- Volume 72, Issue 3, 1991

The reactivities of eighteen monoclonal antibodies with different glycosylated forms of the human respiratory syncytial (RS) virus G protein were tested in Western blots. Only five antibodies recognized the unglycosylated precursor. The majority of antibodies, however, reacted with the O-glycosylated form of the G protein, emphasizing the importance of this type of modification for the antigenicity of the mature molecule. Human antisera, which recognized the RS virus G protein in Western blots, failed to inhibit the binding of anti-G antibodies to the virus but inhibited the binding of anti-F antibodies in the same type of assay. The human antibodies, however, did not recognize the G protein of some neutralization-resistant mutants selected with one anti-G monoclonal antibody. These mutants contain drastic amino acid sequence changes in the C-terminal end of the G molecule. The results are discussed in terms of the G protein antigenic structure.

The complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5′ end of the mRNA mapped using a modification of the polymerase chain reaction technique. The gene contains a single open reading frame, beginning with a 5′-proximal AUG initiation codon, encoding a polypeptide with a predicted M r of 43141. Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide. The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus. Comparisons with the amino acid sequences of N proteins of other paramyxo-viruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure. These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.

We reported earlier that B95a, an Epstein-Barr virus-transformed marmoset B lymphoblastoid cell line, is more susceptible to infection with measles virus than other cells. The cell line also was found to be susceptible to infection with the lapinized Nakamura III (L) strain of rinderpest virus and various strains derived from it. The B95a cell line was therefore the only host cell system available for the propagation and quantification of the L strain. In contrast to the adaptation of the L strain to Vero cells which results in a diminution of virulence in rabbits, the propagation of the virus in B95a cells preserved the virulence and some other properties in rabbits. Furthermore, when Vero cell-adapted variants of the L strain with diminished virulence were serially passaged in B95a cells, virulence in rabbits was gradually regained.

The complete nucleotide sequences of the neuraminidase (NA) genes of two reassortant (H1N2) and two H3N2 influenza A viruses isolated from pigs were determined and phylogenetic relationships between these and previously reported N2 NA genes were investigated. On the basis of pairwise nucleotide sequence identity, the NA genes of two reassortants, A/sw/Kanagawa/2/78 and A/sw/Ehime/1/80, were most closely related to those of human influenza A virus strains isolated in 1972 and the earliest available swine H3N2 influenza A viruses, respectively. Phylogenetic trees showed that the NA genes can be segregated into three groups, including lineages for (i) swine strains, (ii) the earliest human strain and (iii) recent human strains. The evolutionary tree for the 11 nucleotide and amino acid sequences suggested that the NAs of A/sw/HK/4/76 and A/sw/Kanagawa/2/78 belong to the lineage for recent human viruses. In contrast, the NA genes of the A/sw/HK/3/76 and H1N2 reassortant A/sw/Ehime/1/80 viruses were found to be of a swine lineage. The swine virus NA genes were further characterized by the cocirculation of two distinct lineages. Although the rates of synonymous (silent) substitutions for the swine and human viruses were nearly identical (0.00946 to 0.00884 per site per year), the rate of non-synonymous (amino acid changing) substitutions for swine virus NA genes was about 60% of that for the human virus.

Recombinant vaccinia virus expressing the influenza virus haemagglutinin (HA) or nucleoprotein (NP) genes from A/SW/Hong Kong/1/74 (H1N1) under the control of a hybrid promoter containing the P7.5 early promoter element and promoter of the gene encoding the major protein of cowpox virus A type inclusion body was constructed to investigate protective immunity against homologous and heterologous viruses in mice. These recombinant vaccinia viruses produced authentic influenza virus HA and NP in infected cells. The recombinant vaccinia virus-influenza virus HA conferred efficient subtype-specific protection although mice challenged with heterologous influenza viruses underwent initial infection. By contrast, immunization with the recombinant vaccinia-influenza virus NP limited virus multiplication in the lungs against challenge infection with all H1N1 and H3N2 influenza viruses examined, although less efficiently. These results will prompt the re-examination of the possibility of using the recombinant vaccinia virus-influenza virus NP as a cross-protective vaccine

Fever in influenza results from the release of endogenous pyrogen (EP) following virus-phagocyte interaction and its level correlates with the differing virulence of virus strains. However, the different levels of fever produced in ferrets by intracardial inoculation of EP obtained from the interaction of different virus strains with ferret or human phagocytes did not correlate with the levels of interleukin 1 (IL-1), IL-6 or tumour necrosis factor in the same samples as assayed by conventional in vitro methods. Hence, the EP produced by influenza virus appears to be different to these cytokines.

The prevalence of influenza C virus among pigs in Hyogo Prefecture, Japan, was investigated by serological techniques. Out of 240 sera tested, 45 (19%) showed haemagglutination inhibition (HI) to influenza C virus. Pig sera with high HI titres also scored high in neutralization tests and ELISAs. When fractionated by sucrose density gradient ultracentrifugation, the HI/ELISA reactivities corresponded to antibodies of the IgM and IgG classes. Radioimmunoprecipitation tests revealed that some, but not all, of the pig sera with high HI activities precipitated HEF glycoprotein of influenza C virus. These results suggested that the HI activities of pig sera in Hyogo Prefecture were due to the presence of antibody to influenza C virus. Sera with IgM class antibody to influenza C virus were found throughout the year. However, the question of whether or not pigs serve as a natural reservoir for human influenza C virus still remains to be solved.

Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G+C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.

We report the complete nucleotide sequence of a human immunodeficiency virus type 2 (HIV-2) isolate from Guinea-Bissau (HIV-2CAM2). The genomic organization of HIV-2CAM2 is identical to that of other HIV-2 isolates but contains a stop codon in the pol gene. The deduced amino acid sequences of the viral proteins show variation of 20% in the gag, pol and vpx regions, and 25 to 45% in the tat, env and nef regions when compared to other isolates of HIV-2. This is greater than the variation observed between isolates of HIV-1.

Antigenic and genomic relationships between five pairs of non-cytopathic and cytopathic bovine viral diarrhoea (BVD) viruses isolated from cattle with mucosal disease were examined. Antigenic similarity was evaluated by studying the binding characteristics of 10 monoclonal antibodies (MAbs) directed against the gp53 BVD virus glycoprotein. The MAb binding match between members of the same virus pair ranged from 10/10 to 7/10. The genomic relationship was evaluated by studying the hybridization characteristics of two cDNA probes and six complementary 20 base oligomer probes with virus DNA, selected on the basis of conservation between published BVD virus sequences; cDNA probes were hybridized at two stringencies (60 °C and 45 °C). The match of hybridization results between members of the same virus pair ranged from 4/4 to 1/4 with the cDNA probes and from 6/6 to 3/6 with the oligomer probes. The results indicate that members of the same virus pair can differ at both the antigenic and genomic levels.

The human papillomavirus type 6 (HPV-6) E4 gene was expressed in Escherichia coli as a fusion protein with E. coli β-galactosidase (E4-β-Gal), and rabbit antibody against the E4-β-Gal was prepared. By Western blotting with this antibody, we detected E4 gene products in six out of 18 condyloma acuminata specimens. In four specimens (C-1, C-13, C-14 and C-19), the E4 protein was found as a 10K/11K doublet, but in other specimens (C-8 and C-23), only the 11K protein was detected. By Southern blot analysis, it was found that C-13 harboured HPV-6 DNA but that C-1 and C-8 harboured HPV-11 DNA, indicating that the E4 proteins of HPV-6 and -11 have cross-reactive antigenicity. After incubation at 37 °C of the C-23 tissue specimen, the 10K protein was clearly detected. These results suggest that the 10K protein may be derived from the 11K protein by a modification such as proteolytic cleavage before and/or after specimens were taken.

We have investigated the extent of hepatitis delta virus (HDV) genetic variability after serial passages in chimpanzees and woodchucks and between different human isolates. A complete HDV genome, isolated from a woodchuck liver, was cloned after five serial transmissions. The 1679 nucleotide long genome revealed only point mutations and a nucleotide divergence of 0.65% and 0.89% with previously published sequences of two epidemiologically related HDVs. We have obtained partial nucleotide sequences of two unrelated human HDV cDNAs by using the polymerase chain reaction. When compared to the woodchuck HDV strain and other previously reported isolates, a perfectly conserved region of 90 nucleotides was shown in the region encompassing the delta antigen antigenomic self-cleavage site. In woodchuck and human HDV strains, the two forms of delta protein (195 and 214 amino acids) were potentially expressed. Our study indicates that only a limited genetic variability is generated by several passages in animals despite significant modification of pathogenicity during these transmissions.

The culture of parvovirus B19 in foetal liver tissue has been described recently. We have established the technique in our laboratory and studied parameters affecting the yield of B19 virus. Replication of the virus was detected by radioimmunoassay for B19 antigen and dot blot hybridization assay of B19 DNA, and the virus was localized by immunofluorescence and thin section electron microscopy. B19 DNA and antigen production became detectable at day 2 and reached a maximum at day 5. Virus particles were seen mainly in cell nuclei, but some cytoplasmic membranes were lined with virus particles. The amount of virus produced depended on the age of the foetus and the cell culture and the concentration of erythropoietin and interleukin 3 in the culture medium.

The myristoylation of the foot-and-mouth disease virus (FMDV) capsid precursor P1-2A and its amino-terminal cleavage product 1AB, expressed from subgenomic cDNA, has been analysed. The modification reaction is independent of other FMDV proteins and occurs in both mammalian and insect cells. Blocking of the myristoylation site does not prevent efficient processing of the FMDV capsid precursor. A cDNA cassette in which the leader protease sequence is substituted by an ATG codon produces myristoylated 1AB, indicating correct removal of the novel N-terminal methionine residue.

Electron microscopical examination of negatively stained extracellular fluids (ECF) from Spodoptera frugiperda cell cultures infected with a recombinant baculovirus expressing the Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) revealed NDV-like envelopes which resembled the envelopes of authentic NDV. Immunogold staining with anti-NDV HN monoclonal antibodies demonstrated HN antigen in spikes on the NDV-like envelopes. The ECF from the recombinant-infected cultures also contained baculovirus particles which resembled standard baculovirus particles except that some showed polar protrusions of the envelope. It was concluded that the HN of NDV, in the absence of the matrix protein, might be able to initiate and control the production of viral envelopes which are morphologically identical to those of authentic NDV.

Potyviruses produce cylindrical inclusions (CIs) in the cytoplasm of infected cells. Immunogold labelling and electron microscopy of embedded and sectioned wheat and maize cells doubly infected by different potyviruses revealed no mixing of inclusion proteins in CIs. The viruses were wheat streak mosaic virus (WSMV), agropyron mosaic virus and hordeum mosaic virus, in wheat, and WSMV and maize dwarf mosaic virus in maize. The three viruses in wheat were indistinguishable morphologically and in ultrastructural features but can be separated by serology and host range. The absence of phenotypic mixing in CIs showed that in the presence of CI proteins of other potyviruses, assembly was either highly virus-specific, or that no opportunity existed for CI proteins to assemble into hybrid CIs in mixed infections.

The 3′-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3′ untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3′-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from poty-viruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphidtransmitted potyviruses, it should be considered a member of the potyvirus group.

Nicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low M r RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.