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PubMed1985

Molecular cloning of the genome of a cardiotropic Coxsackie B3 virus: full-length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.

The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is characterized by restriction enzyme analysis and partial nucleotide sequencing of the 5' end. The Coxsackie B3 virus cDNA described can now be used to study the molecular basis of human enteroviral heart disease, and it provides a valuable diagnostic means for patients with suspected viral heart disease.

Animals↗

PubMed1996

Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID50) in hamsters was determined. The ID50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10(3) cells. The loss of the 27.5- and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID50 from 10(3) to 10(5) cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID50 > 10(3) cells) occurred with cells that lost the 24.7- and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by two-dimensional polyacrylamide gel electrophoresis.

Animals↗

PubMed1993

Infection of human monocyte-derived macrophages by human immunodeficiency virus mediated by cell-to-cell transmission.

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Base Sequence↗

PubMed2005

Molecular cloning and in vitro evaluation of an infectious simian-human immunodeficiency virus containing env of a primary Chinese HIV-1 subtype C isolate.

Human immunodeficiency virus (HIV) clade C is the most prevalent subtype and accounts for approximately 50% of all HIV infections worldwide. In China, the prevalent HIV strains are B'/C subtypes, in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in non-human primate models, we constructed an infectious simian-human immunodeficiency virus (SHIV) that expresses most of the envelope of a primary HIV strain, which was isolated from a HIV-positive intravenous drug user from XinJiang province in China. The resulting chimeric SHIV-XJ02170 was infectious in human, rhesus monkey and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and used CCR5 exclusively as coreceptor.

Animals↗

PubMed2005

Feline calicivirus VP2 is essential for the production of infectious virions.

The third open reading frame (ORF3) located at the 3' end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5' end and small deletions in the 3' end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3' end.

Animals↗

PubMed2002

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection.

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.

Animals↗

PubMedDate not supplied

Acquisition and transmission of Agrobacterium by the whitefly Bemisia tabaci.

Whiteflies transmit many different plant pathogens. We show here that the whitefly Bemisia tabaci can also acquire Agrobacterium tumefaciens from liquid cultures and from crown galls, and transmit it to plants. Cloned tomato yellow leaf curl virus (TYLCV) DNA and A. tumefaciens tumor-inducing functions were used as reporter genes. Whiteflies were fed through membranes on cultures of Agrobacterium At::pTY4 containing a dimeric copy of an infectious TYLCV genomic clone between the T-DNA borders of a binary vector. One hour of feeding was sufficient for the insects to be able to transmit TYLCV to tomato test plants. Infectious Agrobacterium was recovered from whiteflies that fed on At::pTY4 cultures, indicating that bacteria was acquired and remained intact in the insects. Whiteflies also acquired the virulent A. tumefaciens strain C58 from crown galls and induced tumors in tomato test plants. PCR and Southern blot analyses indicated that the target plant tissues were transformed. These results show that an insect can transfer foreign genes to plants by acquiring and delivering transforming bacteria.

Animals↗

PubMed1997

The yeast Saccharomyces cerevisiae as a genetic system for obtaining variants of poliovirus protease 2A.

The inducible expression of poliovirus protease 2A (2Apro) blocks the growth of Saccharomyces cerevisiae. A number of yeast colonies that grow after 2Apro induction have been isolated. The majority of these clones express 2Apro to control levels, suggesting that their ability to divide is not due to the loss of 2Apro gene inducibility. The sequences of the 2Apro genes isolated from 22 clones were determined. Most of the 2Apro sequences from these colonies contain point mutations in the poliovirus protease. The different variant protease sequences were transferred to an infectious poliovirus cDNA clone. Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2Apro yielded five viable polioviruses with a small plaque phenotype. These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C' and 3D'. All 2Apro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2Apro variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. cerevisiae represents a useful system for obtaining poliovirus 2Apro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.

Amino Acid Sequence↗

PubMed1990

An in-vivo infectivity assay for cloned retroviruses lacking a susceptible cell culture.

The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process. Recently we have molecularly cloned the viral genome. The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome. Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus. According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained. The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia. The applicability of this in-vivo assay for other cloned viral genomes is discussed.

Animals↗

PubMed1989

Molecular and biological characterization of a replication competent human immunodeficiency type 2 (HIV-2) proviral clone.

We obtained complete genomic clones of human immunodeficiency virus type 2 (HIV-2) from the DNA of the neoplastic human cell line HUT 78 freshly infected with a HIV-2 isolate, strain SBL6669. The recombinant phage DNA was transfected into the lymphocytes of CD4-positive HUT 78 cell line to test the replication competence of the proviral DNA. One genomic clone, designated HIV-2SBL/ISY, yielded retroviral particles after a few weeks of culture of the transfected cells. The HIV-2SBL/ISY clone contained a complete provirus and cellular flanking sequence. We obtained the DNA sequence of the provirus and compared it with the published sequence of two other HIV-2 isolates. The degree of variability among HIV-2 isolates is comparable to that observed among African HIV-1 isolates sequenced to date. Immunologically, HIV-2SBL/ISY is similar to the parental virus (HIV-2SBL6669) but differs in the envelope transmembrane protein that is truncated (gp32-34) in the parental virus and not in HIV-2SBL/ISY (gp41). Both the parental and the cloned viruses are infectious and cytopathic for some human T-cell lines, induce syncytia, and infect a human macrophage cell line (U937) in vitro. The availability of a biologically active HIV-2 clone provides the means to study the role and interaction of HIV-2 genes in vitro as well as to assess the functional similarities among HIV-1 and HIV-2 genes. Since HIV-2SBL/ISY cloned virus infects fresh peripheral blood T cells from Rhesus macaques in vitro and infects the same animal in vivo, its use in animals may represent a model for functional study of viral genes in vivo as well as for development of experimental approaches to prevent and cure retroviral infection in humans.

Blotting, Southern↗

PubMed1997

Genetic analysis of the hepatitis C virus (HCV) genome from HCV-infected human T cells.

We recently established a cell culture system for the replication of hepatitis C virus (HCV) by using a human T cell leukaemia virus type I-infected cell line, MT-2, and showed that the quasi-species of the hypervariable region 1 observed in the original inoculum became homogeneous in MT-2 cells 10 days after inoculation of HCV. In this study, we obtained HCV cDNA clones covering the whole viral genome by RT-nested PCR using RNA from HCV-infected cloned MT-2C cells, which support viral replication more efficiently, at 12 days after inoculation. A total of 41 distinct HCV cDNA clones covering almost the whole viral genome were also isolated from a cDNA library derived from the original inoculum. Molecular evolutional analyses comparing the sequences of the HCV clones obtained from both sources revealed that the HCV populations became homogeneous in more than half of the compared regions. This finding suggests that limited HCV populations are able to replicate in MT-2C cells. In addition, we isolated cDNA clones containing a 3' X-tail sequence, which was recently identified as a bona fide 3' terminus of the HCV genome, in the HCV-infected MT-2C cells and confirmed that the nucleotide sequence of the 3' X-tail was highly conserved, suggesting its implication in HCV replication. Finally, on the basis of the sequences of HCV cDNA clones obtained from HCV-infected MT-2C cells, we determined the entire nucleotide sequence of the HCV genome containing the 3' X-tail as a candidate for an infectious HCV molecular clone.

Base Sequence↗

PubMed2003

Epstein-Barr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction.

To assess the impact of the germinal center (GC) reaction on viral spread in Epstein-Barr virus (EBV) infection, we isolated EBV(+) GC B cells from the tonsils of two infectious mononucleosis patients, sequenced their rearranged V genes, and determined expression of the EBV latency genes EBV nuclear antigen 2 and latent membrane protein 1. Most EBV(+) GC B cells belonged to clones of cells harboring somatically mutated V gene rearrangements. Ongoing somatic hypermutation, the hallmark of the GC reaction, was seen only in uninfected GC B cell clones, not in EBV(+) B cell clones. Thus, in infectious mononucleosis, GC and/or memory B cells are directly infected by EBV and expand without somatic hypermutation, whereas the GC passage of EBV-infected naive B cells does not contribute detectably to the generation of infected memory B cells, the main reservoir of EBV during persistence. Most, if not all, EBV-infected cells in GCs exhibited an unusual EBV gene expression pattern in that they were positive for EBV nuclear antigen 2 but negative for latent membrane protein 1. Although the three main types of EBV-associated B cell lymphomas (Burkitt's, Hodgkin's, and posttransplant lymphomas) presumably are derived from GC B cells, EBV(+) GC B cells resembling these EBV(+) GC B cell lymphomas in terms of EBV gene expression and somatic hypermutation pattern could not be identified.

Adolescent↗

PubMed1991

Direct evidence of a role for amino acid 101 of VP-1 in central nervous system disease in Theiler's murine encephalomyelitis virus infection.

The DA virus, a member of the TO subgroup of Theiler's virus, invokes a chronic demyelinating disease in its natural host, the mouse, RNA transcripts from a cDNA clone, pDAFL3, are infectious, and the resulting virus, DAFL3, produces in mice a disease indistinguishable from that caused by the DA virus. Using oligonucleotide-directed site-specific mutagenesis, a single nucleotide, cytosine at position 3305 (viral genome), was changed in this infectious cDNA to a thymine. The mutated nucleotide is located in an area coding for a neutralizing epitope on loop II of VP-1. Virus OSM101, produced from the mutagenized plasmid pDA101, had the same growth characteristics and plaque phenotype in vitro as the virus DAFL3 produced from clone pDAFL3. However, in vivo in the mouse, virus OSM101 was markedly less neurovirulent than DAFL3. Central nervous system tissues from mice infected 4 to 6 weeks previously with the OSM101 virus contained less infectious virus and fewer infected cells than central nervous system tissues from animals infected with the control virus, DAFL3. Thus, we demonstrated that the single nucleotide change resulting in an amino acid substitution at position 101 (threonine to isoleucine) of VP-1 determines one aspect of Theiler's virus persistence and disease in mice.

Animals↗

PubMed1999

Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants.

We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in E. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

Chromosomes, Bacterial↗

PubMed2003

Identification and molecular characterization of a naturally occurring RNA virus mutant defective in the initiation of host recovery.

The host recovery response is characterized by the disappearance of disease symptoms and activation of the RNA silencing virus resistance in the new growth following an initial symptomatic infection. However, it is not clear what triggers the initiation of recovery, which occurs naturally only in some virus-host interactions. Here we report the identification and characterization of a spontaneous mutant of Tobacco streak virus (TSV) that became defective in triggering recovery in tobacco plants. Infectious full-length cDNA clones corresponding to the tripartite RNA genome were constructed from both the wild-type and the nonrecovery mutant of TSV (TSVnr), the first sets of infectious cDNA clones from an Ilarvirus. Genetic and molecular analyses identified an A --> G mutation in the TSVnr genome that was sufficient to confer nonrecovery when introduced into TSV. The mutation was located in the intergenic region of RNA 3 upstream of the mapped transcriptional start site of the coat protein mRNA. Intriguingly, induction of recovery by TSV was not accompanied by virus clearance and TSV consistently accumulated to significantly higher levels than TSVnr did even though TSVnr-infected plants displayed severe symptoms throughout the course of infection. Thus, our findings indicate that recovery of host can be initiated by minimal genetic changes in a viral genome and may occur in the absence of virus clearance. Mechanisms possibly involved in the initiation of host recovery are discussed.

Cloning, Molecular↗

PubMed2001

Recent advances in the molecular biology of hepatitis C virus.

The Hepatitis C virus is a positive-stranded RNA virus which is the causal agent for a chronic liver infection afflicting more than 170,000,000 people world-wide. The HCV genome is approximately 9.6 kb in length and the proteome encoded is a polyprotein of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into structural and non-structural proteins. The functions of most of these proteins have been established by analogy to other viruses and by sequence homology to known proteins, as well as subsequent biochemical analysis. Two of the non-structural proteins, NS4b and NS5a, are still of unknown function. The development of antivirals for this infectious agent has been hampered by the lack of robust and economical cell culture and animal infection systems. Recent progress in the molecular virology of HCV has come about due to the definition of molecular clones, which are infectious in the chimpanzee, the development of a subgenomic replicon system in Huh7 cells, and the description of a transgenic mouse model for HCV infection. Recent progress in the structural biology of the virus has led to the determination of high resolution three-dimensional structures of a number of the key virally encoded enzymes, including the NS3 protease, NS3 helicase, and NS5b RNA-dependent RNA polymerase. In some cases these structures have been determined in complex with substrates, co-factors (NS4a), and inhibitors. Finally, a variety of techniques have been used to define host factors, which may be required for HCV replication, although this work is just beginning.

Animals↗

PubMed2002

Cell clones selected from the Huh7 human hepatoma cell line support efficient replication of a subgenomic GB virus B replicon.

Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.

Amino Acid Sequence↗

PubMed2003

Development and application of a reverse genetics system for Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5' and 3' ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>10(6) PFU/ micro g), and these cells rapidly generated a high titer of synthetic viruses (>5 x 10(6) PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

Animals↗