Persistent infection of human adenovirus type 5 in human monocyte cell lines.

Adenovirus infection of human monocyte hybridoma cell lines and the fusion partner U937 was investigated. Adenovirus adsorbed poorly to these cells as well as primary human alveolar macrophages. The virus-binding experiments showed a 100-fold reduction in apparent viral binding to these cells compared to the permissive HeLa cells. Adsorption of adenovirus to these cells could be enhanced by preincubation of adenovirus with its antiserum. Following entry into the cells amplification of adenovirus DNA was detected starting at 2 days postinfection but few mature virus particles were produced. The infected cultures survived the infection and continued to grow for more than a year. In these chronically infected cultures, linear adenovirus DNA persisted up to 200 copies per cell and a small amount of mature virus was produced. Infectious center assay and cell cloning experiments showed that the majority of the cells in the chronically infected cultures harbor adenovirus genome. These results indicate that restriction of replication of human adenovirus type 5 at the late phase results in persistent infection of U937 and the human monocyte hybridoma cell lines.     

Biologic heterogeneity of human immunodeficiency virus type 2 (HIV-2) strains

Seven HIV-2 isolates recovered from peripheral blood mononuclear cells (PBMC) of patients from the Ivory Coast have been biologically characterized. All seven strains replicated well in primary human lymphocyte and macrophage cultures and in established human T cell lines. They showed differences in infectivity and replicating ability in primary PBMC cultures from chimpanzees, rhesus macaques, and baboons. Moreover, variations in levels of virus replication in PBMC from 13 seronegative donors were observed. Four strains (UC2, UC3, UC7, and UC8) were highly cytopathic and caused extensive surface CD4 antigen depletion in acutely infected PBMC and SupT1 cells. Two strains (UC1 and UC6) showed minimal or no cytopathology, no CD4 down-modulation, and much lower levels of virus protein expression in SupT1 cells. These findings reflect the heterogeneity of HIV-2 strains and suggest that these biological properties could influence pathogenesis in the host.     

Replication of rubella virus in human mononuclear blood cells.

Rubella virus was capable of replicating in both unstimulated and phytohemagglutinin-stimulated cultures of human mononuclear blood cells. Monocyte-derived macrophages were the main cell type responsible for viral replication. The susceptibility of macrophages increased during cultivation. Phytohemagglutinin-stimulated lymphocytes were able to support replication to a limited degree. No viral replication was detected in unstimulated lymphocytes. Both stimulation and viral replication in phytohemagglutinin-treated lymphocyte cultures were enhanced by the addition of murine macrophages. Human leukocyte interferon depressed the production of virus in these combined cultures. The finding that rubella virus is able to replicate in human lymphocytes as well as in macrophages may contribute to understanding the mechanisms of the suppressive effect of the virus on in vitro lymphocyte phytohemagglutinin responsiveness and in vivo immune functions.     

Temperature sensitivity of herpes simplex virus type 1 is a tissue-dependent phenomenon

Summary The temperature sensitivity of herpes simplex virus type 1 (HSV-1) was assessed in primary cultures of mouse central nervous system (MNS) cells and mouse embryo cells (MEC). Infectious yields were determined and the ultrastructural morphogenesis of HSV-1 particles was compared following incubation at 37 or 40.5°C. Yields of infectious virus were significantly reduced for both types of cell cultures following incubation at 40.5°C. However, the effect of supraoptimal temperature (40.5°C) on HSV-1 replication in MEC was significantly greater than the effect of supraoptimal temperature on virus replication in MNS cells. With respect to viral morphogenesis, no significant differences were found in either the quantity or the appearance (empty versus electron opaque core) of intranuclear particles present per infected nucleus, regardless of cell type or incubation temperature. However, complete virus particles (enveloped capsids with dense cores) were never observed in MEC at 40.5°C, either intracytoplasmically or extracellularly. In contrast, complete virus particles were observed in MNS cell cultures at 40.5°C, albeit in reduced numbers. At the permissive temperature (37°C), complete intracytoplasmic and/or extracellular virus particles were associated with every infected cell in the MNS cell or MEC cultures. Thus an interactional effect on HSV-1 replication was found between cell culture type and incubation temperature.     

Immortalized Epstein-Barr virus-positive B-cell lines obtained by prolonged culture of peripheral blood mononuclear cells from human immunodeficiency virus type 1-positive patients.

OBJECTIVES: To study the factors that determine malignant B cell growth in human immunodeficiency virus type 1 (HIV-1)-infected patients. STUDY DESIGN: B-cell lines (lymphocyte cell lines [LCL]) were developed after nonstimulated culture of peripheral blood mononuclear cells (PBMC) from HIV-1-positive (HIV-1(+)) patients. Human immunodeficiency virus type 1 replication in culture, Epstein-Barr virus (EBV) latent oncogene expression, and cell-to-cell interaction were studied after nonstimulated culture of HIV-1(+) PBMC, analyzing their contribution to LCL appearance. METHODS: Nonstimulated PBMC cultures of HIV-1(+) PBMC and controls (N-PBMC) were established. Lymphocyte cell lines were characterized. Epstein-Barr virus latent membrane protein 1 (LMP-1) and Epstein-Barr nuclear antigen 2 were detected by polymerase chain reaction (PCR). Clonality of LCL was determined by light chain restriction (flow cytometry) and immunoglobulin H chain rearrangement (semi-nested PCR). Peripheral blood mononuclear cell phenotypes were studied at different intervals of culture. RESULTS: Lymphocyte cell lines were obtained in 73% of HIV-1(+) PBMC cultures, compared with 6% in N-PBMC. All LCL were EBV-positive (EBV(+)). B-cell lineage was established, and up to 12 different B-cell clones were expanded from the same individual. Occurrence of LCL was more frequent in cultures with HIV-1 replication, high LMP-1 expression in viable B cells, and high CD4:CD8 ratio. Human immunodeficiency virus type 1 replication persisted in 53% of the LCL. CONCLUSIONS: In vitro HIV-1 replication and persistence of viable EBV(+) lymphoblasts favor spontaneous in vitro outgrowth of LCL in HIV-1(+) patients.     

Ribosomal RNA synthesis after infection with adenovirus type 2

The synthesis of mature cytoplasmic ribosomal RNA was preferentially inhibited in comparison to 4S RNA in T6a (hamster) cells infected with adenovirus type 2, but not in uninfected controls. No effect was observed in 3T3–4E (mouse) cells infected with adenovirus type 2. When T6a-3T3-4E hybrids were infected with this virus, no significant change in the proportion of hamster and mouse cytoplasmic ribosomal RNA synthesized was detected, in comparison to uninfected hybrid cells.     

Herpes simplex virus type 1 ICP-0 induces reactivation of pseudorabies virus from latently infected trigeminal ganglia explant cultures

Summary.  Pseudorabies virus (PrV), like other alphaherpesviruses, is a neurotropic virus that can establish a latent infection in swine. Reactivation of PrV from latency may occur spontaneously or after induction with corticosteroids. The mechanisms involved in the establishment of latency and reactivation are currently unknown. Here, we examined gene-specific reactivation of PrV by herpes simplex virus type 1 (HSV-1) immediate early protein, ICP-0. Primary neuronal cell cultures established from the trigeminal ganglia of latently infected swine were superinfected with recombinant adenoviruses expressing ICP-0. Reactivation of PrV occurred in cultures that were superinfected with two different ICP-0-expressing adenovirus recombinants, but not in cultures that were either mock-infected, or superinfected with wild-type adenovirus, or recombinant adenoviruses not expressing ICP-0. Infectious PrV was detected between 4 and 7 days postinfection, regardless of the promoter driving expression of ICP-0. Results from these experiments show that HSV-1 ICP-0, a homologue of PrV EP0, can induce PrV reactivation from explanted trigeminal ganglia of latently infected swine. Accepted October 14, 1997 Received August 4, 1997     

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.     

Production by mixed lymphocyte cultures of a type II interferon able to protect macrophages against virus infection.

In supernatants of mixed mouse spleen cell cultures established for 4 days, a species-specific inhibitor of virus replication with a broad antiviral spectrum was found. The inhibitor was destroyed by trypsin, was nondialyzable and acid labile, and was not neutralized by antibody to mouse L cell interferon. This indicates that in mixed lymphocyte cultures a type II interferon is made that has no immunological relationship with "fibroblast" interferon. This leukocyte product was shown to protect mouse hepatitis viruses. It is suggested that lymphocyte interferon may collaborate with macrophages in host defense against viruses, as a mediator of cellular immunity.     

Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus

C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.     

Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication

Nitric oxide (NO) has been implicated in certain immunopathogenetic mechanisms during the course of infection with human immunodeficiency virus (HIV). We have evaluated the levels of NO release and lymphocyte apoptosis in peripheral blood mononuclear cell (PBMC) cultures from HIV-1 infected subjects and healthy controls. We have also examined these 2 parameters in parallel cultures maintained under conditions where either NO synthesis was inhibited or high level of NO was present. Nitrite contents in culture supernatants were measured as the stable end products of the released NO. Levels of spontaneous apoptosis and activation-induced cell death (AICD) by anti-CD3 or by phytohemagglutinin were evaluated using flow cytometry. Additional experiments were also aimed at addressing a potential link between NO synthesis and HIV-1 replication in human monocyte-derived macrophages (MDMs). Acutely infected MDMs with HIV-1Bal were maintained in culture, without any additional activation signal, for a period of 14 days. Nitrites in the supernatants and mRNA accumulation of the inducible NO synthase (iNOS) in infected cells were assessed over the whole culture period. In addition, the effect of blocking NO synthesis during and after infection of MDMs, using an inhibitor of NO, was evaluated on the level of viral replication as measured by the presence of P24 antigen in the supernatants. Similarly, the effect on HIV replication of high NO levels in MDM cultures, supplied by a donor of NO during the 24 h period of infection, was also studied.We conclude that no elevation in NO release could be detected in PBMC cultures from HIV-1 infected subjects and that modulation of NO content may slightly regulate the level of spontaneous lymphocyte apoptosis but not that of AICD. Infection of MDMs with HIV-1 does not seem to induce detectable NO release or iNOS mRNA accumulation. Similarly, neither inhibition of NO synthesis nor the presence of high NO levels during the infection period could modify the outcome of virus replication in macrophages.     

Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures

The replication of four fowl adenovirus strains (FAV-1, strain Phelps; FAV-5, strains 340 and TR-22; and FAV-7, strain YR-36), in primary chick kidney cell cultures, is described. Differences were found in the distribution of virus particles and virus associated inclusions between viruses belonging to different cytopathology subgroups. Thus in cells infected with FAV-1 (Phelps) and FAV-5 (340) (i.e. subgroup 1) virus particles, as they increased in number, tended to become distributed peripherally, close to the nuclear membranes, with the virus associated inclusions in the centre of the nucleus. With FAV-5 (TR-22) and FAV-7 (YR-36) (i.e. subgroup 2) virus particles and associated inclusions became concentrated initially in the central nuclear area later increasing to fill the whole nucleus, with virus particles and inclusions completely intermixed. The virus-associated inclusions were found to be identical to those described in human adenovirus infected cells and the same nomenclature was adopted. Other inclusions found in infected nuclei, included tubular structures and inclusions composed of granular particles.     

Herpes simplex virus type 2 infection of unstimulated human T-lymphocytes

Unstimulated human leukemia T-cell lines (MOLT-4, MT-4) were tested for their susceptibility to herpes simplex virus type 2 (HSV-2) infection. Permissive infection of MT-4 cells was demonstrated by growth curve and infectious center assays. In growth curve experiments new progeny virus replication was detected by 24 hrs and maximum titers of HSV-2 replication were measured by 72 hrs after infection of MT-4 cells, whereas, MOLT-4 cells did not produce detectable infectious HSV-2 in growth curve experiments. It may be that a T-cell subset is involved with infectious HSV-2 production, since 5.7% of MT-4 cells were scored as infectious centers after HSV-2 infection compared to only 0.06% of MOLT-4 cells. Furthermore, HSV-2 infected MT-4 (45% of cells) and MOLT-4 cells (30% of cells) expressed viral induced antigen(s) detected by immunofluorescence assays. These data provide the first evidence of infectious HSV-2 replication in T-cells not prestimulated in vitro with mitogens, pharmacologic agents or growth factors. The establishment of T-cell systems that permit rapid and efficient replication of HSV-2 could greatly facilitate studies on interactions between human herpesviruses and AIDS retroviruses since recent published evidence indicates possible synergistic interactions between these virus groups.     

Contributions of E1A to mouse adenovirus type 1 pathogenesis following intranasal inoculation

We investigated the role of mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein in adenovirus respiratory infection. Intranasal (i.n.) inoculation of mice with wild type (wt) virus induced chemokine and cellular inflammatory responses in the lung. We observed similar responses in mice infected with an E1A-null mutant virus at the same dose, although the magnitude of these responses was lower. Levels of viral hexon gene expression were lower in the lung following infection with E1A-null virus than with wt virus. When input doses were adjusted so that equivalent viral loads were present following infection with varying doses of wt and E1A-null virus, we observed equivalent chemokine upregulation in the lung. Dissemination to the brain occurred following i.n. inoculation with equal doses of wt or E1A-null virus, but viral gene expression and viral loads were lower and the magnitude of chemokine responses was lower in brains of E1A-null virus-infected mice. CD4 and CD8 T cells and neutrophils were recruited to the brains of mice infected with either wt or E1A-null virus. Together, these data suggest that MAV-1 E1A makes important contributions to viral replication in the lung and the brain following i.n. inoculation. However, E1A is not essential for the induction of inflammatory responses in the lung or for viral dissemination out of the lung.     

High molecular weight virus DNA in KB cells infected with ts mutants of adenovirus type 2 under permissive and non-permissive conditions.

Temperature-sensitive (ts) mutants of adenovirus type 2 (Ad2), which are deficient in virus DNA synthesis at the non-permissive temperature, have been used to investigate whether virus DNA replication is required for the occurrence of high mol. wt. Ad2 DNA (greater than 100S, 50 to 90S) in human cells productively infected with Ad2. The high mol. wt. virus DNA has been previously shown to consist of virus and cellular DNA molecules covalently linked. The present data indicate that after infection with DNA-ts mutants, the production of high mol.wt. virus DNA is much less sensitive to restrictive conditions than the synthesis of unit length (34S) Ad2 DNA. This finding lends further support to the idea that the occurrence of high mol. wt. virus DNA is independent of the synthesis of unit length virus DNA.     

Extracts of hamster cells abortively infected with human adenovirus type 12 are competent to support initiation of viral DNA replication

Baby hamster kidney (BHK-21) cells do not allow replication of human adenovirus type 12 (Ad12) DNA during abortive infection by this virus. However, we have determined that crude extracts of BHK-21 cells abortively infected with Ad12 support in vitro the initiation reaction of Ad12 DNA replication. Synthesis of the Ad12 pTP-dCMP initiation complex by BHK extracts is two- to five-fold less than when crude infected human (KB) cell extracts are used in the reaction. Combining infected KB cytoplasmic and uninfected BHK nuclear extracts in the reaction indicates that the decreased efficiency is probably due to a lesser ability of hamster nuclear extracts to support the initiation reaction, rather than to decreased synthesis of Ad12 pTP and DNA polymerase during abortive infection, or to the presence of an inhibitor in BHK cells.     

The influence of lododeoxyuridine pretreatment of various cell cultures on adenovirus multiplication

Treatment of cell cultures with iododeoxyuridine (IUdR) before virus inoculation may enhance the subsequent virus multiplication. This effect was studied in seven kinds of cell cultures with seven human adenoviruses from six subgenera. IUdR (50 micrograms/ml) was added 24 h after the seeding of cells, left for 2 days, and removed before virus inoculation. IUdR had no effect on cell proliferation. Viral cytopathic effect was enhanced in many instances by IUdR pretreatment. However, virus multiplication was enhanced weakly in only four cases [adenovirus 7 in Vero, African green monkey kidney, cynomolgus monkey kidney; adenovirus 4 (Ad4) in HeLa cells], and strongly only for Ad4 in Vero cells, which are semipermissive for Ad4. IUdR pretreatment of Vero cells shortened the replication cycle for Ad4 and increased sensitivity for minimal virus concentrations about 1000-fold. From immunofluorescence experiments it appears that more than one infectious particle is required to infect an untreated Vero cell.