- Volume 68, Issue 5, 1987

Biophysical characteristics of Vibrio eltor phage e4, a key phage in the Vibrio cholerae typing scheme were studied. This icosahedral phage was found to contain 12 structural polypeptides with mol. wt. ranging from 25 000 to 120000. One of these polypeptides of mol. wt. 50000 accounted for most of the structural proteins present and was probably the major phage capsid protein. The phage genome comprised a single linear, doublestranded DNA molecule, 69·2 kbp in length (45·6 × 106 mol. wt.) as determined by electron microscopy and restriction fragment analyses. The G + C content was 34·6 %. Electron microscopy data indicated that unlike the DNAs of other cholera phages, phage e4 DNA is not circularly permuted. Adsorption under normal conditions was biphasic with rate constants of 1·02 × 10−9/ml/min up to 60 % adsorption and 3 × 10−10/ml/min thereafter. Intracellular phage multiplication was characterized by a latent period of 27 min. The burst size was approximately 100 phage particles per infected cell.

An in vitro replication system for the Choristoneura murinana nuclear polyhedrosis virus (CmMNPV) was established and used (i) to characterize this baculovirus biochemically; (ii) to study the cytoplasmic spindle-shaped inclusions (CSIs) associated with CmMNPV replication; and (iii) to compare the cytopathic changes during CmMNPV replication in vivo and in vitro as well as the properties of virions, polyhedra and CSIs from both systems. It was shown that the processes occurring during, and the products of, CmMNPV replication in vitro closely resemble those in vivo, i.e. in larval hosts. Genome analysis by restriction endonucleases, as well as infectivity studies with polyhedra from both sources did not reveal major differences between virus produced in vivo and that produced in vitro. The CSIs were found exclusively in the cytoplasm of infected cells and were shown to consist of a single protein of M r 50000. Although the biological significance of these spindles, which are produced in large quantities, is not known, they do not seem to be of importance for the infectivity of this baculovirus in vivo.

Infection of L929 cells by Newcastle disease virus (NDV) did not induce cell fusion, whereas addition of anti-L929 cell antiserum to the culture medium did result in cell fusion. When the antiserum was added to the culture medium of NDV-infected cells at the start of incubation, polykaryocytes appeared after 18 h post-infection (p.i.). On the other hand, when the antiserum was added to the culture medium at 18 h p.i., no polykaryocytes appeared, suggesting that the presence of the antiserum at the stage of NDV replication was necessary for the antiserum-induced cell fusion. Since anti-F polypeptide antiserum inhibited the cell fusion, functional F protein was essential for the antiserum-induced cell fusion. All the anti-L929 cell xenogenic sera tested were able to induce the cell fusion, whereas anti-L929 cell allogenic sera did not induce cell fusion. Anti-MCA cell serum also induced the cell fusion. Little replication of NDV was observed in the presence of anti-L929 cell serum and synthesis of virus-specific polypeptides, especially of M protein, was suppressed. However, there was no difference in expression of virus-specific glycoproteins on the surface membranes between cells treated with normal rabbit serum and those treated with anti-L929 cell serum. On the basis of the above results, the mechanism of induction of cell fusion by anti-host cell serum is discussed.

The protein specificities of the antibodies induced during natural respiratory syncytial (RS) virus infection in humans were investigated using immunoblotting of human sera against virus proteins resolved by gel electrophoresis. Sera from 33 patients, who had complement-fixing antibodies against RS virus, were analysed. All the patients showed a response against the virus nucleoprotein (VPN41) and glycoprotein (VGP48). Antibodies against the virus proteins GP90, VPP32, VPM27 and two proteins of molecular weights 24800 and 23700 were also demonstrated. The spectrum of protein specificities of the antibodies varied among patients. There was an increase in the level of antibody for these virus proteins between the acute and convalescent serum samples from five patients. The protein specificities of the IgG and IgM antibodies were determined for five patients; VPN41, VPP32 and VPM27 induced IgG and IgM in all of these sera. Two of the five patients had detectable IgG but not IgM antibodies against VGP48, whereas the remaining three sera had detectable VGP48, specific IgG and IgM antibodies.

The effect of rabbit anti-rubella virus (RV) serum on the course of RV infection of human lymphoid cells has been examined. The antibody was found to abolish viral replication such that no virus progeny could be detected either extracellularly (after removal of the antiserum) or intracellularly, in the treated cells. Viral protein synthesis was also found to be totally inhibited in the presence of anti-RV but resumed immediately on removal of the antibody block at levels suggesting that viral RNA had been accumulating in the cell. Infectious focus assays indicated that the effect could be explained in part by the restriction by antiviral antibody of cell-to-cell spread in the indicator monolayer. Thus by 48 h when 100 % of lymphoid cells infected with virus alone were capable of forming infectious foci, only 40 to 50 % of cells infected in the presence of antiviral antibody produced foci. However this restriction imposed by antibody did not adequately explain the total inhibition of viral protein synthesis that occurred under these conditions. Pretreatment of RV with excess neutralizing antiserum reduced the virus titre > 99·9 % but did not eliminate infectious virus. The ‘non-neutralized’ fraction (102 to 103 p.f.u./ml) brought about a productive infection with final yields of 10 to 25 % of control titres (> 105 p.f.u./ml). Incubation of virus with both anti-RV and goat anti-rabbit Ig eliminated viral infectivity, indicating that the ‘non-neutralized’ fraction was in the form of infectious immune complexes. The results suggest two major modes of action for anti-RV antibody, one in neutralizing extracellular virus, the second exerted on the infected cells, totally inhibiting viral translation and thus preventing the virus from completing its replicative cycle.

Antigenic interrelationships among paramyxoviruses were examined by immunoprecipitation of isotope-labelled virus-infected cell lysates with specific antisera against virions or virus components. Sendai virus, human parainfluenza virus type 1 (Pa-1) and human parainfluenza virus type 3 (Pa-3) belonged to one antigenic group, and human parainfluenza virus type 2 (Pa-2), human parainfluenza virus type 4 (Pa-4), mumps virus (MuV) and simian virus 5 to a second group. Furthermore, the human paramyxoviruses Pa-1, Pa-2, Pa-3, Pa-4 and MuV formed a single antigenic group which overlapped the above groups. Although Newcastle disease virus (NDV) belonged to a separate group it showed some cross-reaction with the human paramyxoviruses. In particular, certain batches of anti-NDV antisera reacted with the fusion (F) polypeptide of Pa-2 and the reciprocal reaction of anti-Pa-2 antiserum with NDV F was also found. The nucleoprotein showed the broadest cross-reactions among these paramyxoviruses, whereas the matrix polypeptide exhibited antigenic individuality. The nucleoprotein of MuV was most cross-reactive. Pa-2 haemagglutinin- neuraminidase (HN) and anti-MuV HN serum showed cross-reactivity with various antisera and antigens.

Reassortment is an important factor in the evolution of segmented genome viruses.For arthropod-borne viruses it is important to determine whether the vertebrate host acts as a site of reassortant virus formation since vertebrates often act as amplifying hosts. Mutants of Thogoto virus, a tick-borne orthomyxo-like virus, were shown to produce wild-type progeny in a dually infected permissive host (hamster), when hamsters were infected with two mutant viruses either by direct inoculation or by oral transmission from infected ticks. Viral dose and time of co-infection of the host affected the incidence of reassortment. This is the first report of reassortment of an arbovirus following infection of a vertebrate host via an arthropod vector.

Tick-borne encephalitis virus codes for two major immunogenic polypeptides, one of which is the major virion envelope protein E, and the other, NV3, does not have a designated function at present. The intracellular forms of both the E and NV3 polypeptides contain at least four types of sugar residues, i.e. galactose, glucosamine, fucose and mannose. The only glycoprotein in the extracellular virion particles is E. Experiments performed in the presence of tunicamycin have demonstrated that most of these sugars are N-linked. The kinetics of synthesis of E and NV3 have been studied and both show a distinct lag period between initiation of protein synthesis and the appearance of either protein. The kinetics of synthesis of these proteins are consistent with the hypothesis that initiation of protein synthesis starts at the 5′ end of a polycistronic genome but the synthesis of the E and NV3 proteins only occurs after translocation of the polyribosome complex to specific areas in the infected cell. No precursors to either the E or NV3 glycoproteins were detected. Synthesis of both glycoproteins can be detected as early as 6 h after infection and rises to a maximum at 15 h after infection.

Amino terminal sequences of the envelope protein E and the three largest nonstructural proteins NS1, NS3, and NS5 of the New Guinea C strain of dengue virus type 2 (DEN-2) were obtained by nucleotide and protein sequencing. Clones were prepared containing cDNA of DEN-2 virus in the plasmid pUC8. The nucleotide sequences of viral cDNA inserts were determined and the cDNA of each clone positioned on the flavivirus genomic map by comparison of the deduced amino acid sequence with that of yellow fever virus. Radiolabelled E, NS1, NS3 and NS5 were purified by lectin affinity chromatography and preparative gel electrophoresis. Purified proteins were subsequently analysed by Edman degradation to establish the origins of the amino termini of these proteins in the deduced DEN-2 amino acid sequence. Thus the amino acid sequences surrounding the likely proteolytic cleavage sites used in the formation of these four proteins were determined. Of particular interest was the sequence containing the amino terminus of NS3, namely Lys-Lys-Gln- Arg-Ala-Gly where Ala is the first amino acid of NS3. Cleavage following one basic residue in the flavivirus polyprotein has not been reported previously.

Human cytomegalovirus (HCMV) purified from cell culture contains two dominant structural phosphoproteins with apparent molecular weights of 65000 and 150000, designated as pp65 and ppl50 respectively. The humoral immune response of infected individuals against pp65 is relatively weak and is not always detectable by Western blot analyses. This report shows that recent clinical isolates of HCMV do not necessarily have pp65 as a prominent constituent, suggesting that the low immune reaction is due to variable expression of the pp65 in natural infections. However, the HCMV strains tested in this study produced the large structural phosphoprotein (ppl50) in about equal amounts. The ppl50 is remarkably immunogenic, if compared with all other virion constituents; serum pools and individual sera from HCMV-infected patients recognized this particular protein intensively in immunoblot assays. Thus, phosphoprotein ppl50 seems to be the primary polypeptide candidate for expression cloning in order to develop reagents for novel ways of HCMV diagnosis.

We report the isolation of a variant (X2D) of herpes simplex virus type 1 strain 17 which has a deletion of 5 × 106 mol. wt. in the long unique and long inverted repeat regions, such that one copy of the immediate early (IE) gene 1 and two unique open reading frames coding for polypeptides of 20K and 22K are deleted. The mutant X2D synthesizes reduced levels of VmwIE110, and also apparently fails to synthesize VmwIE63, at both the protein and RNA levels, despite there being no apparent deletion in the coding or controlling regions of the IE2 gene. X2D also fails to synthesize the thymidine kinase polypeptide but exhibits normal growth characteristics in tissue culture.

We recently reported the expression of human papillomavirus type 18 (HPV-18) E6 protein in bacteria and the production of anti-E6 polyclonal antibodies. This work has now been extended with the production of a panel of monoclonal antibodies against the HPV-18 E6 protein. These antibodies demonstrate that there is little antigenic conservation in the E6 protein between HPV-16 and HPV-18, with only one antibody recognizing a cross-reactive epitope. We have used both the monoclonal and the polyclonal antibodies to look for E6 expression in a number of HPV DNA-containing cell lines. These reagents specifically detected a 16·5K mol. wt. polypeptide in cells derived from a human cervical carcinoma.

A spontaneous variant of adenovirus (Ad) type 3 (subgroup B) was identified, which did not grow in HeLa cells, but grew in 293 cells with a large plaque morphology. The variant (Ad3var100) had a defect in the the early gene region E1a; it could grow in cells that supplied E1a functions and was complemented for growth in HeLa cells by Ad5 wild-type (subgroup C) but not by the E1a deletion mutant Ad5d1312. It also bore a deletion of some 17·5 kb in the E3 region. The loss of these sequences conferred on the variant the ability to inhibit Ad5 wild-type virus, although Ad3 wild-type was dominant over the variant. No transdominance was seen between wild-type Ad3 and Ad5. The E1a mutation was placed in a wild-type background and this E1a mutant had the host range properties of the variant, but did not retain the large plaque morphology and was not dominant over Ad5. The E3 mutation was separated from the E1a lesion by marker rescue; the resulting E3 mutant retained dominance over Ad5, grew in HeLa cells and had a plaque morphology intermediate between wild-type and variant.

An outbreak of poliomyelitis in Finland resulted in the widespread circulation of wild-type 3 poliovirus strains that had antigenic properties distinct from the strains used to produce the attenuated and inactivated vaccines. Considerable variation was observed in the ability of broadly reacting monoclonal antibodies directed against type 3 poliovirus to neutralize the 54 strains examined. Sequential isolates from several persons showed an antigenic drift with these monoclonal antibodies and selected human sera. In addition, some faecal specimens were found to contain more than one antigenic variant. Primer extension sequencing of genomic RNAs of three plaque- purified antigenic variants isolated from one patient showed base substitutions in the region coding for the major antigenic site of poliovirus type 3. The resulting difference in the amino acid sequence in the virion protein VP1 could explain the differences observed in the neutralization of these strains by the monoclonal antibodies. Whether the observed changes in the antigenic characteristics of the sequential isolates represent true antigenic drift under immunological pressure or whether the emergence of the new variants is based on other modes of selection during replication is not known.

By use of monoclonal antibodies, a virus-specific cytoplasmic antigen related to phosphorylated polypeptides specific to serotype 1 of Marek’s disease virus (MDV)- related viruses (MDV1) has been identified in all MD tumour cell lines examined, as well as in infected cells and in tumour lesions of chickens with MD. At least two phosphorylated polypeptides with mol. wt. 39000 (39K) to 36K and 24K (pp39/36 and pp24, respectively) were identified in the MD tumour cell line H10 cultured at 33 °C by immunoprecipitation with monoclonal antibody M21 which reacts with virus-specific phosphorylated polypeptides. These polypeptides were not detected in cells infected with MDV-related viruses of serotype 2 or 3. Immunoblot analysis indicated that these two polypeptides contained a serotype 1-specific epitope recognized with M21. An additional 41K polypeptide appeared in different virus strains of serotype 1. These polypeptides were found to contain phosphorylated serine but no detectable phosphorylated tyrosine or phosphorylated threonine. Cell fractionation indicated that the two phosphorylated polypeptides were mainly associated with smooth and rough endoplasmic reticulum fractions of cells infected with MDV1. Furthermore, the mRNA coding for pp39/36 could be separated from that coding for pp24 on a sucrose density gradient. These results suggest that pp24 and pp39/36 are translated from distinct mRNAs and encoded from overlapping genes or separate regions with partial DNA homology in the MDV1 genome.

A mouse neuroblastoma cell line was successfully infected with scrapie agent. Agent derived from infected mouse brain or spleen infected cultures. However, agent from infected hamsters did not infect mouse cell cultures, suggesting that species specificity influenced the infection process in vitro. Positive cultures supported scrapie replication for as many as 47 passages in vitro. Agent was shown to be cell-associated and between 631 and 7943 unselected culture cells constituted 1 mouse LD50. However, fluctuation analysis indicated that only one of 144 cells in unselected cultures was actually infected. Thus, agent was confined to a small percentage of cells and only 4·4 to 55·1 positive cells were needed to confer a mouse LD50.

By comparative sequence analysis of the herpes simplex virus type 1 DNA polymerase gene of strain Angelotti and a phosphonoacetic acid-resistant (PAAr) derivative, the site of the PAAr mutation was identified as a single nucleotide (C → T) conversion within the mapping limits of the known PAAr mutations of strains KOS and 17. The conservative amino acid change at residue 719 from alanine to valine results in a radical change in the properties of the polymerase, rendering the mutant enzyme resistant to PAA and various antiviral compounds. Amino acid homologies as well as secondary structure analysis reveal that the PAAr mutation is contained in a 14 amino acid sequence which is highly conserved, and detected in the central domain of prokaryotic and eukaryotic DNA polymerases.