the RNA heterogeneity, laboratory ducks were infected with two antigenically distinct avian influenza viruses (Hsw1N1 and Hav1Nav2). Antigenic recombinants (Hav1N1) were readily isolated, without selective antibody pressure, from the feces of these mixedly infected ducks. Comparisons between the RNA migration patterns of the parental viruses and multiple isolates from the feces showed that antigenically identical isolates (as the Hav1N1 recombinants) possessed different gene constellations. These findings show that genetic reassortment between viruses occurs readily in the duck's intestinal tract. Other parameters of virus infection in ducks were also examined. Juvenile ducks, inoculated with influenza virus, shed virus in their feces for 30 days, produced low levels of circulating antibodies (HI titer of 1:20), and were resistant to challenge with homologous virus. These studies illustrate the mechanism by which genetically diverse influenza A viruses may evolve in nature.     

Comparative study of the biophysical properties of the ribonucleoproteins of type A influenza viruses

The sedimentation and density properties of ribonucleoproteins were studied with different strains of influenza type A virus: A/turkey/Wisconsin/66, A/swine/Iowa/15/31, A/horse/Prague/56, antigenic (A/H3Neq1) and biological (MRC-11) recombinants of the A/Port-Chalmers/1/73 strain. In all the viruses under study, ribonucleoprotein (RNP) was found to be heterogeneous and to sediment in a zone of 30-70 S. Variability in the distribution of fragments in different strains was revealed. In A/turkey/Wisconsin/66 and MPC-11 strains the highest radioactivity was found in the zone of "heavy" fragments (60-70 S), in A/horse/Prague/56 and A/swine/Iowa/15/31 strains it was prevalent in the zone of 40-50 S. In recombinant A(H3Neq1) the distribution of radioactivity varied in different tests. The buoyant density in cesium chloride in all RNP compenents was found to be similar, 1.34-1.36 g/ml.     

Characterisation of an avian influenza A virus isolated from a human – is an intermediate host necessary for the emergence of pandemic influenza viruses?

Summary.  The partial sequencing of the internal and the neuraminidase genes of isolate 268/96 obtained from a woman with conjunctivitis showed all seven to have closest homology with avian influenza viruses. The entire nucleotide sequence of the haemagglutinin gene of 268/96 had close, 98.2%, homology with an H7N7 virus isolated from turkeys in Ireland in 1995. This appears to be the first reported case of isolation of an influenza A virus from a human being infected as a result of direct natural transmission of an avian influenza virus from birds. Accepted December 18, 1997 Received December 3, 1997     

Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID₅₀) for each recombinant was approximately 10^5.0 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID₅₀ and approximately 70% of ca or ts vaccinees who received 10 to 32 HID₅₀ of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.     

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

BackgroundDespite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny.Study designWe document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09.ResultsComplete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID₅₀ of 10⁴/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia.ConclusionsIt is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.