Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. A severe cytopathic effect occurred when BHK21 cells were transfected with in vitro-transcribed RNAs from either a DENV2 cDNA clone with multiple silent mutations within the prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of the JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited infectivities similar to those of their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160 to 243) of the core gene of DENV2 infectious cDNA or a subgenomic DENV2 replicon clone. This novel strategy of constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.     

中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT—PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3’末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5a中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到10^7～10^8PFU／ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaⅠ酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。     

A baculovirus-expressed dicistrovirus that is infectious to aphids

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5' and 3' ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.     

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.     

流行性乙型脑炎病毒(JEV)全长感染性克隆的制备及恢复病毒的获得

根据JEV病毒减毒株SA14—14—2基因组序列,设计覆盖全长的4对重叠引物,以提取的活疫苗病毒RNA为模板,RT—PCR扩增出4个片段,并克隆到质粒载体中,进一步构建两个半端分子克隆,然后将全长cDNA序列克隆到一个新改造的低拷贝质粒载体pBR—kpn中,构建我国流行性乙型脑炎病毒(JEV)基因组全长cDNA克隆。经过体外转录后得到的转录子转染BHK-21细胞,重新获得JEV的恢复病毒,通过生物学特性、分子生物学水平、蛋白水平等几个方面对恢复病毒进行鉴定。结果获得了稳定的全长cDNA克隆,转录子转染BHK-21细胞后,第4天开始出现细胞病变(CPE),第6～7天时CPE为 ,经过Vero细胞进一步放大培养后,间接免疫荧光实验和RT—PCR实验均为阳性。证实了构建的JEV的全长cDNA克隆有感染性,为进一步的研究奠定了基础。     

Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK(12). To rescue infectious virus, capped RNA was transcribed in vitro from the pOK(12) clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5' end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.     

Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.

A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV.     

Correlation between point mutations in NS2 and the viability and cytopathogenicity of Bovine viral diarrhea virus strain Oregon analyzed with an infectious cDNA clone

Cytopathogenicity of Bovine viral diarrhea virus (BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5'- and 3'-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast, other mutants with considerably reduced NS2-3 cleavage efficiencies propagated much more slowly and reverted to viruses expressing polyproteins with sequences allowing efficient NS2-3 cleavage. These viruses apparently induced cytopathic effects only after reversion.     

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.     

A Molecularly Cloned,Live-Attenuated Japanese Encephalitis Vaccine SA14-14-2 Virus: A Conserved Single Amino Acid in the ij Hairpin of the Viral E Glycoprotein Determines Neurovirulence in Mice

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA₁₄-14-2, and rescued from the cDNA a molecularly cloned virus, SA₁₄-14-2^MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA₁₄-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD₅₀, <1.5 PFU) from SA₁₄-14-2^MCV (LD₅₀, >1.5×10⁵ PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G¹⁷⁰⁸→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA₁₄-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.     

乙脑/寨卡嵌合病毒包膜蛋白T120A位点突变对小鼠脑内神经毒力的影响

为探索包膜蛋白T120A突变对乙脑/寨卡嵌合病毒JEV/ZIKV小鼠脑内神经毒力的影响,应用重叠延伸PCR和分子克隆技术构建含T120A突变的乙脑/寨卡嵌合病毒全长cDNA质粒,经酶切、测序鉴定后,以其为模板体外转录制备RNA,电转染导入BHK21细胞,收获培养液上清获得突变病毒株JEV/ZIKV (T120A).分别...