Persistent Herpes Simplex Virus Infection In Vitro with Cycles of Cell Destruction and Regrowth.

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.), and Mary Lou Copeland. Persistent herpes simplex virus infection in vitro with cycles of cell destruction and regrowth. J. Bacteriol. 90:205-212. 1965.-The susceptibility of two Chinese hamster cell lines to herpes simplex virus (HSV) was studied from the time of their initiation through successive subcultures. The cells' susceptibility to the cytocidal effects of HSV decreased as the number of cell passages increased. During the early cell passages, the decrease in cell susceptibility to HSV was characterized by an increased time after infection for complete cell destruction to occur, with a concomitant increase in the period when virus could be recovered from supernatant fluids. This was followed by a number of cell passages during which persistent HSV infections were established. The persistent infections were characterized by (i) cycles of virus synthesis and cell destruction followed by regrowth of the cells, (ii) initiation and maintenance under conditions optimal for cell growth in the absence of antibody, (iii) the cells' ability to be passaged while still maintaining their cycling patterns, (iv) a relationship between virus synthesis and cell proliferation, and (v) inability of long-term treatment with antibody to "cure" the persistent infections. The unique characteristics of this HSV infection were compared with other persistent in vitro viral infections.     

Infection of Tumour Cells by Fowl Plague Virus in the Presence of Actinomycin D

INFLUENZA virus is one of the few viruses in which replication is inhibited by the antimetabolite actinomycin D (AM-D)^1–4, which inhibits DNA-dependent RNA synthesis in mammalian cells⁵. It has been reported that the growth of fowl plague virus (FPV) in virus-transformed hamster cells is less sensitive to AM-D^6,7. We have examined the sensitivity of FPV to AM-D to see whether it is related to differences in the oncogenic properties of tumour cells. We found that in cells transformed by polyoma virus (PV) and also in cells transformed by methylcholanthrene, although no infectious virus was produced the cells synthesized viral haemagglutinin (HA). It was only the cell-associated HA, however, that was affected by AM-D and not that released by the cells.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

Initiation of Vaccinia Virus Infection in Actinomycin D-pretreated Cells

The early steps in vaccinia virus infection were studied in HeLa cells which had been treated with actinomycin D (1 μg/ml) and then incubated for several hours in fresh medium prior to infection. Initiation of infection occurred in such cells even though the synthesis of cellular ribonucleic acid and deoxyribonucleic acid (DNA) was severely depressed. Thymidine kinase was synthesized in amounts that exceeded those found in untreated, infected cells. The breakdown of viral “cores” to liberate viral DNA and the synthesis of viral specific DNA-polymerase also occurred but were somewhat delayed. A deoxyribonuclease resembling an exonuclease was made by the infected, pretreated cells. The time course for these events suggested that the genetic code for synthesis of thymidine kinase can be expressed before “cores” are broken down, but the DNA-polymerase can be synthesized only after liberation of the viral DNA. The amount of viral specific DNA-polymerase which was made after infection was proportional to the total number of virus synthesizing sites even beyond the point where all the cells were infected with one infectious particle. A similar relationship was observed for the amount of thymidine kinase formed and for the rate of viral DNA synthesis from ³H-thymidine.     

OX40 Facilitates Control of a Persistent Virus Infection

During acute viral infections, clearance of the pathogen is followed by the contraction of the anti-viral T cell compartment. In contrast, T cell responses need to be maintained over a longer period of time during chronic viral infections in order to control viral replication and to avoid viral spreading. Much is known about inhibitory signals such as through PD-1 that limit T cell activity during chronic viral infection, but little is known about the stimulatory signals that allow maintenance of anti-viral T cells. Here, we show that the co-stimulatory molecule OX40 (CD134) is critically required in the context of persistent LCMV clone 13 infection. Anti-viral T cells express high levels of OX40 in the presence of their cognate antigen and T cells lacking the OX40 receptor fail to accumulate sufficiently. Moreover, the emergence of T cell dependent germinal center responses and LCMV-specific antibodies are severely impaired. Consequently, OX40-deficient mice fail to control LCMV clone 13 infection over time, highlighting the importance of this signaling pathway during persistent viral infection.     

In Vitro and In Vivo Observations on a Murine C-Type Virus

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.     

Effect of Persistent Fibroma Virus Infection on Susceptibility of Cells to Other Viruses

Shope fibroma virus establishes a persistent cytoplasmic infection in primary (RK) and serially cultivated (DRK(3)) rabbit kidney cells which is accompanied by a morphological alteration of the cells. The response of such cells to superinfection by other viruses was compared with that of control cells by determining plaque production and virus yield of superinfecting viruses. It was found that the growth of other poxviruses, myxoma and vaccinia, was greatly inhibited in the fibroma virus-infected cells, but that of pseudorabies and herpes simplex viruses, which are unrelated deoxyribonucleic acid viruses, was virtually unaffected. The ribonucleic acid (RNA) viruses, poliovirus 1 and coxsackievirus B1, did not produce plaques on either RK or fibroma virus-infected (F-RK) monolayers. However, the growth of several other RNA viruses, vesicular stomatitis virus, encephalomyocarditis virus, Sindbis virus, and Newcastle disease virus, was enhanced in F-RK cells. None of these latter RNA viruses produced any infectious progeny in DRK(3) cells, but they all plaqued on and produced good yields in DRK(3) cells persistently infected with fibroma virus. This phenomenon is termed facilitation. Facilitation results from the infection of DRK(3) cells by fibroma virus. Neither interference nor facilitation were due to changes in the adsorption or eclipse of the superinfecting virus.     

Effects of Actinomycin D and Ultraviolet and Ionizing Radiation on Pichinde Virus

Actinomycin D (0.05 μg/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with ³H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 × 10⁶ to 8 × 10⁶ daltons. This value is greater than the 3.2 × 10⁶ daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods.     

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.     

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Infection of Mice,Ferrets, and Rhesus Macaques with a Clinical Mumps Virus Isolate

In recent years, many mumps outbreaks have occurred in vaccinated populations worldwide. The reasons for these outbreaks are not clear. Animal models are needed to investigate the causes of outbreaks and to understand the pathogenesis of mumps virus (MuV). In this study, we have examined the infection of three animal models with an isolate of mumps virus from a recent outbreak (MuV-IA). We have found that while both ferrets and mice generated humoral and cellular immune responses to MuV-IA infection, no obvious signs of illness were observed in these animals; rhesus macaques were the most susceptible to MuV-IA infection. Infection of rhesus macaques via both intranasal and intratracheal routes with MuV-IA led to the typical clinical signs of mumps 2 weeks to 4 weeks postinfection. However, none of the infected macaques showed any fever or neurologic signs during the experimental period. Mumps viral antigen was detected in parotid glands by immunohistochemistry (IHC). Rhesus macaques represent the best animal model for the study of mumps virus pathogenesis.     

Effect of Actinomycin D on Paget's Disease of Bone

Four patients with active Paget''s disease were treated with the RNA inhibitor actinomycin D. Three were clinically improved after treatment; the fourth had multiple collapsed vertebrae and showed no symptomatic improvement. Striking changes took place in urinary calcium and hydroxyproline, in serum alkaline phosphatase, and to a less extent in serum calcium and phosphate. These studies are continuing and are being compared with the effects of mithramycin.     

颗粒体病毒离体复制

迄今 ,尚未建立稳定的颗粒体离体复制系统 ,颗粒体病毒在离体复制中的研究仍处于探索阶段。就颗粒体病毒离体复制的研究进展进行了综述 ,主要包括影响颗粒体病毒离体复制的细胞类型 ,培养温度以及颗粒体病毒离体复制的作用机理和目前仍存在的问题。     

Persistent Infection Promotes Cross-Species Transmissibility of Mouse Hepatitis Virus

Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.     

Studies on the Persistent Infection with Measles Virus in HeLa Cells

HeLa/MV cells which have previously been found to contain measles antigen in more than 90% of cells were very similar to normal HeLa cells in their morphology and growth. Although almost all of HeLa/MV cells may be infected, only 10% of them released infectious virus particles. The hemadsorption test, however, showed that most of the infected cells produced by hemagglutinin. Two kinds of clones were obtained by cloning HeLa/MV cells in the presence of anti-measles serum. One was the virus-releasing clone and the other the uninfected clone which did not contain any measles antigen. The proportions of virus-releasing clones to all clones varied between 20 to 54%, and did not show any increase even after recloning of the virus-releasing clones. The susceptibility of the uninfected clones to the standard measles virus was not different from that of normal HeLa cells.