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.     

Sindbis virus infection of two model insect cell systems--a comparative study

Sindbis, the prototype of the Alphaviruses causes mosquito-borne diseases in mammals and replicates in a wide variety of vertebrate and invertebrate cell cultures. This characteristic can be exploited to use the vast array of Drosophila genetic information available for investigations of the interaction of Sindbis virus with an alternate invertebrate host. For this purpose, a comparative study of Sindbis virus infection of Schnieder-2 Drosophila (S2) cells to cells of the mosquito Aedes albopictus (clone U4.4) was undertaken. After infection, vertebrate cells die within 24-48h, while invertebrate cell cultures survive an acute phase of infection and become persistently infected. In this study, infection of a model Drosophila system, S2 cells, was compared to U4.4 cells. Virus production, the time course of the establishment of persistence and changes in growth properties of the S2 cells upon infection, were studied in comparison to those of the U4.4 cells. S2 cells survived acute Sindbis infection without any significant cytopathology and continued to produce low levels of virus characteristic of persistently infected cells. S2 cells produced 10 PFU/cell on day 1 post-infection, which falls to 2 PFU/cell on day 2. This result is in contrast to U4.4 cells, which produce peak virus titer on day 2 post-infection and establish persistence by day 5. Onset of the persistent phase of infection of either U4.4 or S2 cells did not result in any change in morphology or growth characteristics. This study establishes S2 cells as an additional invertebrate model system to study the interactions of an invertebrate host with Sindbis virus.     

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA is involved in establishing and maintaining a persistent infection, the intracellular viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3′ end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.     

Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system

Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins.     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。     

Mosquito densonucleosis viruses cause dramatically different infection phenotypes in the C6/36 Aedes albopictus cell line

Mosquito densoviruses generally establish persistent infections in mosquito cell lines including the C6/36 Aedes albopictus cell line. In contrast, the closely related Haemagogus equinus densovirus (HeDNV) causes dramatic cytopathic effects in the C6/36 Aedes albopictus cell line. Infection of C6/36 cells by HeDNV causes internucleosomal fragmentation of host chromosomal DNA, changes in cellular morphology (membrane budding, apoptotic bodies), caspase activation and exposure of phosphatidylserine on the cellular membrane. This is accompanied by a higher rate of infection and more vigorous production of virus in these cells. These observations are consistent with the induction of apoptosis during infection. In contrast, expression of AeDNV proteins in C6/36 cells does not cause obvious cytopathic effects although NS1 expression causes accumulation of cells in G2 phase. C6/36 cells persistently infected with AeDNV were not protected from superinfection with HeDNV. Thus, there does not seem to be an antiviral state induced by AeDNV persistent infection.     

Viral RNAs synthesized in cells infected with Germiston Bunyavirus

A rapidly growing strain of Germiston virus was used to study intracellular viral RNA synthesis in BHK cells. The RNAs were separated by electrophoresis into seven bands which fell into three size classes: large (bands L1 and L2), medium (bands M1 and M2), and small (bands S1, S2, and S3). Blot hybridisation established that bands L1, M1, and S1 contained the negative-sense genomic RNAs, while bands L2, M2, S2, and S3 contained positive-sense RNAs complementary to the genomic RNAs within the same size class. After glyoxal treatment the RNAs separated into a large, a medium, and two small bands, indicating that the positive-sense RNAs originally present in bands L2, M2, and S2 are similar in size to their genomic RNAs, while the RNA in S3 is shorter than the small genomic segment. These results suggest that band S2 contains the replicative intermediate RNA and band S3 the messenger RNA of the small genomic segment and also that bands L2 and M2 contain both replicative intermediate and messenger RNAs. Long after virus development had ceased in the infected cells the amounts of RNAs in bands L1, M1, S1, and S2 remained the same, those in bands L2 and M2 were reduced, while only trace amounts of RNAs were observed in band S3, suggesting that the genomic RNAs and the replicative intermediate RNAs form ribonuclease-resistant ribonucleoprotein complexes while the messenger RNAs do not form such complexes. Synthesis of RNA in the infected cells was first evident in bands S3 and M2, after which synthesis was soon observed in all seven bands reaching a maximum rate at the logarithmic phase of growth, suggesting that the pattern of Germiston virus development resembles that of other negative-strand RNA viruses. The presence of defective-interfering particles was indicated by the observation that purified virus preparations contained a minor RNA component originating from the large RNA segment.     

Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus

Replicate cultures of Aedes albopictus cells were infected with Sindbis virus and then maintained for long periods of time by weekly subculture. During the first week the viral titers ranged between 10⁸ and 10⁹ PFU/ml, but then gradually fell and within a few weeks stabilized at about 10⁵ to 10⁸ PFU/ml. Such cultures were followed for the appearance of temperature-sensitive (ts) virus, small-plaque virus, and for the appearance within the cells of small (12–15 S) double-stranded viral RNA (dsRNA). Cloning experiments carried out 6 months or more after the initial infection showed that persistently infected cultures gave rise to both virus-yielding and nonyielding clones. Similar results were obtained when virus-positive clones were recloned. Prolonged treatment of persistently infected cultures with anti-Sindbis virus serum resulted in curing of the virus infection. Cured cultures behaved in every way tested as normal uninfected A. albopictus cells. The ability to cure with anti-viral serum suggests that in this system extracellular virus is needed to perpetuate the long-term infection. After persistently infected cultures were subcultured, viral RNA synthesis and viral yields were maximal during the first 3–4 days. Thereafter, although the cell number continued to increase for several days, viral RNA synthesis and viral yields both decreased sharply. This result strongly suggests that A. albopictus cells have efficient means for the regulation of viral biosynthesis. Although the resistance of persistently infected cultures to superinfection could be accounted for by interfering temperature-sensitive nondefective virus, the presence in these cells of 12 S dsRNA suggests that defective viral genomes are also present.     

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.     

In vitro replication of Macrobrachium rosenbergii nodavirus and extra small virus in C6/36 mosquito cell line

White tail disease (WTD) is a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India and many parts of the world. The pathogenic agents have been identified as M. rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which is 27nm and 15nm in diameter, respectively. Replication of MrNV and XSV was investigated in apparently healthy C6/36 subclone of Aedes albopictus cell line. The results revealed that C6/36 cells were susceptible to these viruses. The replication of these viruses in C6/36 cells was confirmed by RT-PCR, acridine orange staining, infectivity study and electron microscopy. Cytoplasmic ribonucleic acid (RNA) stained by acridine orange increased by 48h, and by 72h larger proportion of cells which indicated alterations in quantity and localization of RNA in the infected cells. Post-larvae, challenged by immersion method using inoculum prepared from infected cells, exhibited lethargy, anorexia and opaqueness of abdominal muscle and 100% mortality was observed at 6 days post-infection. Experimentally infected C6/36 cells and post-larvae showed positive by RT-PCR, whereas control cells and healthy post-larvae showed negative. This is the first study to report the multiplication of MrNV and XSV in C6/36 cell line.     

Detection of bluetongue virus serotype 17 in Culicoides variipennis by nucleic acid blot and sandwich hybridization techniques.

Molecular hybridization techniques were developed for the detection and surveillance of bluetongue virus (BTV) serotype 17 in the insect vector Culicoides variipennis, a biting midge. Radiolabeled RNA and cDNA probes were generated from sequences of the L3 segment of BTV serotype 17. These probes were used to detect BTV RNA in pools of infected C. variipennis by hybridizing the probes directly to analyte immobilized on nylon membranes or by using a nucleic acid sandwich hybridization test. Hybridization procedures were able to detect 1 infected C. variipennis in a pool of 50 and as little as 3.55 log10 50% tissue culture infective doses per ml of virus. These hybridization techniques provide an alternative to virus isolation for the surveillance of BTV in vector populations.     

Structure,restriction map and infectivity of the genomic and replicative forms ofAaPV DNA

Summary We have characterized the genomic and replicative form (RF) DNA of theAedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6/36 clone ofAedes albopictus cell line [22]. The genome ofAaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated from infected C6/36 cells was established. Among the 23 restriction enzymes tested, 14 cleaved theAaPV RF DNA and 30 restriction sites were mapped and oriented with respect to the viral genomic DNA. Both viral and RF DNAs were found infectious when transfected to virus-free C6/36 cells. The asymmetrical encapsidation of the viral genome is a property common to most vertebrate autonomous parvoviruses but rather unusual among densoviruses. Both by its small size, the asymmetrical mode of encapsidation and the restriction map, theAaPV genome resembles that of theAedes Densonucleosis virus [1].     

A novel tetraspanin C189 upregulated in C6/36 mosquito cells following dengue 2 virus infection

Dengue (Den) viruses cause apoptosis in mammalian cells, but usually result in high progeny yields without evident damage in mosquito cells. By using subtractive hybridization, 13 potentially virus-induced genes were selected in Den-2 virus-infected Aedes albopictus C6/36 cells. Based on semi-quantitative and real-time RT-PCR, one novel gene, named C189, was significantly upregulated in infected C6/36 cells. Its full-length of 678 nucleotides (nt) was determined by a combination of 5'- and 3'-RACE products. After alignment, C189 was classified as a member of the tetraspanin superfamily that typically has 2 short cytoplasmic sequences, 4 transmembrane domains, as well as small and large extracellular regions (EC1 and EC2). It contains the hallmark CCG motif in the EC2 region and additional 17 conserved nucleotides as do other tetraspanins. C189 was not upregulated by inoculation of UV-inactivated Den-2 virus to C6/36 cells. This suggests that tetraspanin upregulation is not related to virus binding to the cell surface, and that C189 does not function as a receptor for dengue virus entry. On the other hand, overexpression of C189 was concurrent with viral proteins, targeting the plasma membrane of C6/36 cells infected with Den-2 virus. It is presumably beneficial or essential for cell-to-cell spread of the virus due to the role of tetraspanins demonstrated in intercellular adhesion.     

Long-term effect of elevated temperatures on SSPE virus expression in persistently infected rat glial cells

Summary Cultivation of measles virus (SSPE virus, Lec strain) persistently infected C 6 rat glioma cells at 39°C resulted in the loss of detectable expression of measles virus proteins. Temperature shift-back led to reactivation of measles virus even after maintenance of the cells at 39°C for 15 days. In Northern blot analysis viral mRNA disappeared at 3 days after shift-up whereas 50 S viral genome-sized RNA was detectable until 6 days. The 50 S RNA decreased in quantity in rough correlation with dilution by cell passage at 39°C. The 50 S viral RNA was found in the nucleocapsid fraction. On day 9 after shift-down of persistently infected cells, maintained at 39°C for 15 days, 50 S viral RNA reappeared although mRNAs were not yet detected. Infectious center assays showed that the number of cells in the population at 39°C, which contained an SSPE virus genome that could be reactivated, declined after temperature shift. Moreover, cell cloning experiments, in which single cells of cultures maintained for various lengths of time at 39°C were incubated at 35°C and examined by immunofluorescence, reconfirmed the above results. This indicates that the reactivation of SSPE virus described here was due to re-infection of virus-antigen negative cells with progeny virus produced by a few latently infected cells in the population. The biological significance of this phenomenon in the central nervous system virus infection is discussed.On sabbatical leave from the Queen's University of Belfast, Belfast, Northern Ireland.     

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.     

Influenza C virus persistence depends on exceptional stepsin viral RNA synthesis and transport

Summary.  The cell line MDCK-pi, which is persistently infected with a variant of influenza C/AnnArbor/1/50 virus (C/AA-pi), was studied as a long-term persistence model by means of a strand-specific in situ hybridization assay. As atypical feature of the persistence, we identified a continuous synthesis of anti-genomic positive-strand RNA encoded by segment 7 (NS) during virus production. In contrast, infection with the parental wild-type virus led to a rapid reduction of antigenomic RNA as observed in the late period of replication particularly for RNA segment 7. Furthermore, the replication cycle of the persistent variant did not show the switch from early to late replication events followed by clearance of intracellular virus, but was regulated in terms of productive and nonproductive phases. Nonproductive phases were reversible and characterized by a low level of virus-specific RNA signals. In the productive phase, a difference in cytoplasmic RNA transport was detected for the two viruses: a marked cytoplasmic accumulation of negative- and positive-strand wild-type virus RNAs stood in contrast to a RNA localization in different cellular compartments for the persistent virus. Also, Vero cells infected with the C/AA-pi variant were restricted to a transient, nonpersistent replication cycle and produced a wild-type-like course of virus-specific RNA transport. These data indicate that influenza C virus persistence depends ona distinctly modified and cell type-specific regulation of virus-specific RNAsynthesis and transport. Received July 9, 1998 Accepted October 13, 1998     

Detection of dengue virus RNA using nucleic acid hybridization

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.     

Sindbis virus variants from a persistently infected mosquito cell culture contain an altered E2 glycoprotein

Small plaque temperature-sensitive (SPTS) variants of Sindbis virus were isolated from persistently infected Aedes albopictus cells 73 days after infection with wild-type virus. The viral RNA and protein species synthesized in chick embryo fibroblast (CEF) cells infected by one variant, clone 73-2, were compared with their wild-type virus counterparts. T1 RNase fingerprints of ³²P-labeled intracellular 42 S and 26 S viral RNA species revealed that clone 73-2 virus RNA differed from wild-type virus RNA in four oligonucleotide spots. Three of these were located in the region of the genome coding for structural proteins. Analysis of the virus structural proteins by polyacrylamide gel electrophoresis indicated that clone 73-2 virus glycoprotein E2 had a higher electrophoretic mobility than wild-type virus E2. Analysis of the proteins synthesized in infected cells in the presence and absence of tunicamycin revealed that both the glycosylated and unglycosylated forms of clone 73-2 PE2 migrated slightly faster than their wild-type virus counterparts.     

Detection of dengue virus type 2 in Aedes albopictus by nucleic acid hybridization with strand-specific RNA probes.

A molecular hybridization technique with radiolabeled, strand-specific RNA probes was developed to detect dengue virus type 2 RNA in pools of infected Aedes albopictus mosquitoes. One infected mosquito in a pool of 25 could be detected, corresponding to a dengue virus type 2 titer of 2.75 log10 50% tissue culture infectious doses.