- Volume 81, Issue 2, 2000

In this study, mitochondria migrated to a perinuclear region in the cytoplasm in herpes simplex virus (HSV)-infected cells. HSV infection did not promote the expression of cytochrome c oxidase subunit 2 but did promote that of stress-responsive HSP60, both of which are known to be components of mitochondria. The levels of cellular ATP and lactate and mitochondrial membrane potential were maintained for at least 6 h but decreased at the late stage of infection. It was also found that the UL41 and UL46 gene products, both of which are known to be tegument proteins, accumulated in the perinuclear region. The clustering of mitochondria and the accumulation of tegument proteins were completely blocked by the addition of nocodazole and vinblastine. These results suggest that mitochondria respond to the stimulation of HSV infection, migrating with tegument proteins along microtubules to a site around the nucleus, and maintain function until at least the middle stage of infection.

We have shown that C57BL/6-derived CD8+ CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved Vβ10/junctional sequence combination. This extreme T cell receptor β-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of Vβ10+CD8+ CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of Vβ10+CD8+ T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of Vβ10+CD8+ activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.

Contradictory data have recently been reported on the role of the unique long–internal repeat junction area of pseudorabies (Aujeszky’s disease) virus (PrV) genome in the virulence of the virus. To investigate the basis of the difference, four recombinant PrVs mutated at the outer region of inverted repeats that involved a putative latency promoter (PLAT2) were constructed in this study. Propagation characteristics of mutant viruses in cultured cells were similar to those of the wild-type virus. However, a 757 bp deletion at this location caused significant reduction in the virulence of PrV after intraperitoneal inoculation of mice and a moderate decrease in the virulence after intracranial inoculation. These results indicate that the PLAT2 region is an important virulence determinant that may be implicated in the neuroinvasive capability of the virus.

Murine gammaherpesvirus-68 (γHV-68) induces a lymphocytosis in mice and establishes a latent infection of B lymphocytes following intranasal administration in anaesthetized animals. Because γHV-68 is a gammaherpesvirus, it has been used as a model to understand the pathogenesis of Epstein–Barr virus (EBV) and human herpesvirus-8 (HHV-8) infections. In this study, we investigated the unlikely possibility that γHV-68 could survive the harsh gastrointestinal environment to efficiently infect intestinal epithelial cells, and then disseminate from mucosal sites to cause systemic disease. Surprisingly, oral administration, or gastric instillation which by-passed the oral cavity, readily caused a systemic lymphocytosis and established a latent infection in splenic leukocytes. The finding that γHV-68 could readily infect adult mice following gastric instillation strongly suggested that intestinal epithelial cells could be productively infected. Unlike the more routinely used method of intranasal inoculation, γHV-68 given intragastrically resulted in lytic virus, viral RNA and viral DNA being present in isolated intestinal epithelial cells. Furthermore, γHV-68 RNA and DNA, but not latent virus, could be detected in epithelial cells as long as 30 days post-infection, suggesting that some of these cells might be persistently infected. Taken together, these studies demonstrate that γHV-68 can survive passage through the gastrointestinal tract and infect intestinal epithelial cells. Following infection of gut epithelial cells, γHV-68 can disseminate from mucosal sites to induce a systemic lymphocytosis which is similar to the disease induced following intranasal inoculation.

The expression of antigens or other molecules from recombinant vaccinia viruses requires the insertion of coding sequence at specific sites in the viral genome. Here we investigate the influence of two different sites on the level of protein expressed during a viral infection. The level of immune response in mice to vaccinia virus-expressed murine interleukin 2 (IL-2) or IL-4 varied depending on whether the coding sequence was inserted into the vaccinia virus thymidine kinase (tk) gene or into the HindIII F fragment of the viral genome where herpes simplex virus (HSV) tk was used as a selectable marker. In each case the intensity of the response was greater when the relevant gene was expressed from the HindIII F insertion site. In order to quantify these differences a series of recombinant viruses expressing luciferase was constructed. Luciferase activity from coding sequence inserted into the HindIII F fragment was significantly higher than that from the tk gene insertion, provided HSV tk+ constructs were compared. Insertion of a marker gene (HSV tk) into the HindIII F site with disruption of the F7L open reading frame led to a reduced level of luciferase expressed from the tk insert, despite more than 45 kb of intervening sequence. In mice, luciferase expression was higher from the HindIII F inserted gene than from the tk insert in both lungs and ovaries.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungus Rhizoctonia solani isolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5′ ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem–loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86–2275; molecular mass 86 kDa) and 683 aa (bases 79–2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses from Fusarium poae and Atkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.