Characterization of a full-length infectious clone of bovine foamy virus 3026

The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Construction of Full-length Infectious Clone for Encephalomyocarditis Virus BJC3 and Identification of the Rescued Virus

The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.

Construction Strategy and Affecting Factors of Positive-Strand RNA Virus Infectious Clone

Construction of infectious clones by full-length cDNA is basic and key for recovering RNA virus and is core of reverse genetics.In this article,basic consideration and key technology were viewed and factors affecting infectivity of clones were also summarized.Some research advances were briefly introduced about positive-strand RNA viruses infectious clones.Finally,this article also reviewed the application of infectious clones.

Molecular and biological characterization of melon-infecting squash leaf curl China virus in China

It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.In this study,the pathogen was identified as SLCCNV through biological and molecular characterization.The isolate(SLCCNV-HN)possess a bipartite genome,DNA-A(HM566112.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062251.1)pumpkin and SLCCNV-Hn61(AM260205.1)squash isolates from China,whereas DNA-B(HM566113.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062252.1).Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and-B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin,SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China,forming an independent cluster.Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%,the systemic symptoms in melon showed identical to those of melon plants infected in fields.Additionally,melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95%(19/20)showed leaf curl and dwarf symptoms 15 days post transmission,thereby fulfilling Koch’s postulates.Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.To the best of our knowledge,this is the first characterization of meloninfecting SLCCNV through its genome,infectious clone and transmission.

Complete genome sequence of two strawberry vein banding virus isolates from China

It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated SVBV-AH and SVBV-BJ,that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China,respectively.The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides(nts)and 7,863 nts long,respectively,and both constituted with seven genes typical of the caulimoviruses.Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7%of each other and had 85.7%and 86.0%sequence identity related to SVBV from the United States(SVBV-US),respectively.Phylogenetic trees,based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein(CP),both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates,and other species of caulimoviruses clustered into another tree branch.It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses.We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions.Taken together,our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates,meanwhile providing the necessary vector for studying the gene functions of strawberry.

Construction of chimeric viruses based on pepper mild mottle virus using a modiffed Cre/loxP system

Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo

Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture.In this study,we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China.We constructed a full-length infectious cDNA clone of ToLCNDV,which could induce systemic infection with typical symptoms in Nicotiana benthamiana,Cit-rullus melo,and Citrullus lanatus plants through agrobacterium-mediated inoculation.Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible.We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV.Collectively,the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions,which might provide new insights into breeding resistance germplasm in crops.

Integration of HiBiT into enteroviruses:A universal tool for advancing enterovirus virology research

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications,enabling the development of effective therapeutic and preventive strategies.In this study,our initial attempts to introduce a NanoLuc luciferase(NLuc)reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies.We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus.Therefore,we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71(EV-A71),coxsackievirus A10(CVA10),coxsackievirus A7(CVA7),coxsackievirus A16(CVA16),namely EV-A71-HiBiT,CVA16-HiBiT,CVA10-HiBiT,CVA7-HiBiT,and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency.Notably,the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses.A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics.Moreover,these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages.In addition,the HiBiT-tagged viruses were successfully tested in antiviral drug assays,and the sensitivity of the viruses to drugs was not affected by the HiBiT tag.Ultimately,our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal,convenient,and invaluable method for advancing research in the realm of enterovirus virology.Furthermore,HiBiT-tagged enteroviruses exhibit great potential for diverse applications,including the development of antivirals and the elucidation of viral infection mechanisms.

Improved plasmid-based recovery of coxsackievirus A16 infectious clone driven by human RNA polymerase I promoter

Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone

Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

Construction of an infectious cDNA clone of Tembusu virus isolated from breeder Peking ducks

Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout China(Cao et al.,2011;Su et al.,2011).The causative agent of the disease was identified as Tembusu virus(TMUV),which was classified into the genus Flavivirus,

Reverse genetics systems for SARS-CoV-2:Development and applications

The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.