- Volume 68, Issue 4, 1987

Using both selection enrichment and site-directed mutagenesis, a herpes simplex virus type 1 (HSV-1) strain 17 genome lacking all four XbaI sites has been generated. The site at 0·45 map units which lies within the gene coding for a polypeptide of 28 000 molecular weight was removed by selection enrichment, while the site at 0·29 map units which lies within the gene coding for glycoprotein H was removed by site-directed mutagenesis. The parental virus from which these two XbaI sites were deleted had previously had the sites at 0·07 and 0·63 map units removed through selection enrichment. The variant devoid of XbaI sites (X4) showed normal growth characteristics; its phenotype was normal apart from the absence of the thymidine kinase protein, which is believed to be unrelated to the loss of XbaI sites.

Mouse thymic virus (MTV) is a naturally occurring herpesvirus of mice which produces persistent infection in salivary glands. No transmission study has been reported previously. In the present work, transmissibility of MTV has been studied by close contact between cage-mates, by the transplacental route from experimentally infected pregnant mice to their foetuses at term or delivered by Caesarean section and by nursing mothers to their sucklings. Transmission of MTV was detected between cage-mates after a long period of contact. The virus was also recovered from newborns nursed by infected mothers inoculated 1 day post-delivery. However, no transmission was detected in the foetuses following infection of mothers at different stages of pregnancy.

Preparations of human peripheral blood dendritic cells have been infected with human immunodeficiency virus (HIV). After 5 days in culture they were examined by electron microscopy. Virus was observed budding from the plasma membrane of dendritic cells and mature virions were observed on the cell surface. In addition, a second cell type, similar in morphology to ‘classical’ dendritic cells but containing numerous cytoplasmic granules, was also found to support replication of the virus. We speculate that the growth of HIV in dendritic cells could cause immunosuppression by impairing antigen presentation.

Infection by a human spumavirus of human foetal diploid lung (HFDL) cells was found to be productive with virus titres ranging from 103 to 105 p.f.u./ml. In contrast, infection of recovered amnion (RA) aneuploid cells resulted in a persistent infection with less than 100 p.f.u./ml infectious virus produced. The decreased sensitivity of RA cells to the spumavirus was not due to the failure of virus to penetrate into the cell since infectious virus was not produced even after transfection of infectious proviral DNA. The effect of 5-azacytidine, an inhibitor of DNA methylation, on virus replication was examined. Whereas virus production in HFDL cells was not affected, there was a 100fold increase in virus yield in RA cells treated with the drug for at least 48 h and maximum virus yields were obtained 4 days post-infection.

Genetically athymic and asplenic (Lasat), athymic (Nude), asplenic (Dh) or normal littermate (Hetero) mice with a BALB/c genetic background were injected either intracerebrally or intraperitoneally with a 1% or 10% homogenate of mouse brains infected with the Fukuoka 1 strain of the Creutzfeldt-Jakob disease (CJD) agent. As there were no significant differences in incubation periods among the five groups (Lasat, Nude, Dh, Hetero and BALB/c) inoculated with the same dilution, via the same route, it was concluded that cell-mediated immunity dependent on the thymus plays no significant role in host defence against the CJD agent, and the spleen, a critical site of agent replication, is apparently not an obligatory source from which infection spreads to the central nervous system.

A coxsackievirus A10 strain, isolated from a clinical specimen from a patient with pharyngitis, was characterized with respect to its growth properties in dilferent cultured cells and at different incubation temperatures. This virus multiplied within cultured cells and produced cytopathogenic effects, whereas a prototype strain of coxsackievirus A10 did not. The isolate multiplied efficiently in cultured cells at 37 °C but its replication was markedly restricted at 32 °C. Temperature shift experiments indicated that the cold-sensitive event affected the late function(s) of the virus.

An earlier suggestion that guanidine may inhibit picornavirus replication by interfering with a monovalent cation-mediated event was tested by determining the effect of varying monovalent cation concentration in isotonic medium on the sensitivity of poliovirus replication in HeLa cells to 0·2 mm-guanidine. Lowering [Na+] in the medium to 50 mm had no effect on virus replication. It was found that the degree of inhibition of virus replication by 0·2 mm-guanidine was inversely related to [Na+] in the medium: 99·8 %, 99·1 %, 38% and 0% inhibition in the presence of 50, 75, 100 and 145 mm-Na+ respectively. Likewise, guanidine uptake by HeLa cells was also inversely related to [Na+] in the medium. On the other hand, lowering medium [Na+] to 50 or 75 mm resulted in reduced intracellular [Na+] and [K+]. The increased sensitivity of virus replication to guanidine in the presence of low Na+ medium could be abolished with excess K+ in such medium. Excess K+ in low Na+ medium restored intracellular [Na+] and reduced guanidine uptake. Thus, the increased sensitivity of poliovirus replication to guanidine in the presence of low Na+ medium correlated with reduced intracellular [Na+] and [K+] and elevated guanidine uptake.

The sequence relatedness of ten isolates of the Colorado tick fever (CTF) serogroup of orbiviruses was examined by RNA-RNA blot hybridization. The 12 dsRNA genome segments of each of the isolates were electrophoresed in a 10% polyacrylamide gel, the segments were transferred electrophoretically to membranes and hybridized to radiolabelled genomic RNA from CTF Florio mouse-adapted strain (CTF FMA) or CTF SS-18. All genome segments of the ten CTF viruses exhibited cross-hybridization signals with either CTF FMA or CTF SS-18, under conditions in which ≥74% sequence homology was required to form stable hybrids. Although the dsRNA polyacrylamide gel profiles were unique for each isolate examined, the CTF genes did not exhibit sequence divergence as has been seen among other Orbivirus serogroups. These hybridization analyses suggest that the CTF gene pool is relatively homogeneous which may be a reflection of the lack of multiple serotypes of CTF strains in neutralization tests. Nevertheless, the hybridization signals of segments 4 and 6 were lighter than those of the other genes, indicating that these two genes exhibited the highest degree of sequence variability among these isolates. These data are compared with hybridization data on other Orbivirus serogroups.

A/Taiwan/1/86 is representative of newly emerged antigenic variants of influenza A(H1N1) viruses which are readily distinguishable from all previous A(H1N1) isolates. Nucleotide sequence analysis of the haemagglutinin HA1 coding region of A/Taiwan/1/86 suggests that this virus has evolved from viruses circulating in the Hong Kong region in 1982 to 1983. The considerable alteration in antigenicity of this new isolate is likely to have arisen from five amino acid substitutions in a nine amino acid stretch, situated on the outer surface of a short oc-helix on the tip of the HA molecule in antigenic site Sb.

Respiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23000 in our gel system). Three novel polypeptides with mol. wt. of 24000, 21000 and 17000 were stained in addition to the 23000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under nonreducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24000, 21000 and 17000 polypeptides were not visible, the number of mobility variants of the 23000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.

Immunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h postinfection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.

Sequences of more than 100 residues adjacent to the 3′-terminal poly(A) tract were determined for each of the two genome RNA species of tomato black ring nepovirus (TBRV) strains A and C; in addition a shorter portion of sequence was determined for RNA-2 of strain G. Comparison of these results and the sequence previously determined for RNA-2 of strain S showed that in this region the sequences of RNA-1 and RNA-2 are almost identical and that considerable sequence homology exists between strains. In contrast, a segment of sequence near the 3′ end of RNA-2 of tobacco ringspot virus, another nepovirus, showed no similarity with the corresponding region of TBRV RNA.

Nicotiana clevelandii plants doubly infected with potato leafroll luteovirus (PLRV) and potato Y potyvirus (PVY) developed more severe symptoms than plants infected with either virus alone. Compared with singly infected plants, the PLRV concentration was increased up to eightfold by the double infection and the proportion of leaf parenchyma protoplasts that could be stained with fluorescent antibody to PLRV was increased from 0·2 to 1·4%. The concentration of PVY was unaffected by the double infection. In potato plants, in contrast, double infection with PVY did not increase PLRV concentration and none of the parenchyma protoplasts obtained from singly or doubly infected leaves could be stained with fluorescent antibody to PLRV. PLRV seems restricted to phloem tissue in potato but also invades a few parenchyma cells in N. clevelandii, a process that is accentuated in plants also infected with PVY.

Comparison of the predicted secondary structures of the coat proteins of the potexviruses potato X and papaya mosaic, on the basis of a combination of prediction methods, shows that although the proteins differ at their amino termini, and possibly at their carboxyl termini, they are similar in the distribution of α-helical segments in the central regions of the protein chains. There is an approximate twofold symmetry in the distribution of predicted α-helical segments between the amino and carboxyl halves of the molecules.