- Volume 65, Issue 9, 1984

We have characterized a temperature-sensitive mutant of herpes simplex virus type 1 (HSV-1), 17tsVP1207, that induces a thermolabile ribonucleotide reductase activity. This mutant was derived from the multiple mutant tsG. Fine-structure mapping studies showed that the defect in 17tsVP1207 lies within an 800 bp sequence between genome map coordinates 0.580 and 0.585 in the gene encoding a polypeptide of 140000 mol. wt. (Vmw136, ICP6). Since the mutation in this polypeptide produced a temperature-sensitive ribonucleotide reductase activity, Vmw136 must be a component of the herpes simplex virus-induced ribonucleotide reductase. The mRNA of Vmw136 has a common 3′ terminus with an mRNA encoding a 38000 mol. wt. polypeptide (Vmw38). Although the polypeptide-coding sequences of these mRNAs do not overlap, monoclonal antibodies against Vmw136 immunoprecipitated Vmw38 as well as Vmw136 from wild-type HSV-1-infected cells. Our data do not exclude the possibility that Vmw38 is part of the ribonucleotide reductase complex but suggest that this species on its own is not responsible for the HSV-induced enzyme activity.

We have used a DNA competition binding assay to search for herpes simplex virus (HSV) proteins which are able to bind to specific sequences of the genome of HSV. Cloned DNAs from different regions of the virus genome were tested. Two late polypeptides, one major of apparent molecular weight 21000 and one minor of 22000, were preferentially bound by a variety of fragments containing the HSV-1 400 bp a sequence (a direct repeat present at the ends of the molecule and in inverted orientation between the long and short regions of the genome) but not by other competing DNAs including ones containing an origin of replication. We interpret our result as evidence that the HSV type 1-induced 21K and 22K polypeptides interact specifically with DNA sequences within this 400 bp HSV-1 a sequence.

Zosteriform spread of herpes simplex virus (HSV) infection occurs after primary inoculation of the skin of both outbred and inbred mice. With HSV type 1 strain SC16 few outbred animals died if they were inoculated when 8 weeks old whereas up to 50% of animals died if inoculated when 4 weeks old. However, at either age, zosteriform spread of infection occurred in almost all animals as it did when 4-week-old outbred animals were inoculated with the avirulent strain KOS. Thus, control of zosteriform spread must act by different mechanisms from those controlling the encephalitis which leads to death. During replication in the epidermis virus enters axons and could first be found in sensory ganglia 2 days after inoculation of the skin. Thereafter, it was found in the nerve roots and in skin within the same dermatome but remote from the site of inoculation. When sensory nerves to this latter area were cut during the 4 days after primary inoculation lesions developing as a result of zosteriform spread were either completely inhibited or, with later section, decreased in incidence. Mortality was not affected by such nerve section. Latent infection must be established in neurons serving areas of skin remote from the inoculation site since with HSV-1 strain SC16, recrudescent lesions on the pinna could be induced by stripping the skin of the ear when the original inoculation had been in the skin of the neck. Such recrudescent disease was not demonstrated in animals infected with HSV-1 strain KOS even though this virus efficiently established latent infection in sensory ganglia.

Treatment of bovine alveolar macrophages (BAM) with bovine leukocyte interferon (BoIFN-α1) resulted in reduced bovine herpesvirus type 1 replication and spread. This was demonstrated by reduced virus yields and number of cells infected. BoIFN-α1 treatment of BAM also induced enhanced Fc receptor expression and/or avidity by the cells, increased their activity in antibody-dependent cellular cytotoxicity, and augmented their extrinsic antiviral activity as measured by a reduction in the development of plaques in a susceptible cell line. These results are discussed in the context of the possible role of interferon in activation of AM during the early phases of a virus infection.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase α, and l-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and l-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. l-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of l-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase α plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Monoclonal antibodies were prepared from mice and rats immunized with Friend leukaemia virus and BALB/c xenotropic virus. By immunoprecipitation of 125I-labelled and [35S]methionine-labelled viruses and by protein blotting, ten antibodies were found to react with the viral components p12, p15, p30, gp70 and p15E/p12E. A dot-immunobinding assay was found to be a reliable method to type the antibody reactivity with different murine leukaemia viruses (MuLVs). When tested on a panel of ecotropic and xenotropic MuLVs the antibodies revealed the following antigenic specificities: ecotrop-specific on p15E/p12E; xenotrop-specific on p15E; group-specific on p30 and p15E; FM-specific on gp70; FR-specific on gp70 and p15. Of particular interest is a cytotoxic antibody recognizing an FMR determinant localized on p12.

Persistent infection by the retrovirus caprine arthritis-encephalitis virus (CAEV) induces arthritis in goats which closely resembles rheumatoid arthritis. To examine the relationship between virus expression and development of clinical disease, ten goat kids were inoculated with CAEV and examined at successive intervals through 18 months post-infection. Virus was monitored in cell-free synovial fluid cells, serum and peripheral blood cells by titration, co-cultivation and immunofluorescent techniques. Virus was readily recovered from the synovial cavity of all animals during the first 4 weeks of infection, followed by a reduction and fluctuation in virus titres and ability to detect virus. Recovery of CAEV from peripheral blood cells occurred at low frequency while viraemia was rare. Results obtained over a period of 18 months indicate a positive association between virus expression in the synovial cavity and development of clinically detectable disease.

The characteristics of a reovirus (CRV) recently isolated from channel catfish (Ictalurus punctatus) were examined following purification of virions from infected cell cultures. Virions had double capsids, the inner and outer with diameters of 55 nm and 75 nm respectively. Complete virions had a density of 1.36 g/ml in CsCl gradients and contained seven polypeptides of 132000, 130000, 110000, 68000, 56000, 43000 and 32000 mol. wt. The nucleic acid labelled with [5-3H]uridine had a density of 1.56 g/ml in Cs2SO4 gradients, which suggested that it is double-stranded RNA. The genome was composed of 11 segments that ranged in mol. wt. from approximately 0.4 × 106 to 2.5 × 106. Serum cross-neutralization comparisons of CRV to reoviruses isolated from chum salmon (Oncorhynchus keta) and golden shiners (Notemigonus crysoleucas) indicated that each was distinct although some crossreactions were observed. These viruses seem to represent three serotypes of a new genus within the family Reoviridae.

Full-length measles virus RNA molecules isolated from purified virions or nucleocapsids and examined by electron microscopy were 5.12(±0.12) µm in length, corresponding to a molecular weight of 5.2(±0.1) × 106. Purified virions examined by negative staining in the electron microscope exhibited a pleomorphic range of particle sizes varying in diameter between 300 nm and 1000 nm. Purified nucleocapsids had dimensions of 21 nm (diameter) × 1254(±7) nm (length) and a central core of diameter about 5 nm. Full-length nucleocapsids were composed of 204(±3) protein discs. The pitch of the nucleocapsid helix was calculated to be 6.1 nm and the helix angle, α, to be 8° 16′. Approximate volume calculations indicate that each enveloped virus particle contains multiple nucleocapsids.

Peritoneal macrophages were used to analyse host genetic control of mouse hepatitis virus type 4 (MHV-4) infection. Both infectious centre and immunofluorescence assays indicated that only a subset of macrophages from either susceptible (BALB, C3H or C57BL/6J) or resistant (SJL/J) mice were initially infected with MHV-4 during the first cycle of infection. However, compared to macrophages from susceptible mice, three- to sixfold fewer SJL/J macrophages were infected, and there was no amplification of virus replication by involvement of adjacent cells during the second cycle of infection. Treatment of macrophages from susceptible mice with interferon beta could not duplicate the aborted second cycle of infection that occurred in macrophages from resistant mice.

The structural proteins E1, E2a, E2b and C of rubella virus (RV) were purified by preparative SDS-PAGE. The individual proteins were subjected to amino-terminal sequence analysis by Edman degradation, carboxyl-terminal structure analysis by digestion with carboxypeptidases and quantitative amino acid composition analysis. The partial amino-terminal sequences of E2a and E2b were identical and different from that of E1. The C protein did not yield any consistent results on Edman degradation, suggesting that its amino-terminus is blocked. The amino acid compositions of E2a and E2b were very similar and differed from that obtained for E1 and C, which also differed from each other. Carboxypeptidase digestions showed that E2a and E2b have an identical carboxyl-terminal structure, which differed from that of the C protein. No amino acid residues were released from the E1 protein by digestion with a mixture of carboxypeptidases A and B. These results confirm that the structural proteins of RV are translated from three genes corresponding to C, E2 and E1. E2 exists in virions in two post-translationally modified forms, E2a and E2b, which have an identical apoprotein moiety. The partial amino acid sequence information obtained here should also be sufficient to localize the ends of the individual genes on the 24S mRNA genome once its nucleotide sequence has been established.

Neutralizing monoclonal antibodies raised against intact foot-and-mouth disease virus reacted with intact particles and with isolated VP1 from different strains from the same subtype. Prior treatment of the virus with either trypsin or with arginine-specific protease abolished recognition of both the virus and of VP1, suggesting the presence of a neutralization epitope in the central region of VP1 cleaved by these two enzymes. A synthetic peptide analogue of part of this region showed poor reactivity, however, with neutralizing antibody.

A new inactivation process for foot-and-mouth disease virus (FMDV) has been developed. This process is based on the activation of the FMDV endonuclease by incubation of unfractionated viral suspension or purified virions at 37 °C in the presence of high concentrations of monovalent cations such as K+, Cs+ or NH+ 4 at pH 8.5. This procedure completely inactivated several FMDV vaccine strains yielding preparations having similar amounts of 140S particles to untreated controls. The inactivation followed first-order kinetics and the rate of inactivation was faster than that achieved with other agents, e.g. binary ethyleneimine. Testing in suckling mice or tissue culture revealed no residual infectivity after inactivation. Virus particles purified from inactivated preparations showed (i) the same sedimentation coefficient as non-inactivated preparations, (ii) electrophoretic patterns of their viral capsid proteins identical to those derived from non-inactivated preparations, and (iii) extensive degradation of the 35S viral RNA. This method is safer than inactivation with aziridines because only innocuous chemicals are used in the process.

We have investigated the sensitivity of adenovirus type 2 naked DNA and chromatin at 5 h and 20 h after infection to digestion by DNase I, micrococcal nuclease and endogenous nuclease between map coordinates 11.3 and 18.0 (SmaI-F fragment) using a terminal labelling method. Infected cell nuclei were gently digested with nucleases, DNA was extracted and digested to completion with SmaI and the fragments shorter than the SmaI-F fragment mapped by hybridization with a 708 base pair probe co-terminal with the SmaI-F fragment. Early chromatin contained hypersensitive sites at 16.0 and 14.3. These sites became minor cleavage sites in late chromatin and new hypersensitive sites appeared at 13.5 and 13.0. The change in the location of the hypersensitive sites in the course of infection correlated with the early to late switch in the transcription pattern in this region and the early to late change in the overall structure of adenovirus chromatin.

Infection with mouse hepatitis virus type 3 (MHV 3) of primary cultures of Kupffer and endothelial cells from the livers of resistant (A/J) and susceptible (BALB/c) mice was followed by the appearance of typical syncytia and comparable yields of virus. Using cells from A/J mice there was a delay of about 24 to 36 h in the appearance of the first particles detected by electron microscopy, the maximum viral titre and the number and size of syncytia. The partial resistance to MHV 3 multiplication expressed by cells from A/J mice was also observed when they were infected with a strain of low virulence (JHM strain). The delay in MHV multiplication found in the sinusoidal liver cells, mainly in the endothelial cells, may be an important factor in their resistance, by allowing time for the local and systemic responses to clear the infective particles as well as in determining their degree of hepatotropism.

Infection of baby hamster fibroblasts with pseudorabies virus at high multiplicities resulted in a substantial increase in the phosphorylation of ribosomal protein S6. However, the phosphorylation was still observed with virus that had been completely inactivated by u.v. irradiation. We therefore conclude that expression of the viral genome is not required for the virus to elicit this effect.

Purified herpes simplex virus thymidine kinase has been used to immunize mice for the production of monoclonal antibodies to the enzyme. Monoclonal antibodies were successfully produced against both herpes simplex virus type 1 and type 2 enzymes. These antibodies should prove useful for detecting the enzyme under a variety of experimental conditions. We also demonstrate that the antibodies can provide an alternative method for obtaining large amounts of purified thymidine kinase.

Two phosphorylated and two non-phosphorylated variants of P protein isolated from Newcastle disease virions are known. Here, a fifth form of P was identified using two-dimensional polyacrylamide gel electrophoresis and peptide mapping. P form 5 was phosphorylated; however, unlike the four known variants of P, the new form was not a major protein in virions, which suggested an intracellular function. The subunit composition of four electrophoretically distinct, disulphide-linked multimers of P from virions was determined. Each homomultimer was composed of at least three molecules of a different one of the four virion-associated P variants.