- Volume 79, Issue 11, 1998

Recently a novel DNA virus, designated TT virus (TTV), that possibly accounts for some of the cases of liver disease of unknown aetiology, was identified in Japanese patients. Using specific primer pairs for conserved regions, we detected TTV DNA by PCR in 16/84(19%) German patients awaiting orthotopic liver transplantation because of decompensated liver cirrhosis (of diverse causes); in 4/25 (16%) patients with non-A-G hepatitis; in 1/7 patients with autoimmune hepatitis; and in one intravenous drug user. Sequence analysis showed that in contrast to the findings in Japanese patients only about 37% of our TTV sequences belonged to genomic group 1 but about 58% belonged to group 2, including several sequences belonging to a further subgroup tentatively designated group 2c. Further studies to clarify whether the novel virus has hepatitis-inducing capacity or other clinical significance are needed.

Herpes simplex virus type 1 (HSV-1) infection induces the selective shut-off of host protein synthesis, other than ribosomal proteins, and the successive synthesis of viral proteins. Because viral mRNAs persist in the cytoplasm after viral protein synthesis has been inhibited, we hypothesized that viral gene expression may be under translational control. Expression of genes encoding immediate early ICP27, early DBP and late US11 proteins, together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the course of infection at the level of mRNA and protein synthesis. After an efficient synthesis beginning with the appearance of successive viral mRNAs in the cytoplasm, synthesis of viral proteins was shut off similarly to the synthesis of GAPDH. This shut-off was not achieved by mRNA degradation but by progressive shifts of viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal subunits. Transient expression of the UL41 gene alone, directing synthesis of virion-associated host shut-off (VHS) protein, induced efficient mRNA degradation, but did not impair recruitment of the remaining GAPDH and β-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA translation which is probably the main cause of the shut-off of viral protein synthesis, and which contributes to the repression of host protein synthesis. VHS protein is not directly involved in this repression, at least in the absence of other viral proteins.

The herpes simplex virus type 2 (HSV-2) US2 gene product was identified by using a rabbit polyclonal antiserum raised against a recombinant 6 × His-US2 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 39 kDa protein in HSV-2 strain 186-infected cell lysates. The protein was not detectable in the presence of the virus DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the US2 protein in the cytoplasm and as discrete granules at late times post-infection within and at the periphery of the nucleus, and nuclear fractionation studies showed that the protein was partially associated with the nuclear matrix of infected cells. The protein was easily detected in purified virions. Also, a US2 insertion mutant was constructed which contained an ICP6-lacZ insertion in the US2 gene. This mutant was as virulent as wild-type virus in mice when inoculated by the footpad route. The importance of the US2 protein of HSV-2 in the virus life-cycle may be apparent only in the natural human host.

Nitric oxide (NO), produced in interferon (IFN)-γ- activated murine macrophages by the enzyme inducible nitric oxide synthase (iNOS), has been found to have antiviral properties. We have previously shown that herpes simplex virus type 2 (HSV-2) infection of macrophages synergistically enhances IFN-γ-induced NO production, and we now extend these findings by providing evidence that virus- induced tumour necrosis factor (TNF)-α mediates activation of the transcription factor nuclear factor (NF)- k B, which in turn is responsible for the synergistic effect. HSV-2 infection and IFN-γ stimulation of macrophages synergistically induced TNF-α secretion and nuclear translocation of NF- k B, which bound to a sequence corresponding to a k B site in the iNOS promoter. The effect of HSV-2 on NF- k B and NO production was eliminated when cells were treated with antibodies to TNF-α, and direct inhibition of NF- k B activation with pyrrolidinedithio- carbamate (PDTC) also blocked the effect of HSV-2 infection on NO production. The effect of the NF- k B activation inhibitor was not mediated through inhibition of the production of interferon regulatory factor (IRF)-1 or of TNF-α itself, and a possible alternative mechanism of activation of NF- k B through virus-induced activation of the kinase PKR was also ruled out. Thus, our data indicate that NF- k B activation, through virus-induced autocrine TNF-α secretion, is responsible for the synergistic effect of HSV-2 infection on IFN-γ-induced NO production, and that such activation might constitute a mechanism by which high-output NO production is targeted to infectious foci.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek’s disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek’s disease (line 72) and another resistant (line 61), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 61 and 72, pp38 lymphocytes were detected at 4–6 days in the spleen, thymus and bursa. pp38 cells could not be detected from day 8 onwards. In both lines, pp38 lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38 cells were predominantly B lymphocytes, but CD4 and CD8 TCRαβ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38 lymphocytes was greater in line 72 than in line 61. pp38 lymphocytes were not detected in the peripheral blood at anytime. The data show that the differential susceptibility of lines 61 and 72 to the development of Marek’s disease lymphoma is not attributable to differences in phenotype or location of pp38 lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38 lymphocytes.

Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.

The extent of nucleotide sequence microheterogeneity varies among subgenomic regions of Epstein-Barr virus (EBV). We examined, in EBV- carrying lymphoid cell lines, the extent of polymorphism in EBV DNA fragments amplified from the BamHI E, K, N and Z regions, and then investigated the diversity of the more hypervariable regions in tissues and body fluids. In cell lines, sequence dissimilarities in a genotype-specifying fragment of the EBNA-3C gene varied from < 1–4% within each genotype; dissimilarities in the first intron of the BZLF-1 gene were < 2% within each genotype. By contrast, dissimilarities in a C-terminal unique domain of the EBNA-1 gene, and in a fragment that encompasses and is upstream of the LMP-1 start codon, varied between 2 and 7% and were not genotype-specific. The sequence diversity in BamHI K and N regions was then examined in tissues and body fluids by single-strand conformation polymorphism (SSCP) analysis and cycle sequencing. Extensive inter-host diversity was observed, whether the host was co-infected by human immunodeficiency virus (HIV) or not. In the oral cavity of HIV-infected patients, inter-compartmental EBV diversity could be demonstrated, even between sites that were anatomically proximate. Studies of BamHI K clones derived from EBV in oral lesions revealed infection by multiple variants. Identification of hypermutable loci within the EBV genome such as those located in the BamHI K and N regions should permit fine discrimination of individual EBV variants.

Tobacco plants were transformed with genes encoding the tobacco vein mottling potyvirus (TVMV) P1 or P3 protein. When compared with vector- transformed or P1 transgenic lines, seedlings of P3 transgenic lines (except a low expressor line) developed poorly, suggesting a detrimental effect of P3 on the plant. All P1 and P3 transgenic lines were protected against the homologous TVMV strain and showed variable proportions of two resistance phenotypes: asymptomatic plants or symptomatic plants that recovered from infection. The resistance was effective against a high inoculum dose but had a narrow spectrum. The heterologous strain TVMV-S was able to overcome resistance in most P1 lines but did not break the resistance of most P3 lines. No line showed resistance to another potyvirus (potato virus Y) or to potato virus X. These features and the low levels of transgene expression in resistant plants suggest that protection in P1 and P3 lines is RNA-mediated. In contrast with most reports on virus-activated gene silencing, some P3 lines with the predominant ‘recovery’ phenotype showed silencing of the transgene that was activated at a certain developmental stage of the plant independently of virus infection.

The capsid protein (CP) of tomato yellow leaf curl virus (TYLCV) is the only known component of the virus coat. Here, we identify TYLCV CP as a singlestranded (ss) DNA binding protein. Purified TYLCV CP bound ssDNA in a highly cooperative and sequence-nonspecific fashion. TYLCV CP-ssDNA complexes were resistant to nucleolytic digestion and remained stable at relatively high salt concentrations. Because TYLCV CP is known to contain an active nuclear targeting signal, we propose that its association with the viral genomic ssDNA mediates TYLCV entry into the host cell nucleus during the infection process.

Complete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of athird Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus - Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.

The genome organization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was largely elucidated and compared to those of other baculoviruses. A detailed physical map was constructed for the restriction enzymes BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, XbaI and XhoI. The 120·9 kbp viral genome was cloned as restriction fragments into a plasmid library from which about 43·5 kbp of dispersed sequence information was generated. Fifty-two putative open reading frames homologous to those of other baculoviruses were identified and their location in the genome of BusuNPV was determined. Although the gene content of BusuNPV is similar to that of Autographa californica multiple-nucleocapsid nucleopolyhedrovirus, Bombyx mori nucleopolyhedrovirus and Orgyia pseudotsugata multiple-nucleocapsid nucleopolyhedrovirus, the gene order is, however, significantly different from that observed in the other viruses, which have a high degree of collinearity. A new approach (GeneParity-Plot) was developed to represent the differences in gene order among baculoviruses when limited sequence information is available and to take advantage of the high degree of gene conservation. The data obtained show that BusuNPV is a distinct baculovirus species and the analyses suggest that gene distribution along baculovirus genomes may be used as a phylogenetic marker.

The light brown apple moth, Epiphyas postvittana, is a major insect pest of a variety of fruit crops grown in New Zealand and we are studying a nucleopolyhedrovirus, EppoNPV, isolated from this insect. Restriction endonuclease analysis of EppoNPV DNA shows that this is a single strain of virus with a genome size of approximately 119 kbp and a complete library of the EppoNPV genome has been cloned. A strategy of single-stranded sequencing of the termini of REN fragment clones was employed to map the virus genome. Sequence homologies to NPV gene sequences present in the GenBank database allowed a nearly complete restriction map of the EppoNPV genome to be constructed. The mapping was completed with Southern blotting and restriction analysis. Fifty-five open reading frames (ORFs) with similarity to genes from other NPVs have been identified and placement of these on the restriction map shows that EppoNPV has a nearly identical genome organization to Orgyia pseudotsugata (Op)MNPV. The polyhedrin gene of EppoNPV has been fully sequenced and an ORF of 738 bp encodes a predicted protein of 28·8 kDa. The conserved 12 bp consensus sequence typical of very late baculovirus gene promoters, AATAAGTAATTT, has been located upstream of the ATG initiation codon. An ORF located downstream of the polyhedrin gene shows homology to the 1629-capsid protein from OpMNPV. Phylogenetic comparison to polyhedrin gene sequences from 23 other NPVs shows EppoNPV to be a group I NPV closely related to OpMNPV.

The similarity in histopathological changes seen in scrapie-infected mice and in an uninfected senescence-accelerated mouse strain led to a study in which the mouse strain that is prone to senescence (SAMP8), a strain that is resistant to senescence (SAMR1) and a progenitor strain (AKR) of these two strains were infected with three different scrapie strains, ME7,139Aand 22L. For each scrapie strain, the incubation period was shortest in AKR mice and longest in SAMR1 mice. The induction of obesity was a function of scrapie strain and not mouse strain; ME7 caused obesity in all mouse strains, whereas the average weights of mice injected with 139A and 22L did not differ significantly from mice injected with homogenates of normal mouse brain. The pattern of vacuolation seen in the brain of each mouse strain was primarily dependent on the scrapie strain injected. There were, in general, similarities to the patterns induced in other inbred strains; e.g. ME7 caused extensive forebrain vacuolation, 22L caused prominent vacuolation in the cerebellum, and the 139A strain induced characteristic white matter vacuolation. Vacuolation was also seen inthe medulla and midbrain of SAMP8 mice injected with normal mouse brain, which is consistent with the occurrence of accelerated ageing changes in the brain of this strain. Further analysis of the differences among these mouse strains should provide information relating to the observed differences in scrapie incubation periods.