Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions.

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.

Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.

Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem region.

The effects of monoclonal antibody (MAb) C179 recognizing a conformational epitope in the middle of the hemagglutinine (HA) stem region were examined in a mouse model in the experiments of prevention and treatment of lethal bronchopneumonia caused by influenza A virus of H5 subtype. To model the lethal infection, avian nonpathogenic strain A/mallard duck/Pennsylvania/10218/84 (H5N2) was adapted to mice. This resulted in highly pathogenic pneumovirulent mouse-adapted (MA) variant, which was characterized. Three amino acid changes were found in the HA1 subunit of HA of MA virus. One of these was located inside the region of the conformational epitope recognized by MAb C179. However, this substitution was not significant for the recognition of HA and virus neutralization by MAb C179 in vitro and in vivo. Intraperitoneal administration of two different concentrations of MAb C179 one day before or two days after the virus challenge significantly decreased mortality rate. These results suggest that MAb C179 is efficient not only in the prevention and treatment of H1 and H2 influenza virus bronchopneumonia, as was reported previously, but also of H5-induced bronchopneumonia as well, and demonstrate in vivo the existence of a common neutralizing epitope in the HAs of these three subtypes.

Characterization of adaptation of an avian influenza A (H5N2) virus to a mammalian host.

We have used the mouse model to monitor the acquisition of virulence of a non-pathogenic influenza A virus upon adaptation to a new mammalian host. An avian strain, A/Mallard duck/Pennsylvania/10218/84 (H5N2) (Mld/PA/84) was adapted to mice by 23 serial lung-to-lung passages until a highly virulent mouse-adapted (MA) variant (Mld/PA/84-MA) emerged. This MA variant was characterized and compared to the parental strain as well as some of its intermediate passage variants. MA variant caused bronchopneumonia in mice with a high mortality rate (the virulence of Mld/PA/84-MA measured as log (EID50/LD50) was 1.75), while the parental, avirulent strain Mld/PA/84 did not cause illness and mortality in mice (log (EID50/LD50) was 7.25). Hemagglutination-inhibition (HAI) test with a set of hemagglutinin- (HA) specific monoclonal antibodies (MAbs) revealed antigenic differences between the parental strain and MA variant. Mld/PA/84-MA reacted with HA-specific MAbs in higher titers than the parental strain. The HA genes of the parental strain Mld/PA/84, the 1st, 3rd, 8th, and 15th intermediate passage variants, and Mld/PA/84-MA were sequenced. Three amino acid changes at positions 203, 273 and 320 were determined in the HA of MA variant. The first of them, Leu-->Pro (320), appeared in the HA stem region at the 8th passage. Two other in the HA1 globular region (Ser-->Phe (203) and Glu-->Gly (273)) appeared at the 15th passage. All of these substitutions were associated with the increase of viral infectivity for mouse lungs and changes in the HA antigenicity. The potential role of these changes in HA with respect to the process of viral interspecies transmission and acquisition of virulence for new host is discussed.

Cross-protection and reassortment studies with avian H2 influenza viruses.

In order to assess the degree of immune cross-protection among avian H2 influenza virus strains, mice were immunised with beta-propiolactone-inactivated virus preparations and infected intranasally with mouse-adapted variant of A/Black Duck/New Jersey/1580/78 (H2N3) strain. The experiments with 11 avian H2 strains revealed that both Eurasian and American H2 avian influenza viruses exhibit either high or moderate degree of cross-protection. The grouping of the strains in accordance with their cross-protection efficiency does not coincide with H2 phylogenetic branches. Several reassortant clones were obtained with the use of A/Pintail Duck/Primorie/695/76 (H2N3) strain and high-yield X-67 reassortant as parent viruses, among them a high-yield H2N3 reassortant. Taking into account the data on cross-protection among avian H2 strains, the high-yield H2N3 reassortant may be regarded as a prototype strain to be used for the preparation of killed vaccines in the case of a new appearance of avian H2 haemagglutinin in circulation in humans.

Amino acid changes in the hemagglutinin and matrix proteins of influenza a (H2) viruses adapted to mice.

Mouse-adapted (MA) variants of human and avian influenza A (H2) viruses were generated and characterized with respect to acquisition of virulence in mice. From the nucleotide sequence the amino acid sequence was deduced. The HA1 subunit of the hemagglutinin (HA) contained three amino acid substitutions in the A/black duck/New Jersey/1580/78-MA variant (Glu216-->Asp, Lys307-->Arg, and Thr318-->Ile) and two substitutions in the A/JapanxBellamy/57-MA variant (Lys25-->Thr and Ser203-->Phe). In the M1 protein, there were two substitutions in the A/black duck/New Jersey/1580/78-MA variant (Asn30-->Asp and Gln214-->His) and a single substitution in the A/JapanxBellamy/57-MA variant (Met179-->Lys). The M2 protein amino acid sequences of the parental virus and the MA variants differed by a single identical mutation (Asn93-->Ser). The localization and atomic distances of the observed mutations on the three-dimensional (3D) structure of the HA protein were analyzed for influenza H2 viruses. The obtained results were similar to those published earlier on H1, H3 and H5 subtypes. The amino acid changes in the HA protein could be divided into two groups. In one group the substitutions were situated at the top of the molecule, while in the other group they were clustered in the stem area at the interface region between three HA monomers. The analysis revealed that the substitutions observed in the MA variants probably increase the flexibility of the HA molecule and/or weaken the interactions between monomers or subunits in the HA trimer. The relationships of the observed amino acid changes in the HA and M proteins to the biological properties of the respective viruses and possible mechanisms involved in the acquisition of viral virulence are discussed.

An epitope shared by the hemagglutinins of H1, H2, H5, and H6 subtypes of influenza A virus.

The membrane-inserted hemagglutinin (HA) is the most variable protein of influenza viruses. Here we describe the characterization of a shared epitope in the HA of influenza A virus H1, H2, and H5 subtypes which were completely neutralized by a monoclonal antibody (MAb), directed against this epitope. This MAb (C179) also efficiently precipitated the HAs of these viruses. In addition, MAb C179 did not neutralize H6 subtype strains despite complete amino acid homology of the epitope regions. Furthermore, only the non-glycosylated form of the HA of one of the H6 subtype strains could be precipitated by the MAb. The conformational epitope may be masked by glycosylation, although it could not be excluded that differences in the primary amino acid sequence may cause the decreased accessibility of the epitope in H6 subtype strains.

Effects of ultraviolet laser radiation on Venezuelan equine encephalomyelitis virus.

The effects of usual low-intensity continuous (lambda = 254 nm, I = 10 W/m2) UV radiation and high-intensity laser nanosecond (lambda = 266 nm, tau p = 10 ns, I = 10(9) W/m2) or picosecond (lambda = 266 nm, tau p = 23 ps, I = 10(12) W/m2) UV radiation on Venezuelan equine encephalomyelitis virus (a member of the Togaviridae family) were compared. The quantum yields of infectivity inactivation, pyrimidine dimer formation and RNA-protein crosslinking were determined.

Effect of UV-irradiation on rotavirus.

The effect of UV-irradiation on SAll rotavirus infectivity was followed. The time course of infectivity inactivation in general showed an one-hit pattern. Two basic effects of UV-irradiation on virus particles were investigated: the phenomenon of RNA-protein linkages and the formation of uracil dimers. To determine the number of uridine dimers, 3H-uridine labelled purified rotavirus was exposed to UV-irradiation, subsequently the RNA was extracted and analysed by ascending paper chromatography. Formation of photodimers was found to be an important mechanism of rotavirus inactivation at conventional UV-irradiation; the RNA-protein linkages were registered at high irradiation doses only.

Studies on the genetic basis of human influenza A virus adaptation to mice: degrees of virulence of reassortants with defined genetic content.

A highly virulent mouse-adapted variant of influenza virus A/Aichi/2/68 (H3N2) was crossed either with the original A/USSR/90/77 (H1N1) influenza virus strain or with its mouse-adapted, moderately mouse virulent variant. The reassortants were characterized with respect to their genetic content and pneumovirulence for mice. The reassortants fell into three categories: avirulent, highly virulent (resembling in this respect the parent A/Aichi/2/68 virus) and moderately virulent (resembling the mouse-adapted A/USSR/90/77 parent virus). The analysis of the parental origin of the genes of 6 reassortants allowed to suggest that changes in the HA gene and in a polymerase gene (most likely, PB1) were necessary for the acquisition of virulence by the A/USSR/90/77 virus in the course of adaptation to mice, whereas the changes in two other polymerase genes as well as in the genes NA and NS were not involved. The low degree of pathogenicity characteristic of the mouse-adapted A/USSR/90/77 virus was determined by gene(s) other than HA.

Human-avian influenza virus reassortants: effect of reassortment pattern on multi-cycle reproduction in MDCK cells.

Human-avian influenza reassortants possessing the HA gene of the avian parent virus were tested for their ability to replicate in MDCK cells at 37 degrees C and 31 degrees C. Both avian parent viruses, A/Duck/Ukraine/1/63 (H3N8) and A/Duck/Hoshimin/014/78 (H5N3) induced an efficient multi-cycle infection at 37 degrees C, but replicated poorly at 31 degrees C, whereas the human parent virus, MDCK-adapted variant of A/USSR/90/77 (H1N1) strain, replicated efficiently at both temperatures. The reassortant clone possessing the HA gene of A/Duck/Ukraine/1/63 virus and the other 7 genes of A/USSR/90/77 virus replicated at both temperatures almost as efficiently as the human parent virus. Among the reassortants between A/Duck/Hoshimin/014/78 and A/USSR/90/77, the clones possessing the HA and NA genes of the avian strain, or the HA, NA, NP, and NS genes of the avian strain, and the other genes of the human parent virus, replicated poorly at both temperatures, especially at 31 degrees C, whereas the reassortant possessing the HA, NA, and M genes of the avian virus replicated at both temperatures fairly efficiently. The results are discussed in connection with the limitations imposed by different genes upon avian influenza viruses' ability to replicate in mammalian cells.

Protein-RNA interaction in encephalomyocarditis virus as revealed by UV light-induced covalent linkages.

UV irradiation of encephalomyocarditis virus led to an increase in the buoyant density of the virus in CsCl gradients from 1.34 to 1.46 g/cm3. Heat treatment of the irradiated virus (20 min at 54 degrees C) reduced the density to 1.40 g/cm3 and led to the loss of approximately 55% of the labeled RNA from the virions. The non-irradiated virions were converted by such heating into empty capsids. Irradiation also resulted in an increase in the accessibility of RNA inside the virions to the action of pancreatic RNase. An increase in the UV dose did not enlarge the fraction of RNA molecules covalently linked to protein; this was revealed by the lack of any secondary increase in the apparent RNase resistance of the labeled RNA in the irradiated virions. Destruction of the irradiated virus with sodium dodecyl sulfate and 2-mercaptoethanol allowed the isolation of a 40S structure containing viral RNA and RNA-linked proteins. The latter comprised no more than 2.5% of the whole protein content of the virion. Polyacrylamide gel electrophoretic analysis of the RNase-treated 40S structure revealed at least three viral structural proteins in the same ratio as was present in the intact virions.

Infective substructures of Sendai virus from infected Ehrlich ascites tumor cells.

Increase of infectivity for embryonated eggs was observed in Ehrlich ascites tumor cells after intraperitoneal inoculation of Sendai virus into tumor-bearing mice. Virus-induced actinomycin-resistant ribonucleic acid consisting of 14S, 18S, 22S, 35S, and 48S was synthesized, and S antigen was produced in infected cells. The infectivity was suggested to be due to viral ribonucleoprotein for the following reasons: (i) the infectivity was unaffected by V antiserum but was abolished by whole hyperimmune serum, (ii) the infectivity was resistant to ribonuclease, (iii) virus particles were found neither in cells nor on red blood cell stroma treated with cellular extracts, (iv) structures similar to Sendai virus ribonucleoprotein with a maximal length of 10,500 A were observed in cellular extracts.