- Volume 67, Issue 2, 1986

The isolation of a collection of 44 morphologically T1-like phages is described. It is shown that these phages share some similarity with T1 in terms of cross-inactivation with anti-T1 serum, particle proteins and DNA packaging in vitro by the headful process. Virion DNA extracted from these phages was treated with T1 in vitro packaging extracts and the reaction mixtures were tested for the formation of infectious phage particles. The packaging efficiencies observed varied from about 1 to 100% of that of virion T1 DNA. Phage λ virion DNA was packaged with an efficiency of between 0.01 and 2% (5 × 101 to 3 × 103 p.f.u./µg DNA), the shorter deleted derivative λ L47 being packaged more efficiently than normal length λ CI857 DNA. Virion DNA from phages T3 and T7 was also packaged at an efficiency similar to that for λ. The in vitro packaging of T1 DNA requires the presence of the pac sequence which initiates headful packaging from a concatemeric precursor. The high efficiency of packaging DNA from some of the T1-like phages may indicate the presence of similar packaging sequences. However, in the case of λ L47, which is known not to contain such a sequence, the in vitro DNA packaging reaction must occur by a secondary pathway unrelated to the headful mechanism.

The subgenomic RNAs of the fowl coronavirus infectious bronchitis virus (IBV) form a 3′ co-terminal or ‘nested’ set. The presence of non-contiguous (leader) sequences fused to the 5′ termini of murine hepatitis virus mRNAs has been demonstrated using RNase T1 oligonucleotide mapping and sequencing. The presence of a leader sequence on IBV mRNA A has been demonstrated previously. In this paper the presence of a leader identical to that present on the 5′ terminus of IBV mRNA A is demonstrated to be present on the 5′ terminus of IBV genomic RNA. This has been achieved by sequencing of primer extension products and cDNA clones containing the genomic leader. Analysis of these clones has revealed the presence of a sequence at the leader/genome-length RNA junction which is closely related to regions of homology identified previously within the genomic RNA sequence at the leader/body junctions of subgenomic RNAs. The implications of this finding for mechanisms of coronavirus RNA synthesis are discussed.

Monoclonal antibodies to the envelope proteins (E) of the 17D vaccine strain of yellow fever virus (17D YF) and to dengue 2 virus were examined for their ability to confer passive protection against lethal 17D YF encephalitis in mice. All 13 IgG anti-17D YF antibodies, regardless of neutralizing capacity, conferred solid protection when given in a relatively high dose prior to intracerebral inoculation of virus. Three antibodies with high in vitro neutralizing titres were all protective at a low dose as were several non-neutralizing antibodies. One flavivirus group-reactive antibody to dengue 2 virus conferred similar protection at low dose. Protection was also observed when antibodies were given several days after virus inoculation when peak infectious virus titres and histopathological evidence of infection were present in brains. The ability of a non-neutralizing antibody to protect could not be attributed to complement-dependent lysis of virus-infected cells and did not correlate with avidity or with proximity of its binding site to a critical neutralizing epitope of the E protein. Some antibodies, characterized as non-neutralizing by plaque reduction assay on Vero cells, inhibited the growth of virus in a mouse neuroblastoma cell line, suggesting one possible mechanism of protection. These results may be relevant to the design of prospective flavivirus vaccines and support the possibility of conferring broadened protection among flaviviruses by stimulating the antibody response to appropriate epitopes of the E protein.

Antibody to scrapie-associated fibril (SAF) protein was produced in a rabbit by a primary injection of 50 brain equivalents of native SAF protein emulsified in complete Freund's adjuvant and by a secondary inoculation of polyacrylamide gel which contained 50 brain equivalents of SAF protein emulsified with incomplete Freund's adjuvant. The antibody produced was found to decorate the SAF as demonstrated by the use of goat anti-rabbit colloidal gold second antibody. Immunoblots (dot blots and Western blots) revealed that the antibody reacted with scrapie hamster brain extracts but not with age-matched normal hamster brain extracts. However, the antibody also identified an immunoreactive host cellular protein with an apparent mol. wt. of 28000 from unextracted brain homogenates of both uninfected and scrapie-infected hamster and mouse brains up to a dilution of 1/105 brain equivalent. The SAF purification procedures caused selective enrichment of SAF proteins with selective elimination of this immunoreactive cellular antigen. It may be possible that SAFs are composed of a host cellular protein which has been changed in such a way that it aggregates non-covalently to form fibrils and becomes partially resistant to protease digestion.

After intracerebral (i.c.) infection of hamsters, the 263K strain of scrapie replicated at a nearly constant exponential rate until clinical disease developed when titres in brain averaged 9.8 log10 LD50 i.c. units/g. After intraperitoneal infection, scrapie replication was first detected in spleen, then in thoracic spinal cord and finally in lumbar cord and brain. This pattern suggests that invasion of the central nervous system occurs by spread of infection along certain visceral autonomic nerves. Infectivity was detected in the thoracic cord only 3 to 4 weeks after infection (incubation period 16 weeks) indicating the exceptional neuroinvasiveness of this scrapie model. This observation and the failure of splenectomy to lengthen incubation period raises the possibility of direct infection of nerve tissue in the peritoneum and transport to the thoracic cord with minimal prior replication of scrapie agent extraneurally. After intraocular infection of the right eye, replication (or accumulation) of scrapie was detected in the right optic nerve and left superior colliculus, then in the right superior colliculus and finally in the left optic nerve and medulla. This pattern shows that scrapie infection can spread along nerves, possibly by intra-axonal transport. The duration of agent replication in brain (between detectable onset of replication and clinical disease) was shortest after intraperitoneal infection (51 to 58 days), longer after intracerebral infection (81 to 88 days) and longest after intraocular infection (> 121 days). These differences may reflect the relative efficiency of the neural pathways by which infectivity spreads from different sites of entry in the brain to the postulated ‘clinical target areas’.

Seventy-eight influenza A (H3N2) viruses isolated from a single epidemic in a semi-closed community involving 203 clinical cases were characterized using a panel of monoclonal antibodies to virus haemagglutinin (HA). Thirty groups of antigenically distinguishable viruses were detected but the majority (41%) of the viruses belonged to two serological groupings, designated 11 and 17. Viruses in serological group 11 were present throughout the outbreak. The greatest diversity of antigenic variants occurred at the time of the epidemic peak. Antigenic differences among the HAs of the viruses were also detected using polyclonal human and animal antisera. The electrophoretic mobility of virus-induced structural and non-structural polypeptides and of the RNA of viruses of representative serological groups was very similar or identical, suggesting that new introductions of viruses did not occur during the progress of the epidemic. The evolution of influenza A (H3N2) epidemics even in small communities appears to be complex, although a contribution to the observed antigenic microheterogeneity of the HA by spontaneous variants arising in the laboratory cannot be excluded.

A lapinized rinderpest virus, the L strain, which is virulent in rabbits and had been grown only in rabbits, was adapted to grow in Vero cells by the fusion of Vero cells with virus-infected rabbit spleen cells in the presence of polyethylene glycol, and subsequently passaged in Vero cells by co-culture technique. After several passages, free virus was produced at high titre. The Vero cell-adapted virus acquired the ability to infect several cell lines which were non-permissive to the unadapted virus. Analysis of virus proteins by immunofluorescence using monoclonal antibodies revealed that marked changes occurred in F, P, NP and M proteins by passage in Vero cells. In parallel to the adaptation to cell culture in vitro, the virulence of the virus measured in terms of clinical signs and histological lesions in the lymphoid tissues decreased in its severity whereas its immunosuppressive capacity was maintained unaltered. Thus, rinderpest virus with different degrees of virulence is now available for study in vitro.

Antigenic characteristics of nine mumps virus strains were determined by immunofluorescence and radioimmunoprecipitation assay (RIPA) using a collection of 44 monoclonal antibodies. These antibodies were directed against five different structural components of mumps virus, the haemagglutinin-neuraminidase (HN), fusion (F), matrix (M), phospho- (P) and nucleocapsid (NP) proteins. The nine mumps virus strains could be divided into two groups according to their antigenic characteristics. One group included two strains isolated more than a decade ago and the Jeryl Lynn vaccine strain. These three strains reacted with a wider range of monoclonal antibodies than the second group of six recently isolated strains of different geographical origin. In the F, M and P proteins variations were only found in single antigenic determinants. In the HN and NP components, RIPA revealed variations in three and seven determinants respectively. The Jeryl Lynn vaccine strain showed a unique lack of reaction with one anti-HN antibody clone in the RIPA.

Both whole virus particles and isolated VP1 of foot-and-mouth disease virus type O1 induce neutralizing antibodies. Results obtained with pigs vaccinated with either isolated VP1 or intact particles and subsequently challenged show that neutralizing activity induced by intact virus correlates well with protection in pigs, whereas neutralizing activity induced by isolated VP1 confers little or no protection. Further evidence suggests that the epitope responsible for the induction of neutralizing antibodies by VP1 is located at the C-terminal end of the protein between residues 200 and 210.

Barmah Forest virus, an arbovirus, does not cross-react convincingly with alpha-, flavi- or bunyavirus immune sera. Secondary cytotoxic T cells generated in vitro immune to a number of alphaviruses cross-lyse Barmah Forest virus-infected target cells. Flavivirus (West Nile and Kunjin)- and Bunyamwera virus-immune Tc cells lyse homologous virus-infected target cells, but not alphavirus-infected targets. Using cytotoxic T cell assays Barmah Forest virus can be classified as an alphavirus.

The gene encoding the major capsid polypeptide (MCP 48) of frog virus 3 (FV 3) has been mapped on the viral DNA. Late FV 3 messenger RNA, hybrid-selected by the SalI-F fragment or a subset of these sequences, BamHI-L and -W fragments, directed the synthesis in vitro of a 48000 mol. wt. (48K) polypeptide. This product was recognized by monospecific antibodies raised against the major capsid polypeptide. The RNA complementary to these DNA sequences was about 1350 nucleotides in size. This transcript, encoding MCP 48, was precisely located; S1 nuclease analysis indicated that its 5′ end mapped at 1250 nucleotides to the right and its 3′ end at 160 nucleotides to the left of the BamHI site at the junction between the BamHI-W and -L fragments.

Intravenous inoculation of 4-week-old female NIH (inbred) mice with herpes simplex virus type 1 (HSV-1) strain P2C6 (defective in thymidine kinase) produced bilateral hind limb paralysis in nearly all animals by the 5th day after inoculation; very few mice died. In male mice the incidence and severity of paralysis was considerably lower than in females. The parental strain, CL(101), produced similar paralysis but all mice died by day 7. Observations on paralysis and death after intravenous inoculation are given for other strains of HSV-1 and HSV-2. By day 1 after inoculation of P2C6 significant virus replication had occurred in the adrenal glands but in none of the other organs tested. Titres of virus were similar in the adrenal glands of male and female mice. Histology of the adrenals showed most extensive replication in the cortex with some involvement of the medulla, particularly at the corticomedullary junction. By the 2nd and 3rd days, virus was detected in the lower thoracic spinal cord of both male and female animals but clearance was possibly quicker from males. Adrenalectomy proved that virus reached the cord via the adrenals. In the cord the infection was associated with bilateral demyelination in the ventral white matter as early as day 3.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

Immunoaffinity columns were prepared by covalently binding monoclonal antibodies specific for soybean mosaic virus (SMV) to an agarose support matrix. SMV, purified by binding and elution, contained no contaminating proteins detectable by SDS-polyacrylamide gel electrophoresis. Affinity-purified SMV was essentially identical to virus purified by standard procedures in its infectivity, u.v. absorbance spectrum ratio of A 280/A 260, sedimentation coefficient, electrophoretic pattern of coat protein, morphology and antigenicity. This method of purification is rapid, reproducible, and yields highly purified virus preparations. It may therefore be applicable in the purification of a wide variety of plant viruses.

The process by which Sindbis virus induces the fusion of BHK-21 cells in monolayer cultures after exposure to low pH was examined. The fusion process was found to require an exposure to low pH followed by a return to conditions closer to neutral pH. The rate at which cells fused was affected by the pH to which the monolayers were exposed, subsequent to the initial exposure to acid conditions. Virus-mediated cell fusion was not observed if the virus-cell complexes were maintained at the acid pH required for membrane fusion.

Persistent and latent Japanese encephalitis virus (JEV) infection was studied in pregnant and non-pregnant mice. Following intraperitoneal inoculation into pregnant mice JEV persisted for 16 weeks in contrast to 4 weeks in non-pregnant mice. This was followed by a higher frequency of latent infection in pregnant mice. The virus could be reactivated during pregnancy or by cyclophosphamide treatment, the latter being more effective.

Inoculation of mice (strain C3H/He) with a purified preparation of fixed rabies virus led to the production of interferon with two different peaks of activity detectable in the plasma: an early peak 24 h after inoculation followed by another peak on the 7th day after infection. The level of splenic 2–5A synthetase was enhanced in parallel with the pattern of interferon activity. Neutralization of the first peak of interferon activity by anti-mouse α/β interferon globulin blocked the induction of splenic 2–5A synthetase and modified the development of disease. Infected mice given anti-interferon globulin had a significantly shorter morbidity period than normally infected mice. These results suggest that interferon produced early after virus inoculation plays a role in the onset of rabies disease.

Human adenovirus type 5 is shown to cause a limited infection of the cells lining the ventricles of 2- to 3-week-old newborn rats. By the distribution of fluorescent antibody-labelled cells the infection appears to be confined to the ependymal cells. In these same (ependymal) cells autoradiographic labelling of [3H]thymidine-pulsed brains indicates that viral and possibly cellular DNA synthesis is occurring. Studies with the mutants dl 312 and dl 313 indicate that some cellular DNA synthesis is indeed occurring. No DNA synthesis is observed in cells after inoculation of brains with phosphate-buffered saline or infection of brains with Semliki Forest virus. These results suggest that expression of adenovirus early region genes stimulates differentiated (G0) brain cells to enter a cell cycle.

An in vitro latency system for herpes simplex virus type 2 (HSV-2) in cultured cells has been developed. Virus replication was suppressed by infection of human foetal lung cells at the supraoptimal temperature of 42 °C, and, following transfer of such cell cultures to the normal growth temperature of 37 °C, infectious virus was generally undetectable for at least 6 days. HSV-2 was reactivated by intertypic superinfection at 38.5 °C with temperature-sensitive mutants of HSV-1, or with human cytomegalovirus, but not by superinfection with adenovirus types 2 or 5. The HSV-1 mutant tsKsyn, which produces only immediate early polypeptides at 38.5 °C, was as effective as the late mutant tsIsyn, but tsK which had been irradiated with u.v. light to prevent gene expression did not reactivate HSV-2. The efficiency of reactivation was very high, since 15 to 34% of the theoretical input of infectious HSV-2 particles could be retrieved by superinfection with 0.3 p.f.u. of tsKsyn per cell. Reactivation of latent virus was not induced by cell subculture or by other treatments which alter cell metabolism. The system described here may be important for studies on the molecular basis of HSV latency.

A DNA polymerase induced by Oncorhynchus masou virus (OMV) was isolated from cultured salmon cells infected with OMV using sequential ion-exchange column chromatography steps. The properties of the OMV polymerase were compared to those of human cytomegalovirus (HCMV) polymerase and polymerase alpha from cherry salmon (masu salmon, O. masou) testes. OMV polymerase was clearly distinguished from the other polymerases by its optimum temperature for enzyme activity, 25 °C. In an investigation of the effects of phosphonoacetic acid and aphidicolin, OMV polymerase was found to be more resistant to both drugs than HCMV polymerase.