- Volume 69, Issue 8, 1988

We have studied the interactions of synthetic peptides corresponding to the sequence of the amino terminus of the HA2 subunit of influenza virus haemagglutinin with artificial lipid membranes. The peptides could fuse cholesterol-free liposomes at neutral as well as acid pH; however, liposomes containing cholesterol could only be fused below pH 6. The fusion process caused leakage of aqueous liposomal contents. Peptides with amino acid substitutions had fusion properties similar to whole haemagglutinin molecules with the corresponding sequence changes. Non-fusogenic peptides still interacted with the membrane but did not cause leakage of liposomal contents. A correlation between the α-helical content of peptide and its fusogenicity was noted, but this was not absolute. The results reported here support suggestions for a role of the amino terminus of HA2 in virus-endosome fusion.

Pre- and post-embedding immune electron microscopy techniques employing ferritin and large and small gold markers to detect cell surface and intracellular antigens respectively, have been combined in a study of influenza virus-infected cells. This has permitted, for the first time, the simultaneous detection of intracellular virus matrix protein (M), nucleoprotein (NP) and membrane haemagglutinin (HA). The technique facilitated an investigation of the possible physical interrelationship between these three proteins both in the infected cell, and on the infected cell membrane. Electron-dense bodies uniformly labelled by antibody to M protein were observed in the nucleus and cytoplasm. Similarly, NP was detected in both the nucleus and cytoplasm. Approximately 50% of the nuclear NP was located in close proximity to the M protein-containing dense bodies but mainly on the perimeter of the structures. A similar relationship of NP to the M-containing dense bodies was observed in the cytoplasm. M protein and NP were readily detected in sections of budding virions. Labelling of these proteins was also observed on the cytoplasmic face of the plasma membrane but the density of labelling only occasionally approached that of newly formed virions. These findings suggest that budding occurs very quickly after the internal proteins arrive at the plasma membrane. Double labelling experiments on the cell surface indicate that NP and HA behave as independent molecules and do not form tight complexes with each other.

The IgG subclass and IgA responses were investigated in CBA/CaH mice after inoculation with wild-type (wt) and cold-adapted (ca) derivatives of influenza A/Queensland/6/72 virus, and with purified haemagglutinin (H3) derived from the wt strain of the same virus. Intranasal inoculation of the wt and ca viruses resulted in responses dominated by IgG2a in serum, saliva and lung secretions, whereas an intramuscular injection of purified H3 elicited the production of all four IgG subclasses in serum and IgG2b and IgG3 in saliva and lung secretions. The source of IgG on mucosal surfaces was from local production and was not a transudate from serum, as demonstrated by the lack of albumin in saliva and lung secretions, and by the appearance in saliva and lung samples of IgG subclasses not present in serum at the time of sampling. The level of IgA on mucosal surfaces was influenced by the growth restrictions of intranasally inoculated ca virus, resulting in higher levels of IgA in saliva, whereas wt virus, able to replicate at higher temperatures, induced higher levels of IgA in lung secretions. The purified H3 inoculated by the intramuscular route elicited lower levels of IgA in serum, saliva and lung secretions than either the wt or ca viruses.

The hind foot was chosen for study of the mechanism by which adult mice clear lymphocytic choriomeningitis (LCM) virus. The T cell-mediated swelling that follows the local inoculation of virus allows parallel investigation of the infectious process and delayed-type hypersensitivity in one organ. A dose of 105 mouse infectious units (IU) was optimal, and in all mouse strains tested foot swelling commenced 6 days after injection, with maximal response on days 7 and 8. When mice were sensitized by intravenous (i.v.) infection and challenged locally with infectious virus, the extent of swelling depended on both doses of virus and was most extensive when the interval between primary inoculation and local elicitation was 10 days. The rates of replication of the virus and its clearance were similar in the feet of mice of four strains tested, varying with regard to LCM virus-specific cell-mediated immunity. In CBA/J mice, virus elimination from the foot was followed for a longer time period and was incomplete up to 100 days after infection. A protocol for determining adoptive immunization was established; local inoculation of 105 IU was followed 22 h later by i.v. infusion of 1 × 108 unselected or 4 × 107 T cell-enriched cells from the spleens of syngeneic donors that had been infected i.v. 7 or 8 days previously with 103 IU. The concentrations of virus in the recipients’ feet began to decline 2 to 3 days thereafter. Adoptive immunization by local inoculation of immune spleen cells was less successful, apparently because the virus multiplied in transferred cells.

Lymphocytic choriomeningitis virus (LCMV) infection of most tissue culture cell lines results in a non-cytopathic persistent infection. Persistent infections in vitro share many characteristics with persistent LCMV infection of mice; both are associated with decreased titres of infectious virus, restricted accumulation of viral glycoproteins at the surface of infected cells and the generation of interfering particles. We have used gel electrophoresis and hybridization techniques to analyse LCMV gene expression during persistent infection of a number of tissue culture cell lines. Our study has demonstrated that, although deleted viral RNAs can be detected during persistent LCMV infection in vitro, there may not be an obligatory association between deleted RNAs and persistence. In addition, we have found that LCMV interfering activity can be produced in the apparent absence of deleted intracellular viral RNAs.

The genomes of 21 isolates of Murray Valley encephalitis virus (MVE) from Australia and Papua New Guinea were characterized and compared using RNase T1 oligonucleotide fingerprinting. Most Australian isolates grouped in clusters that were linked with a similarity coefficient of greater than 75%, indicating substantial homogeneity. Two isolates grouped as a cluster that linked with other isolates at a level of 67%. These two isolates, one from the north and one from the south-east of Australia were very similar and could demonstrate the movement of MVE between these areas. This notion is substantiated by genetic homogeneity of isolates from the Kimberley region and from south-eastern Australia. One Australian isolate (OR 156) and the Papua New Guinea isolate (MK 6684) were substantially different from each other as well as from the other isolates. No evidence was found for a poly(A) tract in the genome of MVE.

Two monoclonal antibodies (MAbs) with molecular specificities for either the viral envelope glycoprotein (MAb 541) or the non-structural NS1 glycoprotein (MAb 109) were derived using West Nile and yellow fever (YF) viruses respectively. Their antigenic reactivity with a large number of flaviviruses was tested by indirect immunofluorescence microscopy. Both produced cytoplasmic fluorescent staining patterns with the homologous virus against which they were raised. Additionally, MAb 541 reacted with two substrains of YF virus whereas MAb 109 reacted with Bussuquara, YF and Ntaya viruses. These reactions were exclusively cytoplasmic. Two unexpected patterns of fluorescent labelling were observed when the antibodies were tested with Zika and Langat viruses. MAb 541 produced fluorescent staining of the nuclei, but not the cytoplasm, of cells infected with Zika virus and MAb 109 labelled only the nucleoli of cells infected with Langat virus. Double-labelling experiments showed that the nuclear fluorescent label was confined to virus-infected cells, and antibody absorption experiments with virus-infected cell packs confirmed the virus specificity of the nuclear antigen. The unexpected presence of virus-specific antigen in the nuclei or nucleoli of Zika or Langat virus-infected cells brings into question the role of the nucleus in flavivirus replication.

A cDNA copy of the dengue (DEN) 2 virus genome region encoding the virion capsid, membrane and envelope structural proteins has been inserted into vaccinia virus (VV) DNA under the control of its 11K late promoter. The DEN-2 envelope protein was expressed and processed in cells infected with the VV recombinant (VV/D2S). No DEN-2 virus antibody response was detected in mice, hamsters or monkeys vaccinated with VV/D2S. Furthermore, a viraemia was observed in recombinant-vaccinated monkeys after challenge with infectious DEN-2 virus.

The large Bg/II fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

Antigenic properties of 128 clinical type 3 poliovirus isolates of the 1984 to 1985 Finland outbreak from 95 persons and 45 strains from sewage water specimens were evaluated using five neutralizing monoclonal antibodies (MAbs) directed against an antigenic site (designated site 1) on VP1 at amino acids 89 to 100. All five MAbs neutralized the type 3 poliovirus strains used in the vaccines, P3/Saukett and P3/Sabin, but none of them neutralized the prototype strain of the outbreak (P3/Finland/23127/84). Forty-six percent of the clinical isolates resembled the prototype strain (class A) while the rest of the isolates were neutralized by one or more of the MAbs (classes B to D). Although an antigenic drift from A to one of the other classes was observed in sequential specimens from several individuals, no clear-cut overall change in the class distribution was found within the 3 months time span of the outbreak. Homogeneous virus populations were isolated from the sewage specimens using a microtitre endpoint dilution method. The last positive sewage specimens which were obtained in January to February 1985 still had a majority of the class A strain. Some of the clinical isolates were also tested using MAbs directed against distinct antigenic sites. These studies showed that strains that gave the same pattern of reactivity with site 1 MAbs could be differentiated using antibodies directed against other sites. Fifteen strains belonging to different antigenic subclasses were subjected to partial RNA sequencing of the genome region coding for antigenic site 1. The antigenic variation was usually, but not always associated with corresponding amino acid substitutions in antigenic site 1. These results indicate that the antigenic sites of type 3 poliovirus vary extensively within a given outbreak and even during replication in a given host. This variation may have both pathogenetic and epidemiological significance.

Viruses isolated from the blood of two Korean haemorrhagic fever patients were propagated in cell culture and compared to prototype Hantaan virus which was isolated from Apodemus mice. The antigenic properties of the human isolates were found to be closely related to Hantaan virus by plaque reduction neutralization, haemagglutination inhibition and fluorescent antibody staining with both polyclonal and monoclonal antibodies. The medium genome segment of each human isolate was sequenced and compared to that of Hantaan virus. Nucleotides comprising the Hantaan virus G1 and G2 envelope protein-coding regions differed from those of the other viruses by only 5.4% and 5.7%. The human isolates differed from one another by 1.6%. The nucleotide differences resulted in predicted amino acid variations of 1.3% to 2.3% among the three viruses, with the majority occurring as conservative substitutions in G1.

The physicochemical and antigenic properties of three groups of Marburg (MBG) virus isolates, separated temporally and geographically, were compared to each other and to another member of the same family, Ebola (EBO) virus. Each MBG isolate contained seven virion proteins, one of which was a glycosylated surface protein. Peptide mapping of glycoproteins, nucleoproteins (NP) and viral structural protein (VP40) demonstrated extensive sequence conservation in the proteins of viruses isolated over a 13-year period, but homology was not evident in VP24. Some homology between the NPs of MBG and EBO was observed. A close antigenic relationship between MBG strains was found by radioimmunoassay but no evidence was found of antigenic cross-reactivity with EBO viruses. MBG virion proteins are produced from virus-specific monocistronic mRNA species. Five of the seven viral proteins were produced by in vitro translation of these RNAs. MBG virions contained one RNA species with an M r of 4.2 × 106 and virions had a density of 1.14 g/ml in potassium tartrate. Virus isolates from different outbreaks had distinct T1 oligonucleotide maps, but had approximately 95% homology in base sequence. No two geographically distinct virus pairs were more closely related to each other than to a third virus isolate. MBG viruses are thus similar to EBO viruses in morphology and other physicochemical properties and are very similar to each other in RNA and protein composition. Each of the three geographically and temporally distinct MBG virus outbreaks appears to have been due to a genetically distinguishable, but antigenically closely related virus strain. In addition, these studies confirm the belief that MBG and EBO viruses are members of the new virus family, the Filoviridae.

A temperature-sensitive mutant of vesicular stomatitis virus (VSV), tsG31-KS5 VSV, intracerebrally inoculated into BALB/c (+/+) or Swiss outbred mice yielded a clinically asymptomatic persistent infection of the central nervous system (CNS). BALB/c nude (nu/nu) mice infected with tsG31-KS5 VSV, however, all perished within 26 days of infection. All the nude mice were afflicted with a slowly progressing CNS disorder, with symptoms including lethargy, curvature of the spine, hind-limb paralysis and other neurological disorders, before they succumbed to the infection. Wild-type (wt) VSV infection of either normal or nude mice, on the other hand, invoked a rapidly lethal disease with all animals dying within 4 days of infection. When nude mice were reconstituted with 5 × 106 syngeneic T lymphocyte-enriched splenocytes, over 70% of them not only survived the tsG31-KS5 VSV infection but appeared to be free of any neurological disorders. Only 20% of these reconstituted mice infected for 20 days with tsG31-KS5 VSV endured a wt VSV challenge. In contrast, BALB/c (+/+) mice infected for 20 days with tsG31-KS5 VSV all survived a wt VSV challenge. Reconstitution of nude mice with 5 × 106 T lymphocytes did not elicit a vigorous secondary humoral antibody response against VSV. All the animals reconstituted with 5 × 107 T lymphocytes and infected with tsG31-KS5 VSV, however, had both late and early humoral responses that equalled antibody responses of BALB/c (+/+) mice. Reconstitution with either 5 × 106 or 5 × 107 T lymphocytes afforded the nude mice equivalent protection from the CNS disorder triggered by tsG31-KS5 VSV. Reconstitution with 5 × 106 T lymphocytes, therefore, protected nude mice from the neurological disease induced by the persistent virus without eliciting a robust humoral antibody response. Infectious, temperature-sensitive VSV was retrieved from the CNS of the nude mice that had been reconstituted with 5 × 106 T lymphocytes and infected for up to 30 days with tsG31-KS5 VSV. The CNS-isolated VSV was less temperature-sensitive than tsG31-KS5 VSV. When the CNS-isolated VSV was intracerebrally inoculated into Swiss outbred mice, an aggressive disease ensued with most of the mice developing a CNS disorder. In comparison, Swiss outbred mice were asymptomatically infected with tsG31-KS5 VSV. The VSV isolated from the CNS was more lethal to the mice than tsG31-KS5 VSV possibly because it was less temperature-sensitive.

Treatment of Wag/Rij rats with recombinant rat interferon gamma (rRIF-γ) resulted in complete protection against a lethal pseudorabies virus (PRV) infection. To investigate whether the protection resulted from direct inhibition of virus replication or from a stimulation of immune mechanisms, we tested rRIF-γ activity in naturally immunocompromised and artificially immunosuppressed rats. The antiviral effect of rRIF-γ was not abolished in silica- and carrageenan-treated, phagocyte-depleted rats. Immunologically immature newborn and T cell-deficient nude rats were also protected under a regime of rRIF-γ treatment as well as whole body gamma-irradiated rats. Sera of the protected rats were devoid of PRV-neutralizing antibodies. Our results indicate that the protective activity of rRIF-γ is based on direct inhibition of virus replication; stimulation of the immune system is not required but may be responsible for protection upon challenge several weeks after infection.

The role of antibodies as mediators of genetically determined resistance to murine cytomegalovirus (MCMV) in mice has not been elucidated. The ability of mice with different MCMV resistance phenotypes to produce an antibody response to MCMV was investigated in order to assess whether the host genotypes that control resistance also influence antibody production. Antibodies to MCMV in the sera of resistant (BALB.K, CBA/CaH, B10.BR) and susceptible [BALB/c, BALB.B, C57BL/10ScSn (B10), B10.D2, B10.A, A/J] mice were determined by ELISA and/or a complement-requiring neutralization assay. IgM antibodies were produced by all strains of mice as early as 3 to 5 days post-infection (p.i.) with maximum titres observed after 10 days p.i. for some strains, whilst IgG antibodies were produced by 5 to 7 days p.i. with maximum titres at 20 days p.i. IgA antibodies were not detected in the sera of MCMV-infected mice. Virulent MCMV induced higher antibody titres than either attenuated or u.v.-inactivated forms of the virus. Although high doses of virulent virus delayed the early production of IgM antibody they did not adversely affect the kinetics of IgG antibody production. High titres of neutralizing antibodies were detected as early as day 3 post-inoculation of virulent virus; when attenuated virus was used in the neutralization assay, this was found to be more easily neutralized than salivary gland-derived virus. Interestingly, although guinea-pig complement greatly enhanced antibody-mediated neutralization of MCMV, mouse complement was also effective at enhancing neutralization. Although genetically determined resistance to MCMV is an early event with the resistant phenotype being demonstrable during the first few days of the infection, there was no evidence that antibodies were responsible for this resistance since neither antibody titres nor the time of first appearance of antibody correlated with resistance status. However, these results do not exclude a more general role for antibody in limiting MCMV infection, especially in immunity to re-infection since passively transferred antibodies from resistant or susceptible mouse strains lowered virus titres in MCMV-infected animals.

To define the effect of heterogeneity of murine peritoneal macrophages (Mø) on intrinsic resistance to herpes simplex virus (HSV) infection, several Mø populations were characterized for their response to infection with HSV type 1 (HSV-1) and HSV-2. Steady-state resident Mø (Res Mø) were compared in parallel with Mø activated with Corynebacterium parvum (now designated Propionibacterium acnes) (CP Mø) and thioglycollate-elicited inflammatory Mø (TG Mø). Res Mø were completely non-permissive for productive virus infection and showed no c.p.e. The intrinsic resistance of CP Mø to HSV infection was similar to that of Res Mø, in that the infection was non-productive for infectious virus, but CP Mø showed marked c.p.e. TG Mø showed semi-permissiveness, with virus yields at least 10-fold higher than those in Res Mø and CP Mø, and marked c.p.e. The three distinct intrinsic response patterns were maintained regardless of whether Mø were derived from CD-1 or B6C3F1 mice, or whether the infecting virus was HSV-1 or HSV-2. To define the level at which Mø restrict HSV replication, immunofluorescence assays for viral antigens and hybridization analyses for viral DNA were performed. All Mø populations showed immediate early and early virus polypeptides. Res Mø and CP Mø showed no viral DNA replication, but TG Mø showed moderate levels of viral DNA synthesis that paralleled the infectious virus titres produced. Investigation of the mechanism for the heterogeneous intrinsic antiviral response among the Mø revealed that interferon was not involved, because antiserum to mouse α/β interferon did not alter the intrinsic resistance patterns. Induction of c.p.e. in Mø required live, replication-competent HSV. The involvement of tumour necrosis factor (TNF) in c.p.e. was found to be unlikely; no significant amounts of TNF were detected in the culture medium of the Mø, and inclusion of anti-TNF antibody did not inhibit c.p.e.

In spite of much work, the mechanism of oncogenic transformation by herpes simplex virus (HSV) is as yet unknown. It has been proposed that HSV type 2 (HSV-2) can transform cells by a ‘hit and run’ mechanism. In the past we have demonstrated that several polypeptides can be immunoprecipitated from HSV-2-transformed cells, but not from control cells or adenovirus-transformed cells, by rabbit hyperimmune sera to HSV-2. It is possible that the expression of these proteins might be the result of activation of cellular genes during transformation. We have now isolated cDNAs representing transcripts of genes that are expressed at higher levels in HSV-2-transformed hamster embryo fibroblasts than in the parental cells. Cytoplasmic transcripts and genomic sequences homologous to three clones (pAA8, pHD1 and pLC7) were analysed. Northern blot analyses showed that 0.75 kb transcripts which hybridize to the three cDNAs were present in HSV-2-transformed cells and were completely absent or present at low levels in control hamster fibroblasts. These transcripts were not present in mouse cells transformed by other DNA viruses or by a chemical carcinogen. The expression of these transcripts seemed to be confined to certain HSV-2-transformed cell lines. Southern blot analysis suggested that the 0.75 kb transcripts corresponding to these cDNAs may have arisen from a single gene. Nuclear run-off experiments indicated that activation occurred at the level of transcription. The activation of the gene or genes corresponding to these cDNAs may be an integral part of the mechanism of transformation by HSV-2.

We have analysed Epstein-Barr virus (EBV)- and herpes simplex virus (HSV)-infected cells for evidence of antigenic conservation of virus-coded proteins. Immunofluorescence and Western blot analyses of EBV-transformed cell lines demonstrated the presence of proteins that are antigenically related to the HSV alkaline DNase, infected cell-specific protein 34/35, glycoprotein B, thymidine kinase and the major DNA-binding protein. These proteins were characterized on the basis of M r and possible kinetic class.