- Volume 72, Issue 7, 1991

Neutralizing antibodies against 10 serotypes of rotavirus were measured in sera from different age groups of German cattle. Only five of 143 sera did not neutralize heterologous serotypes. Sera from 64 of 76 calves younger than 1 year neutralized bovine rotavirus NCDV (serotype 6). From these calves, sera 54, 26, 51, 24, 12, 10 and 37, in neutralized addition, the heterologous serotypes 1, 2, 3, 4, 5, 7 and 9, respectively. Thirty-eight of 46 rotavirus isolates from Bavarian calves with diarrhoea were serotyped by neutralization: 22, 2 and 14 isolates were typed as serotype 6, serotype 10 (B223) and a newly defined subtype of serotype 10 (V1005), respectively. All serotype 6 isolates and none of the serotype 10 or V1005-like viruses tested hybridized to a NCDV-specific cDNA probe. Eight isolates gave equivocal results by neutralization. We failed however to identify serotype 1, 2, 3, 4 or 8 bovine rotavirus isolates by neutralization with hyperimmune sera and dot blot hybridization with serotype-specific cDNA probes. Thus cross-reacting antibodies in cattle might not represent an anamnestic response, but the recognition of a cross-reacting neutralization epitope shared by many rotavirus serotypes.

Cytogenetic abnormalities associated with human papillomavirus (HPV) type 16 were studied using primary human and mouse epidermal keratinocytes. The E7 transforming gene of HPV-16 was found to induce chromosome duplication in epidermal keratinocytes; little or no detectable chromosome disorganization was associated with the function of the E6 gene. These results suggest that the E7 gene-linked cytogenetic effect reflects HPV-16-associated pathogenicity in the early phase of transformation.

Epstein—Barr virus (EBV) isolates can be broadly classified as type 1 or type 2 on the basis of allelic polymorphism of the virus-encoded nuclear antigens EBNAs 2, 3a, 3b and 3c, and individually identified based on M r values of their EBNA proteins (EBNA type). Here we have used this natural heterogeneity amongst isolates to re-examine the question of EBV persistence in vivo, asking in particular whether virus carriage in oropharyngeal epithelium and/or in B lymphoid tissues involves infection with a single or with multiple virus strains. Firstly, 76 healthy virus carriers were classified into serotype groups on the basis of preferential antibody reactivity to type 1 EBNAs (serotype 1) or to type 2 EBNAs (serotype 2); 60 of the 76 donors were serotype 1, four of the 76 donors were serotype 2 and 12 of the 76 donors were anti-EBNA 2, 3a, 3b, 3c antibody-negative and therefore could not be serotyped. Representative donors from each group were then selected for virus isolations from blood (by spontaneous in vitro transformation) and from throat washings (by cord blood cell transformation). All 13 serotype 1 donors tested and six of seven non-serotypeable donors gave a type 1 virus isolate, whereas all four serotype 2 donors and one of the seven non-serotypeable donors gave a type 2 isolate. Multiple transforming virus isolates from any one donor, whether from blood or throat washings, were all of the one strain characteristic of that particular donor; sequential isolations showed retention of the same strain over several years. Finally, throat washing samples from these same donors were examined for amplifiable EBV DNA in the polymerase chain reaction using EBV type-specific oligonucleotide primers and probes derived from the polymorphic EBNA 2 and EBNA 3c loci. The results were consistent with earlier virus isolation studies, each individual donor showing amplification either of type 1 or type 2 sequences. We conclude that multiple EBV infections must occur rarely, if at all, in healthy virus carriers; EBV persistence in vivo is characterized by dominance of a single transforming virus strain.

We have shown previously that the EBNA 1 and latent membrane protein encoding regions of the Epstein—Barr virus (EBV) genome are highly methylated at CCGG sequences in the Burkitt's lymphoma (BL)-derived cell line Rael, but are unmethylated in a lymphoblastoid cell line (LCL) harbouring the same virus. To examine whether this is a regular phenomenon, we compared the methylation patterns of selected regions (BamHI C, W, H, M, E, K and N fragments) of EBV DNA in representative EBV-carrying cell types of normal and neoplastic origin. Analysis of HpaII and MspI cleavage patterns showed that all probed regions were highly methylated in all six BL biopsy samples, but hypomethylated in the four LCLs immortalized by the virus. EBV DNA was also highly methylated in the nude mouse-passaged C15 nasopharyngeal carcinoma strain and partially methylated in the C18 strain. Eight BL lines propagated in vitro, ranging from a typical BL group I to a more LCL-like group III phenotype, showed heterogeneous levels of methylation. Rael, the only stable group I cell line, carried highly methylated viral genomes. The other cell lines, which have drifted to an LCL-like blastic phenotype to various degrees, showed more moderate or low viral DNA methylation. Two sublines of the BL cell line Jijoye, which could be classified as groups II and III, respectively, showed a corresponding difference in EBV DNA methylation. To assess the possible influence of hypomethylated linear EBV DNA molecules produced in lytically infected cells, the virus producer P3HR-1 and Jijoye M13 lines were compared for DNA methylation before and after treatment with phosphonoformic acid (PFA), an inhibitor of the viral DNA polymerase. PFA treatment resulted in a shift towards a more methylated pattern in both regions (BamHI W and E) assayed, but had no effect on virus non-producer lines (Rael, CB-M1-Ral-STO and Jijoye p79).

Intravenous (i.v.) injection of u.v. light-inactivated herpes simplex virus type 1 (UV HSV-1) at the time of HSV-1 corneal infection reduced the cytotoxic T lymphocyte (CTL) response to HSV-1, and significantly reduced the incidence of HSV-1-induced corneal stromal disease in A/J mice. The spread of HSV-1 through the eye after corneal infection, detected using engineered HSV-1 (US3::Tn5-lacZ) with the lacZ gene under the transcriptional control of the viral late gene promoter for glycoprotein C, was also markedly reduced by i.v. UV HSV-1 injection. The restriction of HSV-1 corneal invasiveness in i.v. UV HSV-1-injected mice preceded the onset of a detectable specific cell-mediated or humoral immune response to HSV-1, and was accompanied by an elevated serum titre of interferon (IFN-α), reversed by anti-IFN-α/β antibody, and mimicked by systemic IFN-α treatment. IFN-α-treated mice developed a normal CTL response to HSV-1 after corneal infection, but the corneal invasiveness of the virus was markedly reduced and none of the treated mice developed corneal stromal disease. Together with our previous findings that HSV-1-specific CTLs participate in the pathogenesis of corneal stromal disease, these results indicate that i.v. injection of UV HSV-1 at the time of corneal infection may prevent stromal disease by the combined effects of IFN-mediated reduction of the spread of virus in the cornea and inhibition of the activity of the HSV-specific T lymphocytes that induce tissue destruction in the corneal stroma.

By the application of in situ hybridization to thin sections of paraffin-embedded tissues we have been able to determine with high resolution the cell types containing lymphocytic choriomeningitis virus nucleic acid in the tissues of persistently infected mice. We confirm and extend previous observations of virus persistence in the brain, lung, liver, kidney, pancreas, thyroid and reticuloendothelial system. In addition, we demonstrate for the first time persistence of viral nucleic acid in specific cell types in the thymus, lymph nodes, testes and bladder, and the adrenal, parathyroid and salivary glands; the cell types infected were observed in several animals. In lymphoid tissue, viral nucleic acid was predominantly located in the T cell-dependent areas of the spleen and lymph nodes; it was also present in cells of the thymic medulla. This has important implications for the deficiency in T cell function observed in persistently infected mice. In the testes, viral nucleic acid was detected in spermatogonia but not differentiating spermatocytes and therefore, in this tissue at least, persistence is related to the state of differentiation of the cell. Endocrine and exocrine dysfunctions have been described in persistently infected mice and we report that the highest levels of viral nucleic acid were found in the adrenal gland. The infection of endocrine and exocrine tissue was not pantropic, specific cell types expressed viral nucleic acid in each tissue. In the adrenal cortex, cells of the zona reticularis and zona fasciculata but not the zona glomerulosa were positive, whereas in the adrenal medulla viral nucleic acid was predominantly localized to adrenalin-secreting cells. Infection of the renal tubules, transitional epithelium of the bladder and the ducts of the salivary gland indicates the likely sites of virus production for the dissemination of arenavirus infections.

The processing and stability of the non-structural (ns) proteins of Semliki Forest virus were studied in vivo. Virus-specific proteins from infected cells were identified by immunoprecipitation with monospecific antisera. The complete ns precursor, P1234, translated within 7 to 9 min of the start of translation, was processed before the completion of translation into P123 and nsP4. Pulse-chase experiments showed that the mature ns proteins were relatively stable for at least 2 h. Interestingly, the decrease in the amount of the P34 precursor during chase is accompanied by an increase only in the amount of nsP3, which could explain the observed lower amount of nsP4 in infected cells. In cells infected with SFV RNA− mutants ts4 and ts6 maintained at the restrictive temperature, nsP4 but no nsP1, nsP2 or nsP3 accumulated in addition to the ns precursor proteins P1234, P123, P12 and P34. Translation in vitro of mRNAs from a cDNA clone, encoding P1234 with a deletion in the carboxy-terminal proteinase domain of nsP2, did not yield nsP1, nsP2 or nsP3 but only nsP4, indicating that cleavage at the nsP3/4 sites is independent. Evidently, nsP4 is produced by a nascent cleavage of the growing P1234, catalysed by its own proteinase activity; the proteolytic cleavages at the nsP1/2 and nsP2/3 sites are catalysed by the proteinase moiety of nsP2.

Defective interfering (DI) RNAs of Berne virus (BEV) were generated by serial undiluted passaging of the virus in embryonic mule skin cells. Two DI RNAs of 1.0 and 1.4 kb (designated DI1000 and DI1400) were characterized in more detail. Isokinetic sucrose gradient analysis showed that these DI RNAs are specifically packaged into particles with smaller S values than standard virions. Both DI RNAs were cloned and sequenced. Three genomic cDNA clones were identified using probes complementary to the 5′ end of a DI RNA, which are thought to be derived from the 5′-terminal region of the BEV genome. A non-translated region of about 700 nt and the 5′ end of the putative BEV replicase gene were identified in the consensus nucleotide sequence. Both DI RNAs were shown to contain sequences from the 5′ and 3′ ends of the BEV genome. A conserved sequence motif, which has been postulated to be involved in sub-genomic RNA transcription, was also identified just downstream of the extreme 5′ ends of DI1000 and DI1400.

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

A panel of murine hepatitis virus (MHV) surface (S) glycoprotein-specific monoclonal antibodies (MAbs), which recognize either continuous or discontinuous epitopes, were tested in competitive binding assays. The results indicate that the binding site of MAb 30B amino acids 395 to 406 in the amino-terminal S1 subunit, is involved in the discontinuous epitope designated antigenic site A. This site is a major determinant for the induction of neutralizing antibodies. These data define, for the first time, the location of a functionally important domain on the MHV S protein.

The 3′ end of the turkey coronavirus (TCV) genome and the gene encoding the nucleocapsid protein (N) were cloned and sequenced. The gene encoding the membrane protein (M) was obtained by cloning a polymerase chain reaction (PCR)-amplified fragment obtained using bovine coronavirus (BCV)-specific primers. Furthermore, five TCV DNA fragments, obtained by PCR on RNA from clinical specimens and corresponding to either the N terminus of the M protein or the complete M protein were also cloned and sequenced. The sequence revealed a 3′ non-coding region of 291 bases, an open reading frame (ORF) encoding the N protein with a predicted size of 448 amino acids, or an M r of 49K, and an ORF encoding the M protein with a predicted size of 230 amino acids and an M r of 26K. A third ORF, encoding a hypothetical protein of 207 amino acids with an M r of 23K was found within the N gene sequence. The amino acid sequences of both the N and M proteins were more than 99% similar to those published for BCV. Extensive similarity was also observed between the amino acid sequences of the TCV N protein and those of murine hepatitis virus (MHV) (70%) and human respiratory coronavirus strain OC43 (HCV-OC43) (98%) and between the amino acid sequences of the predicted M proteins of TCV and MHV (86%). Such striking identity suggests that BCV, TCV and HCV-OC43 must have diverged from each other only recently. A potential N-glycosylation site was found at the N terminus of the TCV M protein and is situated at the same location in BCV, MHV and transmissible gastroenteritis virus.

We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 °C and 37 °C. The maximum level of attachment was observed at 4 °C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high M r and that the serum glycoprotein, α2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

Fragments of cDNA representing greater than 99% of the entire genome of wild-type hepatitis A virus (HAV) strain AGM-27, isolated from an African green monkey, were obtained by the polymerase chain reaction and sequenced. Comparison with other HAV isolates revealed differences in the predicted amino acid sequence in functionally critical parts of the genome. Comparison of the biological properties of AGM-27 with those of human wild-type and cell culture-adapted HM-175 strains revealed that AGM-27 grew in cell culture significantly better than did wild-type HM-175, but not as well as cell culture-adapted HM-175. AGM-27 and cell culture-adapted HM-175 were distinguishable by their differential growth in CV-1, FRhK-4 and primary AGMK cells.

A new isolate of hepatitis A virus (HAV), CY-145, was isolated from stool specimens obtained from cynomologus macaques naturally infected with this agent. Sequence analysis of the capsid region of the genome indicated that this virus differed from other sequenced HAV strains by about 20% at the nucleotide level and 7% at the amino acid level. Two amino acid residues (residues 70 of VP3 and 102 of VP1), previously identified as constituting an immunodominant site and conserved in all sequenced HAVs, were changed in the CY-145 virus. Sequence analysis of a second cynomolgus HAV isolate (CY-55), which came from a different geographical location, showed the same amino acid replacement at these two sites. In addition both isolates had an amino acid substitution at the VP3-VP1 cleavage site. These data suggest that the cynomolgus HAV differs genetically and antigenically from all other sequenced HAVs.

The immune response to foot-and-mouth disease virus (FMDV) elicited by infection or immunization with inactivated virus in adult mice was examined. A model of adoptive transfer of immunocompetent cells was used for this purpose. The results presented here indicate that both short- and long-term secondary immune responses elicited by high doses of inactivated virus are indistinguishable, at the humoral or cellular level, from that observed after infection. The responses to inactivated or infectious virus were both efficiently mediated by B cells. However, immunization with low doses of inactivated virus induced a response which, although effective in aborting infection, was fully dependent on FMDV-specific T cell cooperation. These findings suggest that the different immune responses observed after infection and immunization are mainly the result of the different viral mass presented to the immune system in each case.

The distribution and clearance of viral RNA (vRNA) and mRNA has been analysed for the acute and recovery stages of the pneumonia induced by intranasal infection of C57BL/6J mice with H3N2 influenza A viruses. Amplification of viral genomic material by the polymerase chain reaction showed that the influenza haemagglutinin (HA) gene was eliminated from the lungs of immunologically intact mice by day 14 post-infection, whereas in vitro depletion of the CD4+ T cells delayed clearance by at most 4 days. Viral RNA encoding the HA gene was first demonstrated in the regional mediastinal lymph nodes at 48 h, and continued to be present until day 6 or day 10 after infection of the intact and CD4-depleted mice, respectively. Evidence for the presence of vRNA in the thymus, but not in the mesenteric lymph nodes or the spleen, was found in some situations. Otherwise, the distribution and clearance of vRNA was as would be predicted from earlier studies using virus isolation procedures to monitor localization patterns, and shows a lack of long-term persistence of the influenza virus genome.

Both the supernatant of influenza virus-infected chick embryo cells and allantoic fluid containing influenza virus were shown to contain non-virion nucleoprotein (NP), which reacted readily with anti-NP monoclonal antibodies. Adsorption onto erythrocytes and centrifugation at 70000 g for 2 h resulted in the removal of about 20% of the extracellular NP, whereas centrifugation at 100000 g for 4 h eliminated about 50%, and practically all [3H]uridine-labelled virions. These results suggest that of the extracellular NP about 30% exists in the form of ribonucleoprotein, about 20% is precipitated with virions and about 50% occurs as free molecules. Comparative analysis of the kinetics of the accumulation of NP in the supernatant of infected cells, on the cell surface and inside the cells in relation to virus production, showed that there is a significant correlation between them.

A temperature-sensitive mutant of Sendai virus, ts271, when grown at restrictive temperature is known to produce virions lacking integral haemagglutinin—neuraminidase (HN). In this study, it is shown that the transmembrane-cytoplasmic tail of HN is not detected either. This apparent complete lack of HN does not affect budding efficiency.

Newcastle disease virus (NDV) virions possess two proteins which react strongly with monoclonal antibody 688 following separation by high resolution two-dimensional (isoelectric focusing/SDS) PAGE and detection by Western blotting. One is the phosphorylated nucleocapsid-associated 53K [P (NAP)] protein, the other comigrates with the 36K protein detected by radiolabelling NDV-infected chick embryo fibroblasts. [35S]Cysteine/[3H]leucine dual-labelling experiments show that the 36K protein is very rich in cysteine compared to the P (NAP) protein. In the Beaudette C strain it comigrates on one-dimensional SDS-polyacrylamide gels with the matrix protein (M); however, it is resolved from the slower migrating M protein from the Ulster strain of NDV. The size, strain-specific isoelectric point, high cysteine content and antigenic relatedness to the P (NAP) protein suggest that the 36K protein is the ‘V’ protein of NDV, the counterpart of which is found in other Paramyxoviridae.