- Volume 65, Issue 1, 1984

A series of cell lines was constructed by transformation of murine LTK− cells with a family of deletion mutants of the herpes simplex virus (HSV) thymidine kinase (tk) gene. These mutants, differing in the extent of 5′ sequence flanking the coding region for tk, varied in the frequency with which they were able to convert tk− cells to the tk+ phenotype. Converted cell lines were analysed for tk DNA sequences, tk mRNA sequences, the 5′ terminus of tk-specific transcripts and for their ability to respond to a signal provided in trans by infecting tk− virus (transactivation). The results of these analyses reveal that transformation efficiency correlates inversely with the extent of 5′ flanking information. Thus mutants retaining less than 109 bp of 5′ sequences transform less efficiently than those that retain at least 109 bp. Cell lines established by transformation with mutants retaining the proximal 109 bp contain relatively few copies of tk DNA whereas those which arose as a result of transformation with mutant DNA containing less than 109 bp generally contained multiple copies of tk DNA. Analyses of tk-specific transcripts revealed that cell lines derived from plasmids that transformed efficiently synthesized an mRNA which was indistinguishable by its size or 5′ end from infected cell mRNA. Cell lines established by plasmids that were inefficient at transformation accumulated truncated mRNAs that initiated at aberrant start sites. The presence of the 5′ 109 bp block was required for transformants to increase the level of tk mRNA and enzyme when infected with a tk− deletion mutant of HSV. We also show that transactivation does not alter the initiation site of the tk mRNA synthesized by transformants.

The selective and potent anti-herpesvirus drug, (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU), has been examined for its inhibitory effects on several parameters of Epstein—Barr virus (EBV) infection in the lymphoblastoid cell lines Raji, P3HR-1, B-95-8 and P3 hybrid cells (a human embryo oropharyngeal cell line fused with a nasopharyngeal carcinoma cell line). At a dosage of 0.03 to 0.1 mm, BVdU caused a marked inhibition of (i) spontaneous viral capsid antigen (VCA) expression in B-95-8 and P3 hybrid cells, (ii) VCA expression and DNA synthesis in B-95-8 cells induced with croton oil and n-butyrate, (iii) early antigen (EA) expression and DNA synthesis in Raji cells superinfected with EBV, and (iv) VCA expression and DNA synthesis in B-95-8 cells superinfected with EBV. In its inhibitory effects on these various parameters of EBV infection, BVdU appears to be comparable to acyclovir [9-(2-hydroxyethoxymethyl)guanine], another selective anti-herpesvirus drug which has been previously recognized as an effective inhibitor of EBV replication.

Four nucleoside analogues, 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5-methyluracil(FMAU), -5-iodouracil (FIAU), -5-methylcytosine (FMAC) and -5-iodocytosine (FIAC), were studied for their effect on human cytomegalovirus (HCMV) replication in vitro. FMAU, FIAU, FMAC and FIAC showed antiviral activities for four strains of HCMV (Major, Clegg, D550 and Towne) in a plaque reduction asssay, with a dose required for 50% inhibition (ED50) in the range of 0.1 to 0.65 µm. At a concentration of 1 µm-FMAU or -FIAC, the synthesis of five virus-specific late polypeptides of molecular weights 150000, 120000, 67000, 54000 and 27000 was entirely blocked. Quantification of Towne viral DNA synthesis, using complementary RNA-DNA hybridization with a Towne-specific cRNA probe, demonstrated a complete inhibition of HCMV DNA replication at 1 µm of FMAU or FIAC. After the removal of the inhibitors, however, viral DNA synthesis resumed, and infectious virus reappeared, indicating that the inhibition of HCMV replication by these nucleoside analogues was of a virostatic reversible type.

Attachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

An ultrastructural examination of various tissues from CFW/D mice, neonatally injected with Moloney murine leukaemia virus (Mo-MuLV) revealed the presence of type C virus particles budding from the membranes of various bone marrow-derived cells, pancreatic acinar cells, beta cells, and submandibular gland acinar cells. These observations indicate that Mo-MuLV has the potential to replicate in non-lymphoid cell types even when acquired post-partum. In the pancreas, a large accumulation of immature and mature viral particles was observed between the cell membranes of the acinar cells and the basal lamina. A similar accumulation of viral particles was observed in the lumina of the acinus of submandibular glands. The role of murine leukaemia viruses as potential mediators of aberrations of non-haemopoietic tissues and an alternative mode of viral transmission are discussed.

The role of immune mechanisms in the pathogenicity of the M9 mutant of Semliki Forest virus (SFV) has been examined by the use of immune-deficient and immune-suppressed mice. In immune-competent BALB/c and C57BL/6 mice, the lesions in the central nervous system (CNS) were characterized by acute demyelinating meningo-encephalomyelitis. Myelin vacuolation and demyelination were more severe in BALB/c mice than in mice with a C57BL background. The mortality was 12% and 29% respectively. Treatment with cyclophosphamide or sodium aurothiomalate did not greatly alter the type of lesion produced, although mortality was increased. Lesions were less severe in nude (T-lymphocyte-deficient) mice and more severe in beige (natural killer cell-deficient) mice than in most immune-competent mice. Mortality was marginally increased in nude mice but not in beige mice. Demyelination in nude mice was followed rapidly by remyelination. Immune modification did not significantly alter the titres of virus in the brain at 7 days post-infection and infectious virus had been cleared from the brain by 14 days in all cases. Degenerating oligodendrocytes were detected in the CNS of all immune-modified mice examined at day 7. This study therefore suggests that both immune mechanisms and destruction of oligodendrocytes play a role in the production of demyelination by M9.

The antigenic relationships between seven members of the tick-borne encephalitis complex of flaviviruses (group B arboviruses) were examined by raising a library of 16 monoclonal antibodies against one of them and examining their biological and antigenic properties. These clones reacted with only one of two intracellular, virus-specific polypeptides. One polypeptide [mol. wt. 58 × 103 (58K)] is related to the major envelope protein E, but the identity of the other is at present unknown, even though it is a major immunogen in experimental infections and vaccinations. Only those clones specific for the 58K polypeptide contain either neutralizing or haemagglutinin-inhibiting activity, but these epitopes are not identical. In general, epitopes on the 51K polypeptide were more heavily conserved than those on the 58K polypeptide, although both conserved and variant epitopes were found on both polypeptides. One epitope was present on the 51K polypeptide which was conserved on all seven isolates studied and another epitope on the same polypeptide was specific for all the western isolates including one isolate of louping-ill virus. Using the monoclonal antibodies raised in the study, it was shown that louping-ill virus was closely related antigenically to isolates of the Western subtype of tick-borne encephalitis virus.

The growth cycle of sigma virus in Drosophila melanogaster cells was studied: optimal virus production was reached about 40 h after infection; virus release declined thereafter and then remained approximately constant (carrier state). In the presence of DEAE-dextran during virus adsorption, more cells became infected and sigma virus production was enhanced. Sigma virus was partially purified by a gentle procedure. Five presumptive virus-specific proteins with molecular weights 210K, 68K, 57K, 44K and 25K were observed. The p68 polypeptide was glycosylated and formed the spikes of the virion particles. The nucleocapsid contained a single major protein, p44, and one or two minor proteins (p210 and probably p57); another protein, p25, was more loosely associated with the nucleocapsid. None of these proteins was found to be phosphorylated.

If interferon is produced by the gut-associated lymphoid tissue of healthy rabbits under normal conditions and is not all bound in situ, it may spill over and be detectable in lymph. We therefore measured antiviral activity in lymph and plasma simultaneously. We found that antiviral activity was consistently measurable in abdominal and thoracic lymph but not in plasma or in the lymph collected from the hind leg duct. The antiviral activity was neither due to lipoproteins, nor immunoglobulins, nor to cell-produced viral inhibitors other than interferons. Extensive characterization of the antiviral activity indicated that the inhibitor is either rabbit interferon gamma or an acid-labile interferon alpha or a mixture; appropriate antisera will be necessary to resolve this uncertainty. The results support the view of the existence of a physiological interferon response.

Preparations of purified poliovirus type 1, strain Mahoney, and empty viral capsids contain a protein kinase activity. The γ-phosphoryl group of [32P]ATP is transferred to all of the capsid proteins. Viral proteins phosphorylated in vitro are recognized by antiserum directed against isolated viral capsid proteins, indicating that phosphorylation does not alter the antigenic sites to such an extent that the recognition by antibodies is abolished. Viral capsid protein phosphorylation exposes new antigenic sites and leads to a destabilization of the virions. The transfer of 32P to viral proteins is linear for 90 min at 37 °C in the presence of 10 mm-Mg2+ at pH 8.1. Reducing agents or virus-stabilizing agents (such as arildone) reduce the kinase activity and result in a different pattern of capsid protein phosphorylation.

The gp70s isolated from normal mouse tissues by radioimmune precipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were shown to be highly pleomorphic. Their apparent molecular weights calculated from SDS-PAGE ranged from 75000 for thymus gp70 to 65000 for epididymal secretion gp70. Differences in the M r of tissue-associated gp70s were confirmed by double-label co-electrophoresis studies. In addition, a high degree of primary structural pleomorphism among tissue-associated gp70s was demonstrated using two-dimensional tryptic peptide fingerprint analysis. These studies showed that most of the conserved peptides of tissue-associated gp70s were also common to xenotropic murine leukaemia virus (MuLV) gp70s. Thus, tissue-associated gp70s are probably encoded by endogenous xenotropic MuLV env genes or gene fragments. Tissue-associated gp70s also showed a very high level of primary structural pleomorphism. These phenomena were observed for gp70s derived from the tissues of several strains of mice. Tissue-associated gp70 pleomorphism may arise as a consequence of at least two simultaneously operating mechanisms. First, the expression of pleomorphic forms of gp70s on murine tissues may be regulated by mechanisms that also determine the differentiated state of the tissues. Second, endogenous xenotropic env genes may be modified by recombinational or mutational events among these genes, or among cellular genes that regulate the expression of endogenous proviral genes.

Spliced transcripts of influenza A (fowl plague) virus (FPV) RNA (vRNA) segments 7 and 8 accumulate to a much greater extent during non-productive infection of mouse L cells, than they do during productive infection in primary chick embryo fibroblasts (CEF). Virus-specific protein synthesis, or a consequent event in virus replication appears necessary to promote splicing of vRNA segment 8-encoded mRNAs in both cell types, and of vRNA segment 7-encoded mRNAs in CEF. In L cells, however, splicing of the segment 7-encoded mRNAs seems to be independent of such virus-specific control. This observation is discussed in relation to the defect in expression of vRNA 7 which has been observed previously in FPV-infected L cells, and which is thought to account for the failure of virus replication.

A recombinant (R3/3) has been obtained which inherited the gene coding for the haemagglutinin from human influenza virus A/Hong Kong/1/68 (H3N2), and seven other genes from influenza virus A/duck/Ukraine/1/63 (H3N8). The recombinant R3/3 had properties, a ts + phenotype and a capability of reproducing in chick embryos, similar to those of the duck influenza virus, but, in contrast to this parent, had lost its ability to reproduce in organ cultures of colon of ducks and chickens as well as in monolayer cultures of chick embryo fibroblasts.

The small chain of influenza virus haemagglutinin, HA2 was isolated by a selective enzymic removal of HA1 or by preparative SDS-polyacrylamide gel electrophoresis. Anti-HA2 specific antisera and monoclonal antibodies were subtype-specific in immunodiffusion tests and radioimmunoassays. These antibodies did not inhibit haemagglutination or haemolysis, did not prevent virus release, did not neutralize infectivity, and HA2 did not induce a protective immunity. HA2-specific antigenic determinants could not be demonstrated on the surface of infected cells. Lymphocytes from pre-immunized mice could not be stimulated by HA2 to exert a cytotoxic effect.

Three newly established monoclonal hybridoma antibodies to the haemagglutinin molecule of HVJ, designated A7, B3 and F11, recognize operationally non-overlapping antigenic determinants and have neutralizing activity. Using these antibodies, the frequencies of occurrence of neutralization-resistant antigenic variants were analysed in virus populations released from four cell lines persistently infected with HVJ, namely GM2-HVJ, LLCMK2-HVJ, Vero-HVJ and GEsl-HVJ at various passage stages. Antigenic variants were selected from culture fluids of these HVJ carrier cells at a total frequency of 10−3.3, 10−3.8 and 10−3.6 by monoclonal antibodies A7, B3 and F11, respectively. These values were considerably higher than those of 10−4.7 to 10−5.2 detected in a stock preparation of wild-type virus with these antibodies. All the variant viruses isolated as above were negative in neutralization, haemagglutination inhibition and immunofluorescent staining tests with each monoclonal antibody used for their isolation, but were positive with the other antibodies.

Administration of the protease inhibitors, ε-aminocaproic acid or aprotinins, to mice infected with mouse-adapted influenza virus strain A/PR/8/34 (H0N1) and A/Aichi/2/68 (H3N2) reduced virus replication in the lungs. Up to 100-fold reduction of virus titre and virus-induced neuraminidase activity were revealed in mouse lungs under protease inhibitor treatment. As a result, drug-treated mice rapidly cleared the virus from their lungs. The predominant synthesis was of non-infectious virions with uncleaved haemagglutinin in the lungs of drug-treated mice, in contrast to the production of highly infectious virions with proteolytically cleaved haemagglutinin in untreated mice. These observations suggest that protease inhibitors suppress influenza virus replication in mouse lungs due to prevention of haemagglutinin cleavage and virus proteolytic activation.

Monoclonal antibodies to poliovirus type 3 secreted by 51 hybridoma cell clones have been characterized in terms of (i) virus-neutralizing properties, (ii) reactivity in antigen-blocking tests with infectious, 155S (‘D’ antigen) and empty 80S (‘C’ antigen) poliovirus particles and (iii) reactivity in immunoblot tests with the isolated protein components of the poliovirus capsid. The antibodies could be separated into three groups on the basis of their reactivities with ‘D’ and ‘C’ antigens. All antibodies that reacted with both ‘D’ and ‘C’ antigen had potent neutralizing activity. Only a proportion of antibodies that reacted uniquely with ‘D’ antigen possessed neutralizing activity. Unexpectedly, one of 24 ‘C’ antigen-specific antibodies inhibited virus growth. None of the antibodies that possessed virus-neutralizing activity reacted with isolated poliovirus capsid proteins, although the majority of these have been shown in previous studies to be specific for VP1 on intact virus particles. These findings suggest that antigenic determinants involved in virus neutralization do not survive the denaturing conditions required for the isolation of poliovirus capsid proteins and consequently are likely to be specified by the structural conformation of VP1 rather than by amino acid sequence alone. However, several of the antibodies which bound uniquely to ‘C’ antigen reacted in immunoblot tests, five with VP1 and one with VP3. Some of these antibodies also possessed heterotypic reactivity with the corresponding capsid proteins separated from other poliovirus types.

The isolation of monoclonal antibodies directed against the trypsin-sensitive site on the 140S particle of foot-and-mouth disease virus (FMDV) has enabled the demonstration of at least three distinct epitopes within this site. Reaction with two of these resulted in neutralization of virus infectivity. None of the epitopes appeared to be present on the 12S particles, and one of the neutralizing epitopes was sensitive to even milder configurational changes of the particle.

The deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity which is induced in KB cells infected with herpes simplex virus type 2 (HSV-2) and expressed in biochemically transformed HeLa cells was separated from the host dUTPase activity using either gel filtration or chromatofocusing chromatography. The HSV-2-induced dUTPase differed from the host dUTPase activity in its molecular weight (53000 versus 46000) and isoelectric point (pI 5.9 versus 6.4). However, it was similar to the host enzyme in that only dUTP served as a substrate for the enzyme.