- Volume 72, Issue 11, 1991

Detailed analysis of the selection process in serial co-infections of cell cultures by wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) strain E2 (AcNPV/E2) and Ac360-β-gal, a genetically engineered strain, shows that the unaltered strain was clearly dominant even when it initially constituted the minority component in the inoculum. A method of calculating a selection coefficient that quantifies the relative advantage of one strain of virus over the other under specific culture conditions is described. Calculated selection coefficients were relatively homogeneous and almost exclusively favoured the progenitor. Selection pressure was not influenced by the relative proportions of the two strains in the population. Selection coefficients, as determined in the present study, may be useful for evaluating the effect of a genetic alteration on viral fitness under specified conditions. Unexpected high frequencies of mixed phenotype plaques were observed during infectivity titrations of media from early serial passages of co-infected cultures. Statistical evaluation implicates some non-heritable combinational phenomenon. Virus plated from mixed phenotype plaques show high segregation of phenotypes implying that genetic recombination does not contribute in a major way to the high mixed phenotype frequencies. Electron microscopic examination of virion pellets from infected 72 h cell culture media similarly argue against co-envelopment as a major contributory factor to the high frequency of mixed phenotype plaques. The cause remains undetermined.

To determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 °C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 °C, the mutant virus produced severalfold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts+ revertants of ICRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 °C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the down-regulation of the three polymerase genes and the up-regulation of the HA gene during secondary transcription.

We used the polymerase chain reaction to amplify the HA1 coding region of influenza A (H1N1) viruses present in clinical material from recent cases of influenza in the U.K. Previously, we have demonstrated that isolation of human influenza viruses in embryonated hens’ eggs selects variants which have amino acid substitutions in their haemagglutinin (HA) clustering around the receptor-binding site. Such egg-selected variants are often antigenically distinct from each other and from corresponding viruses isolated on mammalian cells. Since in general the virus used for vaccine production is an egg-adapted virus, it is important to determine the extent to which these variants are present in the natural virus which causes disease in man. To achieve this, amplified products from clinical material were cloned and many individual clones sequenced. Our results indicate that the HA of the naturally occurring virus is relatively homogeneous and represented by virus isolated in the laboratory on MDCK cells, whereas the variants isolated in eggs are present only at low levels in clinical material.

Processing of the measles virus haemagglutinin (H) protein was analysed by the pulse-chase method, immunoprecipitation with an anti-H monoclonal antibody and SDS-polyacrylamide gel electrophoresis, combined with the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or monensin (inhibitors of intracellular processing of secretory proteins) to cultures and digestion of the protein with endoglycosidase H or neuraminidase. The apparent M r of the H protein was increased from 74K to 78K during the chase period. Addition of either CCCP or monensin to the chase medium inhibited the appearance of the 78K H protein, but not the immunoreactivity of the H protein or dimer formation, suggesting that these two events occur in the rough endoplasmic reticulum. The 74K H protein processed in the presence of CCCP was fully sensitive to endoglycosidase H digestion, whereas the 74K H protein processed in the presence of monensin was partially resistant to endoglycosidase H. In experiments using 3H-labelled sugars, [3H]galactose was incorporated into the 74K H protein in the presence of monensin. Neuraminidase treatment increased the electrophoretic mobility of the 78K H protein to 74K. Only the 78K H protein was detected on the surface of untreated cells, and it was resistant to endoglycosidase H digestion. These data suggest that after galactose addition sialic acid is added to the H protein in the trans-Golgi complex and then the mature 78K H protein is transported to the cell surface.

We have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV-1) by liposomes composed of the cationic lipid N-[2, 3-(dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4− K562 and Raji cell lines.

The complete nucleotide sequence of a hepatitis C virus derived from plasma of a human carrier in Japan was determined. The cDNA of the isolate (HC-J6) contained 9481 nucleotides and an additional T stretch of 30 to 108 nucleotides at the 3′ end, and had one large open reading frame coding for a polyprotein of 3033 amino acids. It differed by 31.8 to 32.1% in the nucleotide sequence and by 27.4 to 27.7% in the amino acid sequence from an American isolate and two Japanese isolates previously reported. Among these four isolates, the 5′ non-coding region of 329 to 341 nucleotides was well conserved (>93% identity), whereas the 3′ non-coding region of 39 to 45 nucleotides (T stretches not included) was more variable (>30% identity). An excellent degree of conservation of the 5′ non-coding region would reflect its pivotal role in replication, and primers deduced from this region could be applied for the sensitive and specific detection of viral RNA by polymerase chain reaction. Due to a high degree of similarity in the amino acid sequence of the putative core protein (>90%), antigen probes deduced from it would be suitable for the serological diagnosis of HCV infection. Low sequence similarity in the putative envelope protein (>53% identity), however, would have to be taken into account in considering the immunoprophylaxis of HCV infection.

Defective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmunoprecipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 °C and empty capsids at 37 °C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.

The 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is only 16 amino acids in length. During synthesis of the FMDV polyprotein a primary proteolytic processing event occurs between the 2A and 2B regions of the polyprotein. The activity responsible for this cleavage is not known but it is thought that either an unidentified virus-encoded proteinase may be responsible, or that 2A acts as a substrate for a host cell proteinase. A series of recombinant FMDV polyproteins has been constructed in which sequences to the N- or C-terminal side of the 2A region have been deleted. Analysis of the processing of these polyproteins shows that a 19 amino acid sequence spanning 2A is sufficient to mediate polyprotein cleavage at a site immediately C-terminal to 2A, whereas deletions extending into the 2A region prevent cleavage.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

Twenty commercial batches of calf serum, obtained from several suppliers, were tested for the presence of bovine polyomavirus (BPyV) DNA and antibodies against the virus. Using polymerase chain reaction (PCR) technology, BPyV DNA was detected in 70% of the batches; no BPyV was detected in any of the negative control samples. The specificity of the amplification reactions was proven by hybridization. PCR results were confirmed by virus isolation experiments performed with five PCR-positive and five PCR-negative serum batches. The results indicate that the use of calf serum to supplement tissue culture media involves a serious risk of contaminating cell cultures with BPyV. No correlation was observed between the presence or absence of anti-BPyV immunoglobulins and the detection of BPyV-specific DNA sequences in the serum batches.

The Epstein—Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.

Human cytomegalovirus (HCMV) purified from urine or tissue culture supernatant has been reported to contain β2-microglobulin (β2m), which forms the light chain of HLA class I molecules. It has been postulated that HCMV covered with β2m binds to HLA class I α-chains at the cell surface. In the present study we used transfected human and mouse cell lines expressing distinct allelic forms of HLA class I and β2m to determine whether HLA class I molecules could act as cellular receptors for HCMV. The susceptibility of cells to HCMV infection was estimated by calculating the percentage of cells expressing HCMV immediate early antigens. Although the results showed some variation between different transfected cell clones, no correlation was found between expression of HLA class I on the cell membrane and HCMV infection. Preincubation of HLA class I-positive cells with antibodies against HLA class I antigens inhibited HCMV infection after binding and adsorption of HCMV. Trypsin prevented HCMV infection of both class I-positive and class I-negative cells. We conclude that these results do not support the assumption that HLA class I molecules are functional receptors for HCMV.

Human cytomegalovirus (HCMV) induction of growth factors in fibroblasts was investigated after establishing serum-free culture conditions conducive to viral replication. HCMV infection induced a type II heparin-binding growth factor which stimulated human endothelial cell proliferation.

VP13 and VP14, major tegument proteins of herpes simplex virus type 1 (HSV-1) and the products of the UL47 gene, have been shown by partial proteolytic mapping to have closely related protein sequences. These proteins are phosphorylated in virus-infected cells, but not in preparations of purified virus. They also contain O-linked oligosaccharide units which include β-1,4-N-acetyl galactosamine residues, as demonstrated by the binding of Dolichos biflorus lectin. This modification was detected only in purified virus and appears to be restricted to VP13/14 and VP22, another HSV-1 tegument protein.

We have characterized a spontaneous variant of herpes simplex virus type 2 (HSV-2) strain HG52, 2616, which has 786 bp of both copies of RL deleted, but which retains 782 bp upstream of the 5′ end of immediate early gene 1 and 463 bp downstream of the ‘a’ sequence. Variant 2616 is avirulent following intracerebral inoculation of BALB/c mice, thus showing in vivo characteristics similar to those of variant 2604, described previously (a 1488 bp deletion incorporating the DR1 element of the ‘a’ sequence), but not to those of variant 2701, which has an intermediate neurovirulence phenotype. The deletions in variants 2616 and 2604 remove the conserved HSV-1 and -2 RL open reading frame entirely and extend downstream from it, whereas the deletion in variant 2701 removes only the 5′ part. The data show that deletion of part of the ‘a’ sequence is not required for the production of the RL avirulence phenotype in HSV-2 strain HG52.

Conserved amino acid sequences within the L1 open reading frame of the human papillomavirus (HPV) genome were used as a basis to design two degenerate primers (GP17 and GP18) and one general probe (GPR22) which direct polymerase chain reaction (PCR) amplification and subsequent detection of a 620 to 660 bp DNA fragment. The conserved nature of the primers and probe was tested experimentally on a panel of 24 cloned HPV DNAs isolated from cutaneous and mucosal lesions, including HPV-2a and -57, which are known to be associated with lesions at both anatomical sites. The sensitivity of this PCR test was at the level of genomic Southern blot analysis, indicating that HPV infections producing high copy numbers can be detected. Positive results were obtained with DNA extracted from clinical samples of genital and cutaneous origin.

The skin of animals of a laboratory strain of Mastomys natalensis carrying endogenous, latent papillomavirus genomes was irritated by scratching with glasspaper. Hyperproliferation of the epidermis and amplification of viral DNA followed this treatment, and in approximately 27% of the animals virus-producing papillomas were induced.