Ultrastructural localization of herpes simplex virus RNA by in situ hybridization

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Comparative Studies of Wild-Type and “Cold-Mutant” (Temperature-Sensitive) Influenza Viruses: Polypeptide Synthesis by an Asian (H2N2) Strain and Its Cold-Adapted Variant

The structure and replication of a cold-adapted, temperature-sensitive (TS) mutant of an Asian (H2N2) influenza virus was compared with that of its wild-type (WT) parent. Viruses were grown in a chicken kidney cell system, and at the nonpermissive temperature of 40 C, production of infectious TS virus was about 100,000-fold less than at 35 C, in contrast to WT virus. Major structural polypeptides of each virus grown at 35 C were similar, except that the hemagglutinin glycopolypeptide (HA) of the TS virions was slightly more heterogenous than that of WT virions. Synthesis of viral polypeptides was examined by sodium dodecyl sulfate acrylamide gel electrophoresis of pulse-labeled infected cells. This revealed a defect in the synthesis of TS viral hemagglutinin that was most pronounced at the nonpermissive temperature. Other TS viral polypeptides appeared to be synthesized normally at 40 C. A defect in the TS virus hemagglutinin was also indicated by serological studies that demonstrated that TS virus hemagglutinin had lost antigenic sites present on the WT virus. Thus, it is concluded that the virus mutant examined contains lesions in the hemagglutinin gene, although the possibility of additional unrecognized lesions is not excluded.     

Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines.

Clinical studies previously demonstrated that live influenza A virus vaccines derived by genetic reassortment from the mating of influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus with epidemic wild-type influenza A viruses are reproducibly safe, infectious, immunogenic, and efficacious in the prevention of illness caused by challenge with virulent wild-type virus. These influenza A reassortant virus vaccines also express the ca and temperature sensitivity (ts) phenotypes in vitro, but the genes of the ca virus parent which specify the ca, ts, and attenuation (att) phenotypes have not adequately been defined. To identify the genes associated with each of these phenotypes, we isolated six single-gene substitution reassortant viruses, each of which inherited only one RNA segment from the ca parent virus and the remaining seven RNA segments from the A/Korea/1/82 (H3N2) wild-type virus parent. These were evaluated in vitro for their ca and ts phenotypes and in ferrets, hamsters, and seronegative adult volunteers for the att phenotype. We found that the polymerase PA gene of the ca parent specifies the ca phenotype and that the PB2 and PB1 genes independently specify the ts phenotype. The PA, M, PB2, and PB1 genes of the ca donor virus each contribute to the att phenotype. The finding that four genes of the ca donor virus contribute to the att phenotype provides a partial explanation for the observed phenotypic stability of ca reassortant viruses following replication in humans.     

Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms.

The development of severe childhood asthma may be influenced by several factors including environmental and infectious stimuli. The causal relationship between infectious viral responses, such as respiratory syncytial virus (RSV), and severe asthma during early childhood is unclear. In these studies, the ability for an initial RSV infection to exacerbate and promote a more severe asthmatic-type response was investigated by combining established murine models of disease. We examined the ability of RSV to induce exacerbation of allergic disease over a relatively long period, leading to development of severe airway responses including airway inflammation and hyperreactivity. The preferential production of IL-13 during a primary RSV infection appears to play a critical role for the exacerbation of cockroach allergen-induced disease. The depletion of IL-13 during RSV infections inhibited the exacerbation and acceleration of severe allergen-induced airway hyperreactivity. This was indicated by decreases in airway hyperreactivity and changes in lung chemokine production. These data suggest that the airway responses during asthma can be greatly affected by a previous RSV infection, even when infection occurs before allergen sensitization. Overall, infection of the airways with RSV can induce an IL-13-dependent change in airway function and promotes an environment that contributes to the development of severe allergic asthmatic responses.