Intestinal influenza: replication and characterization of influenza viruses in ducks.

Influenza A viruses isolated from the cloaca of naturally infected feral ducks replicate in the lungs and in the cells lining the intestinal tract of feral and domestic ducks. Despite the low pH of the gizzard, the duck influenza viruses reach the intestines via the digestive tract and are found in high concentration in the feces. The viruses retain infectivity in fecal material for at least 30 days at 4° and for 7 days at 20°. The morphology of one strain of intestinal duck influenza virus (Hav7 Neg2) that had never been passed in embryonated eggs and was isolated from the feces was roughly spherical and fairly uniform in size and shape. However, another strain of duck influenza virus studied (Hav3 Nav6) was predominantly filamentous, suggesting that the morphology of influenza viruses in their natural hosts varies from strain to strain. After passage in the chick embryo each strain retained the morphological characteristics found in the feces. In contrast to duck influenza viruses, representative human influenza viruses of the HON1, H3N2, and Hswl Nl subtypes replicate only in the upper respiratory tract of ducks. The duck influenza viruses are more stable to low pH than human strains and retain infectivity for over 30 days in nonchlorinated river water at 0° and for 4 days at 22°. The susceptibility of ducks to infection with human and avian strains of influenza virus and the possibility of transmission to animal species through the water supply suggests that ducks may be important in the ecology of influenza viruses. The possibility of “intestinal influenza” virus vaccines is considered.     

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.     

Genetic diversity among avian influenza viruses

The RNAs of a series of avian influenza viruses of the subtype Hav7Neg2 were examined to determine if their antigenic similarity reflected an overall conservation of their RNA sequences. Genetic analysis by gel electrophoresis showed a marked variability in all of the RNA segments of the isolates. Analysis by competitive hybridization indicated that with some genome segments this variability represented major genetic differences. These differences were present even among concurrent isolates from one geographical area. In contrast, similar analysis of human H3N2 influenza virus isolates showed that viruses isolated 9 years apart were much more similar than the cocirculating avian viruses. The genetic diversity in avian influenza viruses may result from the cocirculation of many different influenza A viruses in ducks and their ability to recombine in nature.     

Similar frequencies of antigenic variants in Sendai, vesicular stomatitis, and influenza A viruses

The effects of three inhibitors of glycosylation (2-deoxy-d-glucose, d-glucosamine, and tunicamycin) on the formation of infectious extracellular snowshoe hare virus and the synthesis of intracellular virus-induced polypeptides have been examined. The inhibitor 2-deoxy-d-glucose, used at concentrations of up to 10 mM, did not significantly affect either infectious virus release or viral-induced polypeptide synthesis and maturation. By contrast, both d-glucosamine (40 mM) and tunicamycin (1 μg/ml) prevented the release of infectious virus and inhibited the maturation of the two viral glycoproteins (Gl and G2), but not the viral nucleoprotein (N). Two new polypeptides, Gl₀ and G2₀, of molecular weights 105 × 103 and 33 × 103, respectively, were found in infected cells treated with tunicamycin or D-glucosamine. These two polypeptides were identified as virus specific since they could be precipitated by virus immune serum. Tunicamycin was found to inhibit the release of extracellular virus particles at 33 and 38°, which correlated with the observation that no intracellular virus particles were detected in electron micrographs of cells treated with the drug.     

Categorization of nucleoproteins and matrix proteins from type A influenza viruses by peptide mapping

Relationships between the nucleoprotein (NP) and matrix protein (M) of 16 human and 3 lower vertebrate strains of type A influenza virus were investigated by two-dimensional mapping of [³⁵S]methionine peptides. By regarding certain common groups of peptides as taxonomic criteria, all NP proteins could be classified as belonging to one of two basic patterns. Differences existed between maps of either category but these have not been investigated in detail. The chymotryptic-tryptic peptide maps of the M proteins were too similar overall to warrant dividing them into subgroups but differences between individual strains were clearly apparent. Both patterns of NP protein occurred within a subtype. The change from one NP pattern to another, and back again, within a subtype is consistent with the hypothesis that genetic assortment of NP genes occurs during the reign of a particular subtype. This and other implications are discussed.     

Antigenic variants of influenza viruses: marked differences in the frequencies of variants selected with different monoclonal antibodies

Monoclonal antibodies against the hemagglutinin of two influenza A viruses and one influenza B virus were used to analyze the frequencies of antigenic variant subpopulations in cloned virus seeds. Antigenic variants selected with monoclonal antibodies specific for different antigenic determinants were obtained over a wide range of frequencies. With one monoclonal antibody directed to the hemagglutinin of A/PR/8/34 virus antigenic variants were isolated at a frequency of 10-^4.4, whereas another monoclonal antibody, specific for the hemagglutinin of X-31 virus, completely neutralized all infectious virus in the X-31 virus seeds examined, indicating a frequency of antigenic variation, for one seed at least, of less than 10^?8.1.     

The large P proteins of influenza A viruses are composed of one acidic and two basic polypeptides

Six strains of influenza A virus, grown and labeled with [³⁵S]methionine in tissue culture, were purified and their proteins were analyzed in a two-dimensional system combining non-equilibrium pH gradient electrophoresis (NEPHGE) with NaDodSO₄-polyacrylamide gel electrophoresis. The P polypeptides separated as one acidic polypeptide (pI 6) and two basic polypeptides (pI above 8). This method facilitated the identification of P polypeptides, especially of P² and P³ whose separation was difficult in a one-dimensional system.     

The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses.

Comparative studies on naturally occurring avian influenza viruses have been carried out in order to investigate the determinant(s) for pathogenicity for chickens. At least one virus isolate from each of the nine different hemagglutinin (HA) subtypes was included. The polypeptides of these viruses were studied by analyzing infected cell extracts on SDS-polyacrylamide gels. Both viral glycoproteins, HA and neuraminidase, showed remarkable variation in their electrophoretic mobility even among serologically closely related viruses. Pulse-chase experiments revealed that most avian influenza virus strains had an HA which was not susceptible to proteolytic cleavage in MDCK, turkey (TEC), and chicken embryo cells (CEC). Only viruses belonging to the subtype Hav5 and some strains of the subtype Hav1 possessed a cleaved HA in these cells. Only the virus strains with cleaved HA were produced in infectious form in MDCK, CEC, TEC, as well as in duck embryo cells (DEC) and quail embryo cells (QEC). The other virus strains produced plaques in these cells only in the presence of trypsin. There was a strict correlation between the cleavability of the HA, the potential of the virus to be produced in infectious form in a wide range of host cells, and their pathogenicity for chickens. No evidence was obtained for an involvement of the neuraminidase in determining pathogenicity. For the nonpathogenic viruses it could be shown that they can replicate and produce infectious progeny in some organs of the chicken. The results obtained permit the conclusion that in naturally occurring avian influenza viruses the structure of the hemagglutinin, that is its susceptibility to proteolytic cleavage in a broad spectrum of host cells, is the determining factor for pathogenicity.     

Analysis of antigenic drift in recently isolated influenza A (H1N1) viruses using monoclonal antibody preparations.

Monoclonal antibodies to the hemagglutinin (HA) and neuraminidase (NA) of A/USSR/ 90/77(HlN1) were prepared and used to study antigenic drift in the H1N1 subtype of influenza viruses. The results obtained with five different monoclones to each molecule were compared with the results obtained with postinfection ferret sera. Monoclonal antibodies and postinfection ferret sera detected antigenic drift in the hemagglutinin and neuraminidase molecules of H1N1 viruses and monoclonal antibodies showed that the A/USSR/90/77, A/ Roma/1/49, and A/Fort Warren/1/50 viruses were identical at five sites on both the hemagglutinin and neuraminidase molecules. To further evaluate monoclonal antibodies, they were used in hemagglutination inhibition tests with recently isolated influenza A(HlNl) viruses, including some shown in tests with postinfection ferret sera to have undergone antigenic drift. Three patterns of reactivity with the monoclonal antibodies were detected with the variants: two variants differed at two of the five sites identified by the monoclonal antibodies and a third variant differed at only one of the five sites. Two of these variant groupings had not been distinguished from each other in HI tests with ferret sera, whereas ferret sera were capable of distinguishing between two groups of variants that had similar reaction patterns with the five monoclonal antibody preparations. Monoclonal antibodies failed to detect antigenic drift in the NA molecules of the recent isolates of H1N1 influenza viruses, suggesting that antigenic changes occur less frequently in this molecule. The observation that antigenic differences could be detected between the recent variants with such a small panel of monoclonal antibodies (less than 10% of the number obtained to the HA of A/PR/8/34) suggests that monoclonal antibodies may provide an exquisitely sensitive method for antigenic mapping of influenza viruses.     

Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: nonrandom reassortment of genes during preparation of live virus vaccine candidates by recombination at 25 degrees between recent H3N2 and H1N1 epidemic strains and cold-adapted A/An Arbor/6/60.

Genetic compositions of 35 recombinant cold-adapted influenza A(H3N2 and H1N1) candidate live attenuated vaccine strains have been determined. The viruses, which had been obtained by recombination (reassortment) at 25° between contemporary epidemic wild-type strains and cold-adapted A/Ann Arbor/6/60(H2N2), followed by selection for growth at 25° of virus with wild-type HA and NA, have a highly restricted genetic composition. Eighteen of the thirty-five recombinants had RNAs coding for the three polymerase (P) proteins, NP, M, and NS, from the cold-adapted mutant A/Ann Arbor/6/60 had only the HA and NA of the wild-type strains. Only 4 out of 64 theoretically possible combinations of genes coding for nonglycoprotein viral products were detected. The restricted genetic composition of cold-adapted recombinants produced at 25° supports the evaluation of this method of preparing live vaccine strains to determine whether recombinants with constant gene composition have predictable levels of attenuation for man.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Evidence for antigenic variation in influenza A nucleoprotein.

Nucleoprotein (NP) antigens isolated from sodium sarcosyl detergent-disrupted influenza A virus particles by cellulose acetate electrophoresis were used to prepare specific immune sera. Antigenic analysis of the nucleoproteins by immuno-double-diffusion and antibody absorption tests revealed antigenic differences in the nucleoprotein antigens of human A/PR8/34 (HON1) virus and viruses of the Hong Kong (H3N2) subtype. The nucleoprotein antigens of avian A/duck/Ukraine/1/63 (Hav7Neg2) and A/swine/Iowa/15/30 (Hsw1Nl) viruses resembled more closely the NP of A/PR8/34 virus than the NP of human H3N2 strains. Antigenic analysis of recombinant strains prepared from A/PR8/34 and H3N2 parent viruses indicated that the parental origin of the NP antigens could be clearly identified. Identification of the nucleoproteins of parental and recombinant influenza A viruses by migration rate analysis of NP polypeptides and RNA genes by electrophoresis on polyacrylamide gels gave results which correlated with the antigenic characterization of their NP antigens. The findings suggested that antigenic “drift” occurs in the nucleoproteins of human influenza A viruses.     

Characterization of an influenza A virus from seals

An influenza A virus antigenically similar to A/FPV/Dutch/27 (Hav1Neq1) [H7N7] was isolated from harbor seals (Phoca vitulina) that had died of acute hemorrhagic pneumonia on Cape Cod Peninsula, beginning in the winter of 1979–1980. High titers of virus were obtained from the lungs and lower titers from the brains of the seals. Although antigenic analyses and characterization of the RNAs show that all of the genes and gene products are closely related to different avian influenza viruses, biologically the virus behaves more like a mammalian strain. The seal virus replicated and produced pneumonia in experimentally infected harbor seals, but the clinical course and pathology were less severe than in the natural infection; the virus also replicated in ferrets, cats, and pigs but produced no disease. In avian species, the seal influenza virus replicated poorly, produced no disease signs, and was not shed in the feces. Although the seal influenza virus can cause conjunctivitis in humans who have known contamination of the eyes from infected animals, serological studies detected no evidence of seroconversion among persons working with infected seals or with the virus. Preliminary studies detected antibodies to this virus in harbor seals on the New England coast but not in harbor seals, gray seals, or fur seals from other areas, suggesting that this virus may be a new introduction to this species. An Hav1Neq1 [H7N7] virus was also isolated from feral ducks in Iceland in 1980, but the two viruses could be distinguished by analysis of their RNAs and host range. The A/Seal/Mass/1/80 influenza virus provides the first evidence suggesting that a strain deriving all of its genes from one or more avian influenza viruses can be associated with severe disease in a mammalian population in nature. Whether this breach of species specificity represents a unique event in influenza evolution remains to be determined, but raises the possibility that human or animal influenza viruses may be derived directly from avian strains.     

A comparison of the granulosis viruses from Pieris brassicae and Pieris rapae

The granulosis viruses of Pieris brassicae and P. rapae have been compared biochemically and biologically. No differences were found between virus capsules when examined by immunodiffusion, ELISA, or SDS-polyacrylamide gel electrophoresis. The virus particles were identical by immunodiffusion but distinguishable by ELISA. Comparison of virus particle polypeptides on SDS-polyacrylamide gels indicated small differences in molecular weight in three of the polypeptides of the virus envelope but no difference between the nucleocapsids. There were several differences between the DNA fragments produced by digestion with EcoRI, BamH1, and HindIII restriction endonucleases from which the homology between the two DNAs was calculated to be 97.7%. The small structural difference between the two viruses is associated with a very large difference in their virulence for P. brassicae; the LD50 with P. rapae GV being at least 1000 times greater than with P. brassicae GV. P. rapae was much more susceptible to either virus than P. brassicae and in this case the LD50 values were not significantly different for the two viruses.     

Gene 8 of influenza virus: Sequences of cDNA transcribed from the 3′ ends of viral RNA of influenza A and B strains

Nucleotide sequences of cDNA transcribed from the Tends of RNA segment 8 of influenza strains A/PR/8/34 (HONI), A/RI/5-/57 (H2N2), and B/Lee/40 have been obtained by the dideoxy method, with depurination analysis of specific dideoxy-terminated products used to partially confirm the sequences. In the first 80 nucleotides there are 3 differences when the sequences of segment 8 from PR8 and R15^? are compared, whereas a comparison of the first 130 nucleotides of cDNA transcribed from R15^? and B/Lee segment 8 shows no similarities either in the nucleotide sequences or predicted amino acid sequences of the “nonstructural” proteins coded by segment 8.     

Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response

The cellular immune competence of lymphocytes obtained from birds infected with acute or chronic leukemia viruses was studied. The in vitro blastogenic response of thymus-derived lymphocytes (T cells) to the mitogens phytohemagglutinin (PHA) and connanavalin A (Con A) correlates with the ability of birds to elicit a cellular immune response (C. K. Naspitz and M. Richter, 1968, Prog. Allergy12, 1–85; P. Toivanen and A. Toivanen, 1973, J. Immunol.111, 1602–1603). The PHA response of splenic lymphocytes from birds infected with the avian acute leukemia viruses, myelocytomatosis virus strain 29 (MC29), avian erythroblastosis virus (AEV), avian myeloblastosis virus (AMV), or reticuloendotheliosis virus (REV-T), was completely suppressed within 1 week after virus infection of chickens by avian acute leukemia virusus. Spleen cells from chickens infected with the chronic leukemia viruses, Rous-associated viruses (RAVs) types 1 and 3, exhibited PHA responses similar to those of uninfected birds. In contrast, lymphocytes from RAV-2 infected birds had suppressed PHA responses. Acute leukemia viruses are replication-defective and are associated with replication-competent nontransforming helper viruses. When chickens were infected with helper viruses isolated from the acute leukemia virus stocks by endpoint dilution, the T-cell response to various T-cell mitogens was completely suppressed. The helper viruses, therefore, contribute to the immunosuppression which occurs during the development of avian acute leukemia. The rapid lethality of the acute leukemia viruses could be, in part, the result of the immunosuppressive activity of the associated helper viruses. Though all the acute leukemia viruses severely immunosuppress their hosts, the mechanism by which the immunosuppression was induced by these viruses differed. REV-T was the only acute leukemia virus which induced a population of splenic suppressor cells. All the viruses of the leukosis-sarcoma complex tested which had subgroup B specificity were immunosuppressive while those of subgroup A were not. These results suggest that immunosuppression by these viruses may be related to subgroup specificity.     

A mutant of influenza virus with a temperature-sensitive defect in the posttranslational processing of the hemagglutinin.

A mutant of fowl plague virus (ts 227) with a temperature-sensitive defect in the hemagglutinin [Scholtissek, C., and Bowles, A. L. (1975). Virology67, 576–587] has been analyzed. At the nonpermissive temperature, the hemagglutinin is synthesized and incorporated into the rough endoplasmic reticulum, but is not transported to the smooth endoplasmic reticulum and to the cell surface. As a result of this defect in migration, proteolytic cleavage of the hemagglutinin does not occur and glycosylation is incomplete, in that mannose and glucosamine are incorporated into the glycoprotein, whereas galactose and fucose are attached only at the permissive temperature. Another step involved in glycosylation, which occurs also only at the permissive temperature, is partial removal of mannose in a trimming process. The data suggest that the hemagglutinin has a structural feature which, after insertion of the molecule into the rough endoplasmic reticulum, promotes its transport to the cell surface. The hemagglutinin incorporated into the rough endoplasmic reticulum at the nonpermissive temperature does not show hemagglutinating activity. After solubilization with detergent, it reacts with antiserum specified against wild-type hemagglutinin. When migration of the hemagglutinin is blocked, synthesis of the neuraminidase proceeds normally, indicating that there is little interdependence in the processing of both envelope glycoproteins.     

Homologies among the nucleotide sequences of the genomes of C-type viruses

DNA was synthesized with detergent-disrupted virions of several C-type viruses and used to measure the extent of homology among the genomes of these viruses by molecular hybridization. The DNA was reacted with viral RNA under conditions which permit saturation of most if not all complementary nucleotide sequences in the RNA. This technique provides a quantitative estimate of the extent of homology among viral RNAs and is superior to current procedures that measure the fraction of DNA hybridized to an excess of viral RNA. The genomes of RD-114 and Crandell virus are at least 85% related, whereas there is no detectable homology among the genomes of RD-114 virus, feline sarcoma-leukemia viruses, murine leukemia virus, avian sarcoma virus, and visna virus. The genome of feline sarcoma-leukemia viruses, like that of RD-114 virus, is at least partly homologous to DNA from normal cats, suggesting that normal cats harbor endogenous genes coding for components of at least two classes of C-type viruses.     

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.     

Sequence of RNA segment 7 of the influenza B virus genome: Partial amino acid homology between the membrane proteins (M1) of Influenza A and B viruses and conservation of a second open reading frame

The complete nucleotide sequence of a cloned full-length DNA copy of genome RNA segment 7 of influenza B/Lee/40 virus has been determined. The messenger RNA (～ 1175 viral nucleotides) for the nonglycosylated membrane protein of the virus (M₁) is capable of coding for a protein of 248 amino acids, of which 63 are conserved between influenza A and B viruses. An internal hydrophobic domain, which includes three cysteine residues in the same positions, is also conserved between the M₁ proteins of the two viruses and may be of functional significance. A second coding region overlaps that of the influenza B M₁ protein by 86 amino acids. This region could code for a maximum of 195 amino acids in the +2 reading frame, depending on whether or not the mRNA is spliced and the location of the initiating methionine residue. This region is likely to code for a protein analogous to the influenza A virus M₂ protein. No obvious homology has been detected in the deduced amino sequences of the M₂ proteins of the two viruses. The size of the M₁ coding regions is conserved between influenza A and B viruses, however the B virus segment 7 is larger, with the difference largely due to a longer M₂ coding region.