Viral genome synthesis in BHK 21 cells persistently infected with Sendai virus

We have carried out daily analysis of the viral RNA species replicated by nucleocapsids present in the cytoplasm of two different BHK 21 cell lines persistently infected with Sendai virus. At each day of analysis (both before and after crises of cytopathology, and during long periods of stable growth) DI particle size RNA was the major species of viral RNA replicated within these nucleocapsids. The molar ratio of DI RNA to standard virus (50 S) RNA species within intracellular nucleocapsids was always extremely high (from 10/1 to 340/1). Thus, there is no evidence for cyclic variations in relative predominance of standard virus and DI nucleocapsids in populations of these persistently infected cells, although “cycling” could occur at the individual cell level at infrequent intervals. Furthermore, challenge of persistently infected cultures with standard Sendai virus or incubation at 33° also failed to significantly decrease the ratio of DI nucleocapsids to standard virus nucleocapsids. It was also found that multiple DI RNA species are present (most are not resolvable by sucrose gradient analysis), and that these evolve with time—some arising and increasing with time, while others are outcompeted. No selective advantage for DI RNA of any particular size was observed. Finally, by measuring the total amount of nucleocapsids present in these persistently infected cells (as estimated by level of NP protein in purified nucleocapsids), and the degree of synthesis of viral nucleocapsid (as determined by [³H]uridine incorporation in the presence of actinomycin D), we found that the rate of viral genome replication was 30- to 40-fold lower in the persistently infected cells than it was in St acutely infected cells. These findings may indicate that the accumulated nucleocapsids in persistently infected cells are built up over a period of many weeks of slow synthesis, although turnover times for NP protein and RNA of nucleocapsids have not yet been determined in persistently infected cells.     

Evidence for the synthesis of defective interfering particles by Aedes albopictus cells persistently infected with sindbis virus

The species of double- and single-stranded viral RNA in Aedes albopictus cells persistently infected with Sindbis virus have been analyzed. In addition to small amounts of 42-S and 26-S single-stranded and 23-S double-stranded viral RNA, persistently infected mosquito cells contain several new species of single-stranded RNA with molecular weights ranging from 0.65 x 108 to 1.0 x 108. New double-stranded RNA forms with sedimentation coefficients in the 12- to 15-S range are also detected. Similar double- and single-stranded RNA forms are synthesized in BHK cells after infection with virus released from persistently infected cells. Treatment of the virus from persistently infected cells with anti-Sindbis antiserum inhibits the synthesis of all the viral RNA species in BHK cells. These results suggest that DI particles of Sindbis virus may be synthesized and released from persistently infected A. albopictus cells.     

Transmission of viral persistence by transfection of human cultured cells with RNA of a persistent strain of echovirus 6.

A cloned line of persistently infected (PI) human cells has been established with a strain of the normally lytic, echovirus 6. All of the cells contained non-lytic viral RNA and synthesized defective viral particles. The present study was undertaken to determine whether replication of non-lytic viral RNA occurred after transfection. Uninfected human WISH cells were transfected with viral RNA recovered either directly from persistently infected PI cells or from virus particles produced by PI cells. Cytoplasmic extracts were prepared at various times after transfection and examined for presence of viral RNA and protein. The viral RNA was detected by hybridization of Northern blots of cellular RNA extracts with a cDNA clone of wild-type, lytic echovirus 6. Viral proteins were isolated by immunoprecipitation with specific anti-viral serum and analysed by polyacrylamide gel electrophoresis. Increased concentrations of viral RNA were detectable in cellular extracts at 48 h after transfection. Replicate transfected cultures retained viral RNA and produced viral proteins after cultivation for 287 days. RNA extracts from the transfected cells did not produce cytopathology or lytic virus. Thus, conversion of uninfected cells into a persistently infected cell line was accomplished by transfection with the non-lytic genome of echovirus 6.     

Viral RNA and protein synthesis in two LLC-MK2 cell lines persistently infected with human parainfluenza virus 3.

Two lines of LLC-MK2 cells persistently infected with human parainfluenza virus 3 (HPIV-3) have been maintained in culture for approximately 3 years. Subgenomic RNAs (putative defective interfering particle genomes) were detected in virions released from both persistently infected cultures. In one of the persistently infected cell lines cyclic variation in the production of virions containing standard virus genomic-size (50S) RNA and subgenomic RNA was observed. The molar ratio of subgenomic RNA to 50S RNA ranged from less than 0.1/1 to 8.7/1. Northern blot analyses revealed that the patterns of viral mRNA synthesis in persistently infected cells from both cultures were similar to those of standard virus infected cells. Furthermore, the intracellular viral-specific proteins had electrophoretic mobilities similar to the corresponding proteins in standard virus-infected cells. Nucleotide sequence analysis of cloned M gene from virus after 29 months of persistence (147 passages) revealed only one variable conservative amino acid change in two clones analyzed from each cell line, indicating that the M protein is not likely to be involved in the maintenance of the persistent infections. The possible mechanisms by which the persistent state is maintained are discussed.     

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.     

Oxytocin and prostaglandins F<Subscript>2α</Subscript> and E<Subscript>2</Subscript> do not enhance HIV antigen production <Emphasis Type="Italic">in vitro</Emphasis>

Summary.  Oxytocin and prostaglandins (PGs) are hormones involved in labor and are used clinically for its induction. In this study the effect of oxytocin, PGF_2α, and PGE₂ on Humour immunodeficiency virus-1 production in acutely and persistently infected cells was measured. No significant effect on p24 antigen production was found with oxytocin or PGs, except for a transient decrease in persistently infected cells treated with 1 μM PGF_2α. These results showed that oxytocin and PGs could be used clinically for labor induction without any direct enhancement in viral production. Besides, the results with PGF_2α at the highest concentration studied may indicate a pharmacological effect. Received October 10, 2002; accepted October 28, 2002     

Virus carrier state suppresses tumorigenicity of tumor cells in athymic (nude) mice.

Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.     

Persistent infection of Aedes albopictus C6/36 cells by Bunyamwera virus

Two cell lines persistently infected with Bunyamwera virus have been established from the C6/36 clone of Aedes albopictus cells. The cells express Bunyamwera virus antigens as detected by immunofluorescence and are resistant to superinfection with Bunyamwera virus and other bunyaviruses, but not Dugbe virus (Nairovirus) nor vesicular stomatitis virus. The virus released from the persistently infected cells developed an altered cloudy or "bull's-eye" plaque morphology with increasing passage level, and a greater temperature sensitivity at 39.5 degrees than standard virus. The persistent virus interfered strongly with the replication of standard Bunyamwera virus in normal C6/36 cells and to a much lesser extent in BHK cells. Interference was not noted with other bunyaviruses or vesicular stomatitis virus. The persistent virus from one cell line, C6/36-PI LO, had a slower migrating nucleocapsid protein on polyacrylamide gels. Analysis of the RNA in persistently infected cells or in persistent virus by Northern blot hybridization with cloned cDNA probes showed that the major viral RNA species was the S segment, while the L and M RNA segments were barely detectable. Our results indicate that Bunyamwera virus can readily establish persistent infections in mosquito cells, and that persistence is accompanied by the generation of viruses with variable genetic and phenotypic characteristics.     

Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman)

Real time quantitative RT-PCR (Taqman) identified specifically BVD virus in freshly processed formalin-fixed paraffin-embedded tissue sections and archival samples up to 7 years old. Samples included tissue from both acutely and persistently infected animals. To assess RNA degradation due to tissue handling and processing, freshly collected tissues from a calf persistently infected with BVD virus were stored at 4 degrees C or room temperature for up to 1 week prior to 24 h formalin fixation and routine histologic processing. Samples stored at 4 degrees C for up to a week prior to fixation were positive while samples stored at room temperature were positive at 74 h but became negative after 96 h. Fixation of fresh tissue in formalin for 1 week prior to processing resulted in a mild decrease in signal strength compared with tissue fixed for 24-48 h. Real time RT-PCR improves diagnosis of BVD infection by allowing prospective and retrospective identification of BVD virus in tissues processed routinely and stored for histologic evaluation.     

Heterologous resistance to superinfection by louping ill virus persistently infected cell cultures

Summary Louping ill virus, a tick-borne arbovirus readily established a persistent infection in porcine kidney (PS) cells after initially inducing minor cytopathic changes. Nucleotide sequence analysis of the envelope glycoprotein of the viral RNA recovered from the persistently infected cells showed no changes as compared with the virus used to establish persistent infections. More than 80 per cent of the cells contained virus specific antigen when analysed by indirect immunofluorescence microscopy. This persistently infected cell line resisted superinfection with either homologous or most heterologous flaviviruses. However, the yellow fever French neurotropic virus (YF FNV) multiplied in the persistently infected cells and evidence of dual infections in these cells was obtained using specific monoclonal antibodies in double labelling immunofluorescence tests. The relevance of these observations is discussed in the light of other evidence that tick-borne viruses can survive for long periods in wild animal species.     

The localization of persistent foot and mouth disease virus in the epithelial cells of the soft palate and pharynx

After contact with foot and mouth disease virus (FMDV), cattle may become persistently infected, regardless of their pre-existing immune status or whether they develop clinical disease. The cellular sites of FMDV persistence have not previously been determined. The use of in-situ hybridization in combination with tyramide signal amplification (TSA) provided the first direct evidence that FMDV RNA is localized within the epithelial cells of the soft palate and pharynx during persistent infection, indicating that these cells remain persistently infected after contact with FMDV. Copyright Harcourt Publishers Ltd.     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Lack of vertical transmission of Hantaan virus from persistently infected dam to progeny in laboratory mice

It is unclear how the hantaviruses are transferred from infected to uninfected rodents. We studied the status of persistently infected laboratory mice and examined the frequency of viral transmission to their offspring. Expression of Hantaan virus nucleocapsid protein was detected in the lungs of persistently infected dams. None of the progeny displayed viral antigen, although they were strongly positive for IgG antibodies against hantavirus. There was neither hantavirus RNA nor virus-specific IgM antibodies or virus-specific CD8(+) T cells in the progeny. These results did not show any indication for a vertical transmission of hantaviruses, at least in the laboratory mouse model studied.     

Site of suppression of Banzi viral replication by an antiviral factor released from Aedes albopictus cells persistently infected with Banzi virus

The ability of the antiviral factor present in culture medium of Aedes albopictus cells persistently infected with the flavivirus, Banzi virus, to inhibit the replication of Banzi virus was examined. The anti-Banzi viral factor did not inhibit the uncoating of the virion. Levels of viral RNA were markedly reduced in mosquito cells treated with the antiviral factor. Syntheses of negative-strand and of positive-strand viral RNA species were inhibited to approximately the same extent. This inhibition was virus-specific in that the anti-Banzi viral factor had no effect on the synthesis of viral RNA in mosquito cells infected with either Japanese encephalitis or Eastern equine encephalitis viruses. The anti-Banzi viral factor inhibited the in vitro Banzi viral RNA synthesis but not that of Eastern equine encephalitis virus or of Japanese encephalitis virus.