- Volume 80, Issue 2, 1999

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a protein expressed consistently in EBV-infected cells and EBV-associated malignant tissues. A panel of monoclonal antibodies (MAbs) was generated against the C terminus of EBNA-1 and evaluated for the detection of EBNA-1 in different cell lines. The epitopes recognized were mapped. Since sequence variations of EBNA-1 have been reported in nasopharyngeal carcinoma (NPC) tissues and in infected healthy individuals, the ability of these MAbs to recognize a recombinant protein derived from an NPC biopsy was also analysed. MAb 4H11 appeared to react with EBNA-1 sequences from different sources, whereas MAbs 5C11, 5F12 and 8F6 failed to recognize a recombinant EBNA-1 protein cloned from an NPC patient. Using different recombinant EBNA-1 fragments in an immunoblot format, this study demonstrates that the domain bounded by amino acids 408 and 498 is very immunogenic in mice in that epitopes in this region are recognized by various MAbs. Amino acid sequences of EBNA-1 were also deduced from nucleotide sequences amplified from three Burkitt’s lymphoma cell lines, two spontaneous lymphoblastoid cell lines, two NPC biopsies and one NPC hybrid cell line, NPC-KT, and compared to the sequence from B95-8. The amino acid sequence of EBNA-1 in Akata is almost identical to that in an NPC biopsy, except for amino acid 585. The results of this study indicate that the immunogenic epitopes of EBNA-1 are highly variable.

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3′ sequences. Furthermore, a new transcript, initiated at an unidentified site 5′ of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.

Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposi’s sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans.

Adult thymectomized C57 BL/6J mice were depleted of T cell subsets by MAb treatment either prior to, or after, respiratory challenge with murine gammaherpesvirus-68. Protection against acute infection was maintained when either the CD4+ or the CD8+ T cell population was greatly diminished, whereas the concurrent removal of both T cell subsets proved invariably fatal. The same depletions had little effect on mice with established infection. The results indicate firstly that both CD4+ and CD8+ T cells play a significant part in dealing with the acute infection, and secondly that virus-specific antibody contributes to controlling persistent infection with this gammaherpesvirus.

The baculovirus enzyme ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, EGT has been overproduced using a recombinant Autographa californica nucleopolyhedrovirus and an antiserum has been raised against the purified protein. This antiserum was used to visualize the kinetics of expression of EGT by wild-type AcMNPVL-1 and by the overproducing recombinant virus. The inclusion of tunicamycin during these time-course experiments suggested that EGT is glycosylated. This was confirmed by Endo F treatment, which showed that glycosylation increased the apparent subunit molecular mass by approximately 11 kDa. These sugars do not appear to be required for enzyme activity. EGT activity invariantly elutes from gel-filtration columns as a single peak corresponding to a 260 kDa (+/-50 kDa) protein. This suggests that the enzyme is an oligomer of three to five subunits, since the subunit molecular mass is 56 kDa.

LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

Bean yellow dwarf virus (BeYDV) and maize streak virus (MSV) belong to the geminivirus genus Mastrevirus and have host ranges confined to dicotyledonous and monocotyledonous species, respectively. To investigate viral determinants of host range specificity, chimeras were constructed by exchanging their coding and non-coding regions. BeYDV chimeras containing MSV ORF V1, ORF V2 or small intergenic region sequences, either individually or in various sequential combinations, replicated and produced virus particles in Nicotiana tabacum protoplasts. BeYDV chimeras containing MSV ORFs C1 and C2 and/or the large intergenic region were unable to replicate. None of the chimeras was able to systemically infect either N. benthamiana or maize. Complementation experiments using BeYDV chimeras containing MSV ORF V1 and/or ORF V2 suggest that expression of MSV movement protein and/or coat protein prevents BeYDV movement. The results demonstrate that factors involved in both viral DNA replication and virus movement are exclusively adapted to either monocotyledonous or dicotyledonous host backgrounds.

The development of tomato spotted wilt tospovirus (TSWV) infection in the midgut and salivary glands of transmitting and non-transmitting thrips, Frankliniella occidentalis, was studied to elucidate tissue tropism and the virus pathway within the body of this vector. Immunohistological techniques used in this study showed that the midgut, foregut and salivary glands were the only organs in which virus accumulated. The first signals of infection, observed as randomly distributed fluorescent granular spots, were found in the epithelial cells of the midgut, mainly restricted to the anterior region. The virus subsequently spread to the circular and longitudinal midgut muscle tissues, a process which occurred late in the larval stage. In the adult stage, the infection occurred in the visceral muscle tissues, covering the whole midgut and foregut, and was abolished in the midgut epithelium. The infection of the salivary glands was first observed 72 h post-acquisition, and simultaneously in the ligaments connecting the midgut with these glands. The salivary glands of transmitting individuals appeared heavily or completely infected, while no or only a low level of infection was found in the glands of non-transmitting individuals. Moreover, the development of an age-dependent midgut barrier against virus infection was observed in second instar larvae and adults. The results show that the establishment of TSWV infection in the various tissues and the potential of transmission seems to be regulated by different barriers and processes related to the metamorphosis of thrips.

Goats are susceptible to experimental challenge with bovine spongiform encephalopathy (BSE). This study set out to investigate whether the transmission of BSE could occur in goats following the transfer of embryos from experimentally infected donor females into uninfected recipient females. The results showed no evidence of transmissible spongiform encephalopathy disease in any of the offspring which developed from embryos from infected donors, nor indeed in any of the recipient females used as surrogate dams. In addition, there was no indication of experimental BSE spreading as either a venereal infection to males used in mating or by maternal transmission to offspring born naturally to experimentally infected donors, although numbers were small.