- Volume 70, Issue 6, 1989

B/Singapore/222/79-like influenza viruses isolated from three patients during the winter of 1981 to 1982 and cultured in either embryonated hens' eggs or MDCK cells were studied. Sequence analysis indicated that the haemagglutinin (HA) genes of the six virus preparations contained at least four distinct HA1 sequences which differed by up to six amino acids. Only one pair of viruses had amino acid differences between the egg- and MDCK cell-derived viral subpopulations and this change did not affect a glycosylation site. Mice infected with previously described recombinant vaccinia viruses expressing either the egg- or MDCK cell-derived HA of B/England/222/82 developed neutralizing antibodies against all of the 1982 type B viruses and were protected against intranasal challenge with these viruses. Therefore, in this model system, the minor sequence variation between the HAs of egg- and MDCK cell-derived influenza B/England/222/82 virus had no detectable effect on the induction of cross-protection.

A 2330 nucleotide sequence spanning the 1B (NS2), IC (NS1) and N genes and intergenic regions of human respiratory syncytial virus strain 18537, representing antigenic subgroup B, was determined by sequencing cloned cDNAs of intracellular mRNAs. Comparison with the previously reported sequences for strain A2 of subgroup A showed that 1B, 1C and N were highly conserved at the nucleotide level (78, 78 and 86% identity, respectively) and at the amino acid level (92, 87 and 96% identity, respectively). The gene-start signals were exactly conserved between subgroups, and the gene-end signals contained only a single nucleotide substitution each in 1B and N. In most cases intergenic and non-coding gene sequences that were not part of presumed transcriptive signals were much less well conserved (generally 50 to 71%) than sequences that were part of translational open reading frames (82 to 86%). The nucleotide and deduced amino acid sequences of the N gene and protein of the Long strain of subgroup A were determined by sequencing cDNA clones of intracellular mRNA; the nucleotide sequence (representing all but the first 10 nucleotides of the gene) contained 15 differences from that of the A2 strain, but the deduced amino acid sequences were identical.

The nucleotide sequences of two monoclonal antibody-resistant mutant viruses predict changes from the wild-type in the number of potential glycosylation sites (Asn-X-Thr/Ser) in the mutant haemagglutinin–neuraminidase (HN) glycoproteins of the Beaudette C strain of Newcastle disease virus. The HN glycoproteins of these mutants, F5 and Z18, migrate either slower (F5) or faster (Z18) than that of the wild-type in SDS–PAGE. HN proteins synthesized in chick embryo fibroblasts following infection by either mutant or wild-type virus in the presence of tunicamycin (an inhibitor of glycosylation), comigrate on SDS–PAGE. These results confirm that the HN protein of the mutant virus, F5, has gained a glycosylation site at Asn(323)-Ser-Ser and that the conserved potential glycosylation site at Asn(481)-His-Thr is indeed glycosylated in the HN protein of the wild-type Beaudette C strain of Newcastle disease virus but is lost in that of the mutant virus, Z18.

Glycoprotein gp52 exists within the mature human cytomegalovirus (HCMV) envelope in heterodimeric, disulphide-linked complexes with glycoproteins gp95 and gp130. Biochemical studies involving immunoprecipitations and Western blots have demonstrated that gp52 is the glycoprotein B (gB) homologue of HCMV but that gp95 and gp130 are probably separate gene products. The distribution of this putative gB on extracellular HCMV particles was revealed by high resolution electron microscopy of preparations labelled with a monoclonal antibody, F5, directly coupled to colloidal gold. F5–gold probes, specific for HCMV gp52, bind to the distal end of 12 nm long, slender spikes projecting from virion and dense body envelopes. Labelled spikes were most often present in closely packed, homogeneous clusters and were frequently present on envelope protrusions. The degree of labelling on individual HCMV particles was highly variable. Both the morphology and distribution of HCMV gp52 show strong similarity with that previously reported for the gB of herpes simplex virus. Other morphologically distinct spikes occur in the HCMV envelope but these were not recognized by F5–gold probes.

We expressed the bovine herpesvirus type 1 (BHV-1) glycoproteins, gI and gIII, in bovine cells using a bovine papillomavirus vector. The proteins expressed by these cells had the same M r as the native BHV-1 proteins and monoclonal antibodies detected no differences in their antigenic structure. Cells expressing gI were infected with either BHV-1 or herpes simplex virus type 1 (HSV-1). The number of plaques in gI-expressing cells was similar to that seen with normal fibroblasts infected with BHV-1 or HSV-1. However, BHV-1 or HSV-1 plaques produced in gI-expressing cells were smaller and darker than those seen in normal fibroblasts indicating an interference with cell-to-cell transmission or cellular lysis. Virus growth curves and [35S]methionine labelling of BHV-1-infected gI-expressing cells showed no difference in virus production, virus protein synthesis or cellular protein shutdown when compared to BHV-1-infected normal cells. This led us to conclude that the gI protein may interfere with a cellular protein(s) responsible for the cytopathic effects of BHV-1 infection. Cells expressing gIII were fully susceptible to BHV-1 infection.

Three monoclonal antibodies (MAbs) have been raised against cell membrane-derived herpes simplex virus type 1 glycoprotein C (gC-1). By using different DNA constructs of gC-1 expressed in Escherichia coli the sites recognized by these antibodies could be assigned to a peptide in the more hydrophobic and probably non-glycosylated middle third of the gC-1 molecule. This peptide segment corresponds to a 571 bp segment on the gC-1 gene located between the NcoI and the NruI restriction sites. None of the three MAbs interfered with the binding of the human serum complement component C3b to gC, which has been shown to be rather glycosylation-dependent. Since the data of other groups have suggested that antibodies directed against all regions of gC-1 inhibit C3b binding to gC-1, whereas our results suggest that the central part of gC-1 is not actually involved in C3b-binding activity, it can be inferred that the N- and C-terminal segments are involved in C3b binding by gC-1.

A temperature-sensitive mutant (ts13) of herpes simplex virus type 2 (HSV-2) has a mutation which causes in vitro thermolability of the virion. This mutation lies within the gene encoding a virion structural protein of M r 65K which is known to stimulate immediate early transcription (the trans-inducing factor, 65K tif ). The results presented here show that the structural role of 65K tif is essential. The electrophoretic mobility of the 65K tif encoded by ts13 and a revertant of ts13 differed from that of the wild-type HSV-2 parent. Two monoclonal antibodies directed against 65K tif were shown to react with two different epitopes on this polypeptide, one of which was altered by the mutation in ts13. No differences were observed in the phosphorylation status of 65K tif from mutant- and wild-type-infected cells.

We have screened 78 genital tract tumours from Italian female patients for the presence of human papillomavirus type 16 (HPV-16) and HPV-18 DNA. Dot and Southern blot hybridization experiments revealed that DNA sequences of HPV-16 and/or HPV-18 are present in 66% of tumour samples. HPV-18 was detected in 80% of vulvar carcinomas. In these tumours the integration of HPV-18 DNA seems to occur in the E1/E2 region of the virus genome with long deletions in almost all samples. The invariable retention of the non-coding region and the E6/E7 open reading frames suggests that the integration event and the possible expression of these sequences could play a role in some stages of cellular transformation.

Seventeen independent cell clones were isolated from C127 cells transformed by bovine papillomavirus type 1 (BPV-1). Transformants showed differing degrees of expression of the transformed phenotype as monitored by saturation density, doubling time, growth in medium with a low serum concentration and colony-forming efficiency in soft agar. The degree of expression of the transformed phenotype did not correlate with either the BPV-1 copy number or levels of BPV-1-specific RNA in the transformed cell clones. A characteristic transformed cell clone, T1c, showed the lowest degree of expression of the transformed phenotype but contained the highest copy number of BPV-1 DNA and the highest level of BPV-1-specific mRNA. When we analysed different transformants by two-dimensional gel electrophoresis, we found that a set of six proteins changed quantitatively. Changes in the expression of these proteins were most consistent in clones expressing the greatest number of parameters of transformation, e.g. clone T4a. These data indicate that changes in the expression of cellular genes may correlate with the degree of expression of the transformed phenotype.

We have investigated the influence of anti-rabies vaccination on the onset of the disease as well as the delay of death in foxes previously infected with rabies virus. A live vaccinia recombinant virus expressing the rabies virus glycoprotein (VVTGgRAB) was used as vaccine. Foxes were divided into six experimental groups of four animals. On day 0, each fox was experimentally infected with a rabies virus suspension. VVTGgRAB was administered by the oral route to each animal of three groups on day 0, 3 or 14. Foxes of other groups were used as unvaccinated controls or received, on day 0, the parental Copenhagen strain of vaccinia virus. The length of post-challenge survival and duration of clinical disease were recorded for each animal tested. All foxes, vaccinated or not, died from rabies as confirmed by fluorescent antibody tests. Animals vaccinated on days 0 and 3 died after a shorter period of incubation than unvaccinated controls. On the other hand, animals vaccinated on day 14 post-challenge died later than control animals. Foxes administered vaccinia virus died at the same time as unvaccinated controls. These results demonstrate that early and late death phenomena, as consequences of interactions between oral vaccination with VVTGgRAB and rabies infection, can occur in foxes.

Antibody-dependent enhancement of yellow fever virus neurovirulence, as measured by a reduction in the average survival time of groups of mice, was demonstrated with wild-type or vaccine strains of yellow fever virus and with Japanese encephalitis virus using intraperitoneally administered monoclonal antibodies specific for the viral E glycoprotein of yellow fever virus. Enhancement of virulence could be induced by neutralizing, non-neutralizing or protective antibodies if the virus was allowed to establish a productive infection in the mouse brain before the antibody was administered. The implications of antibody-dependent enhancement in flaviviruses are discussed.

Viroids have been isolated from plum trees (Prunus salicina Lindley) affected with plum dapple fruit disease and from peach trees (Prunus persica Batsch) showing dapple fruit symptoms. The viroids were inoculated mechanically to cucurbitaceous plants, in which symptoms typical of hop stunt viroid (HSV) infection appeared. The complete nucleotide sequences of an isolate from plum and an isolate from peach (AF isolate) were shown to be identical, consisting of 297 nucleotides with a 93·6% sequence homology to HSV-hop. Another isolate from peach (A9 isolate) also consists of 297 nucleotides, but the sequence homology to HSV-hop is 99·7%, showing only one nucleotide replacement. These results indicate that these three viroids are strains of HSV, which we designate HSV-plum, HSV-peach (AF) and HSV-peach (A9), respectively. Comparative analysis of the nucleotide sequences of HSV strains from hop, grapevine, citrus, cucumber, plum and peach revealed variable and conserved regions in the HSV molecule. In Japan, these viroids are closely related not only to dapple fruit disease in plum cv. Taiyo, but also to dapple fruit symptoms on peach cv. Asama-Hakutou.

In mixed infections, the potato spindle tuber viroid (PSTV) does not complement the transport function of the tobacco mosaic virus (TMV) Lsl mutant, which has a temperature-sensitive transport function. This appears to be due to the fundamentally different mechanisms of virus and viroid transport. However, PSTV significantly enhances the accumulation of temperature-resistant TMV in mixed infections at a temperature non-permissive for Lsl.