Persistent Varicella-Zoster virus infection in a human rhabdomyosarcoma cell line and recovery of a plaque variant.

Varicella-zoster virus (VZV) has been found to persistently infect the human rhabdomyosarcoma cell line A204. Infectious center assays and fluorescent antibody staining demonstrated continuous production of infectious VZV and viral antigen. The level of infection determined by fluorescent antibody staining was variable, and usually only a small percentage of the cells were capable of producing plaques in permissive fibroblasts. The extent of infection was similar in cell cultures passaged at split ratios of 1:2 or 1:10 and grown at 33 or 37 degrees C. VZV recovered from A204 cells several months after establishment of the persistent infection had markedly increased syncytia-forming activity as compared with the parental VZV grown in human diploid fibroblast cells and the three monkey kidney-derived cell lines Vero, CV-1, and MA104. The expression of this altered phenotype continued after serial passage of the cell-associated virus in human diploid fibroblast and Vero cells. Consequently, we designated the reisolated VZV as plaque variant A. The buoyant densities of VZV plaque variant A and VZV DNAs in CsCl gradients were indistinguishable.     

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.     

Dependence of the synthesis of virion structures in the tick-borne encephalitis virus on the cell culture type

Variations in synthesis of antigenic structures are observed in tick-borne encephalitis virus replication in cell cultures of different origin. In a number of cell cultures: pig and Syrian hamster embryo kidney cells, as well as in Chinese striped hamster cells and Tasmanian rat kangaroo cells, virion antigens (VA) are synthesized which differ in the direction of movement in the electric field, namely, anode and cathode VA. In other cell cultures: green monkey kidney, barking deer kidney, and human fibroblasts, only cathode VA is synthesized. In chick embryo fibroblast cultures, in addition to the above-mentioned VA, considerable amounts of a VA which does not move in the electric field are synthesized. In all the cultures, a low molecular nonvirion antigen (NA) is actively produced, the virus-containing fluids of human fibroblast cultures and Chinese striped hamster kidney cell cultures contain lower amounts of high molecular NA, while rat kangaroo and green monkey kidney cell cultures have no high-molecular NA of tick-borne encephalitis virus.     

Biological properties of simian virus 40 host range mutants lacking the COOH-terminus of large T antigen

Three mutants of simian virus 40 (SV40), with deletions near the 3' end of the A gene, displayed a host range phenotype for growth and virus production in various African green monkey kidney cell lines. The mutants formed plaques in CV-1P cells at 40.5 degrees, in BSC-1 cells at 37 and 40.5 degrees, and in Vero cells at 32, 37, and 40.5 degrees. Virus yields in these three lines were cold sensitive: the burst size was greatest at 40.5 degrees and least at 32 degrees, but some progeny was produced under all conditions examined. Mutant yields never exceeded 10% of wild-type yields under the most permissive conditions (Vero cells at 37 or 40 degrees) and were less than 1% of wild type under the most restrictive conditions (CV-1P cells at 32 degrees). These mutants can be complemented by any SV40 mutant which produces a large T antigen containing a normal COOH-terminus. Mutants whose T antigens could not be transported to the nucleus were most efficient at complementation. Mutant virus production in a line of rhesus monkey kidney cells and in primary cultures of African green and rhesus monkey kidney cells was also substantially below wild type. These mutants were also completely defective for adenovirus helper function. Our data suggest that the host range property and adenovirus helper function represent the same activities of large T antigen.     

Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype.

Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.     

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus

Summary Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P_7·5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.     

Advantages of multiple cell culture systems for detection of mixed-virus infections.

Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients. In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory. Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture. For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation. During 1989, nine cases of multiple-virus isolations were obtained in this laboratory. In all nine patients, CMV was detected in MRC-5 cells. Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus. All four adenovirus isolates were obtained in A549 cells. Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g. MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only. For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only. Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.     

Helper function for adenovirus replication in monkey cells by BK human papovavirus.

CV-1 monkey kidney cells are semipermissive for BK human papovavirus at 37°. Although infected cells synthesize T antigen at this temperature, only a small percentage of the cells (less than 5%) produce viral antigen. However, when infected cells were incubated at 40°, characteristic CPE was observed with high virus yields. The inhibition of BKV growth in CV-1 cells was thus shown to be a temperature-dependent phenomenon. Experiments were then conducted to determine whether BKV provided a helper function for adenovirus growth in CV-1 cells at temperatures which were either permissive (40°) or semipermissive (37°) for BKV replication. At 37°, there was a low level of adenovirus enhancement of 0.5 to 1.0 log increase. However, at 40°, there was a 1.5 to 2.5 log increase in adenovirus yields, comparable to those obtained by coinfection with SV40 and adenovirus. These data provide additional information on common viral functions shared by BKV and SV40.     

Reproduction of Lassa virus in different cell cultures

Sierra Leone strain of Lassa virus was growing to high titres of 10(5)-10(6) plaque forming units (PFU) per ml in Vero, L and swine kidney cell lines as well as in diploid human cells and primary human embryo kidney cells. As many as 80% of the cells became infected as demonstrated by the immunofluorescence (IF) technique. In BHK-21, CV-1, HeLa, FL, HEp-2 and dog kidney cell lines, the virus reproduced to lower titres (10(4)-10(5) PFU per ml), whereas in primary chick embryo fibroblasts it did not multiply at all. The virus formed plaques under agar overlay only in CV-1 and Vero cells.     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

Alternative cell line for virus isolation.

A human lung carcinoma cell line (A549) was compared with various other cell lines to determine susceptibility to viral growth. In the first phase of the study, A549 cells were compared with human embryonic kidney (HEK) and cynomolgus monkey kidney (CMK) cells for isolation of upper-respiratory disease viruses by using 1,248 throat swab specimens from basic-combat trainees. Of the 552 virus isolates, 507 were adenoviruses, 41 were polioviruses, and 4 were herpes simplex viruses (HSV). Of the isolates, 518 (93.8%) were isolated in A549 cells, 480 (87.0%) were isolated in HEK cells, and 262 (47.5%) were isolated in CMK cells (P less than 0.001). In the second phase of the study, A549 cells were compared with a human diploid fibroblast cell strain (MRC-5) and Vero monkey kidney (VMK) cells for the isolation of HSV from 1,157 specimens submitted for culture. Of the 227 HSV isolates, 210 (92.5%) were isolated in A549 cells, 202 (89.0%) were isolated in VMK cells (P greater than 0.1 for A549 versus VMK cells), and 167 (73.6%) were isolated in MRC-5 cells (P less than 0.001 for A549 versus MRC-5 cells). These results suggest that A549 cells are more susceptible to adenovirus infection and at least as susceptible to HSV infection compared with the other cell cultures evaluated. Detracting factors for the use of A549 cells were a slight loss of sensitivity to adenovirus at passage 120 and a concurrent change in the morphology of the cells. The A549 cell line proved to be an efficient, practical, and economical alternative cell system for the isolation of adenovirus and HSV in particular. Initial indications are that other clinically significant viruses may be grown in A549 cells; however, additional studies need to be performed.     

HBV cccDNA在2.2.15细胞表达的动态观察

目的研究2.2.15细胞中是否存在HBVcccDNA,探讨2.2.15细胞内HBV产物的动态表达规律。方法采用PCR方法检测2.2.15细胞内cccDNA,Taqman定量PCR技术检测2.2.15细胞内及培养上清中HBVDNA含量,EIA方法检测培养上清中HBsAg、HBeAg的动态表达,并进行定量资料相关性统计学分析。结果2.2.15细胞及培养上清中存在cccDNA,2.2.15细胞培养上清中HBVDNA与HBsAg、HBeAg之间存在相关性(r=0.833,P<0.05和r=0.939,P<0.01),而细胞内HBVDNA与培养上清HBVDNA及HBsAg、HBeAg之间无相关性(r=0.024,P>0.05和r=0.177,P>0.05)。结论为阐明2.2.15细胞内HBV的复制规律提供一定依据。     

Ultrastructural changes induced by influenza viruses in permissive and nonpermissive cells

Human diploid fibroblast (LEP) cells and hamster embryo fibroblast (HEF) cells were infected with four influenza viruses, viz., A/NWS, A/WS, and their recombinants r₁₂ and r₁₄. NWS virus could be propagated in both cell systems and the recombinants could be propagated in HEF cells only, while WS virus could not be passed in either type of cells. After 24 hr, or earlier, all of the cells in both types of culture infected with any of the four viruses produced hemagglutinin and a great majority of them also produced ribonucleo-protein antigen. Electron microscopic examination revealed all of the ultrastructural changes typical of influenza virus replication in HEF and LEP cells when these were infected with NWS virus and in HEF cells when these were infected with WS virus or either of the recombinants. In LEP cells infected with the latter three viruses, only a low percentage (less than 5%) of cells exhibited such alterations; a great majority were free of any detectable change. These results indicate that cells can be infected with influenza virus and synthesize some virus-specific products without being morphologically altered.     

Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics.     

Hepatitis B virus (HBV) antigen-pulsed monocyte-derived dendritic cells from HBV-associated hepatocellular carcinoma patients significantly enhance specific T cell responses in vitro

To investigate whether hepatitis B virus (HBV) antigen-pulsed monocyte-derived dendritic cells (MoDC) could mount a T cell response in hepatocellular carcinoma (HCC) patients associated with chronic HBV infection, peripheral blood mononuclear cells (PBMCs) from 36 HBV-associated HCC patients were induced into MoDC and pulsed with hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg), alone and in combination. Co-stimulatory molecules CD80, CD86 and CD40, as well as human leucocyte antigens D-related (HLA-DR) were found to express at the highest level on MoDC pulsed with HBcAg or HBsAg + HBcAg, at a median level on MoDC pulsed with HBcAg or HBsAg alone, and at the lowest level on non-antigen-pulsed MoDC. Interleukin (IL)-10 and IL-12 cytokines were released by antigen-pulsed MoDC at increased levels in the order: no-antigen < HBsAg < HBcAg < HBcAg + HBsAg. MoDC pulsed with HBcAg or HBsAg + HBcAg also had the strongest ability to stimulate autologous T cell proliferation and intracellular interferon (IFN)-gamma production. HBcAg- or HBsAg + HBcAg-pulsed MoDC could also induce HBV core peptide-specific CD8(+) T cell proliferation determined by tetramer staining. In addition, the antigen-pulsed MoDC were found to have a stronger capacity to produce IL-12 and induce T cell response in vitro for patients with higher alanine transaminase (ALT) levels than those with lower ALT levels, indicating that antigen pulse could substantially reverse the impaired function of MoDC in primary HCC patients with active chronic hepatitis B. In conclusion, HBV antigen-pulsed MoDC from HCC patients with chronic hepatitis B could induce HBV-specific T cell response in vitro.     

Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy

The prevalence of hepatitis B virus vaccine escape mutants has increased as a consequence of the introduction of global vaccination programs. Furthermore and as a consequence of the organization of the genome of hepatitis B virus (HBV) into overlapping reading frames, the selection of polymerase mutants during long-term lamivudine therapy can select viruses with changes in the overlapping S gene coding for the hepatitis B small antigen (HBsAg). We have investigated the role of lamivudine in selecting HBV mutants with antigenically altered HBsAg protein using pooled human vaccine sera in enzyme immunosorbent assays and radioimmunoassays. HBsAg proteins containing the vaccine escape mutations G145R and D144E/G145R demonstrated markedly reduced binding to anti-HBs antibody. HBsAg mutants including E164D, W196S, I195M, M198I, and E164D/I195M (corresponding to the polymerase protein changes of V519L, M550I, L526M/M550V V553I, and V519L/L526M/M550V) selected during lamivudine treatment also demonstrated reduced binding to anti-HBs antibody. These findings raise the possibility of lamivudine-resistant mutants arising that possess antigenically distinct HBsAg proteins.     

Growth of SV40, adeno 7 and SV40-adeno 7 viruses in monkey and human cells at 29° and 37°C

Adeno 7 virus replicated well in human diploid (LEP) cells but only to a low degree in green monkey kidney (GMK) cells at 37 degrees C; it did not replicate in either system at 29 degrees C. At 37 degrees C SV 40 virus replicated well in GMK cells but only moderately in LEP cells; at 29 degrees C it did not replicate in either system. SV 40-adeno 7 hybrid grew in both GMK cells and LEP cells at 37 degrees C. At 29 degrees C this virus replicated in GMK cells but not in LEP cells. While the formation of V-antigen generally corresponded to the infectious virus production in the respective system, considerable differences were encountered in the T-antigens production. Adeno 7 T-antigen was detected earlier and in a higher percentage of GMK cells than in the fully permissive LEP cells and its formation was only slightly influenced by the incubation temperature. SV 40 T-antigen was more efficiently formed in GMK cells than in LEP cells. At 29 degrees C SV 40 T-antigen was only found in GMK cells and was detected later than at 37 degrees C. The difference in the formation of SV 40 T-antigen in GMK cells infected with SV 40 and SV 40-adeno 7 hybrid virus was further analyzed. The results obtained suggest that an early step of the virus-cell interaction, but neither virus attachment nor penetration, was involved.     

CV-1 and MRC-5 mixed cells for simultaneous detection of herpes simplex viruses and varicella zoster virus in skin lesions.

BACKGROUND: Culture for varicella zoster virus (VZV) is relatively insensitive. Herpes simplex viruses (HSV) culture methods, which rely on primary rabbit kidney (pRK), mink lung (Mv1Lu) or the ELVIS HSV culture system fail to detect VZV. Culture of atypical vesicular skin lesions should be able to detect both HSV and VZV. OBJECTIVES: In this study, we evaluated the sensitivity of a newly developed mixture of CV-1/MRC-5 cells for the concurrent detection of both HSV and VZV. STUDY DESIGN: The CV-1/MRC-5 mixed cells were compared with pRK cells and Mv1Lu cells for the detection of HSV and to MRC-5 and A-549 cells for the detection of VZV. Fresh clinical samples submitted for HSV culture, VZV culture, and/or direct immunofluorescent assay (DFA) as well as frozen clinical samples previously positive for VZV were used for these comparisons. RESULTS: This preliminary study suggest that CV-1/MRC-5 mixed cells are as sensitive as pRK and Mv1Lu cells for the detection of HSV and appear to be more sensitive than MRC-5 and A-549 cells for the detection of VZV. Although the sample size is small, pre-CPE staining with VZV specific monoclonal antibody (Mab) at day 2 post-inoculation may provide a rapid detection of VZV with these mixed cells, but not with MRC-5 or A549 cells. In addition, culture of VZV in mixed cells from fresh clinical specimens appears to be as sensitive as antigen detection by DFA. Finally, 1% of specimens from skin lesions submitted for HSV culture grew VZV, highlighting the importance of culturing for both VZV and HSV, particularly in the case of atypical lesions. CONCLUSION: CV-1/MRC-5 mixed cells are highly sensitive for the simultaneous culture of HSV and VZV. The ability to detect either HSV or VZV from skin lesions is important for patient management.     

Altered DNA binding and replication activities of JC virus T-antigen mutants.

Ten mutations were introduced into the JC virus (JCV) T antigen within a region corresponding to the SV40 T-antigen DNA binding domain (SV40 amino acids 131 to 220); nine of these increased homology between the two proteins in sequences critical for SV40 T antigen DNA binding. All mutant JCV T antigens bound to JCV and SV40 origins of DNA replication. Binding efficiency relative to the of wild-type JCV T antigen ranged from 83 to 301% for the JCV binding sites and from 44 to 240% for the SV40 binding sites. Nine mutant proteins promoted viral DNA replication in primary human fetal glial (PHFG) and CV-1 cells. In PHFG cells, promotion of DNA replication ranged from 26 to 220% relative to that of wild-type T antigen; in CV-1 cells it ranged from 14 to 522%. Coding sequences for five mutant proteins were transferred into the hybrid virus M1 (SV40) [M1(SV40) contains coding sequences from JCV and regulatory sequences from SV40]. Wild-type T antigen promoted replication weakly from the SV40 origin in these hybrid viruses in CV-1 cells (2% that from the JCV origin); replication driven by the mutant proteins ranged from 110 to 412% of that induced by the wild-type protein. Efficient specific DNA binding by a mutant T antigen was not a reliable indicator of that mutant protein's ability to promote DNA replication.