Full-length Infectious cDNA Clone Research Articles

Molecular and biological characterization of infectious full-length cDNA clones of two viruses in Paris yunnanensis, including a novel potyvirus

Paris yunnanensis, also named as Rhizoma Paridis in the Chinese Pharmacopeia, is a perennial Chinese medicinal herb commonly grown in Southwest China. However, several viruses have been found infecting this plant in recent years. Using high-throughput sequencing (HTS) and Sanger sequencing, this study obtained the complete genome sequences of three capillovirus isolates and one potyvirus isolate. Genomic and phylogenetic analyses revealed that these three capillovirus isolates are the same virus as the newly reported capillovirus, Paris polyphylla chlorotic mottle virus. The newly found potyvirus isolate shares 52.4–68.9% nucleotide sequence identity with other known potyviruses and thus, is grouped into the bean common mosaic virus subgroup. Based on the nucleotide sequence identity, we consider this virus a novel potyvirus species and propose ‘Paris potyvirus 5’ (ParPV-5) as its common name, and ‘Potyvirus shilinense’ as its species name. To characterize their biological features, two infectious clones, representing the two viruses, have been constructed through homologous recombination or yeast homologous recombination, and inoculated to several species plants, respectively. The results showed both of the viruses can infect P. yunnanensis and Nicotiana benthamiana. In addition, Paris polyphylla chlorotic mottle virus (PpCMV) can infect N. tabacum var. Xanthi nc, Cucurbita moschata, and Capsicum annuum, and ParPV-5 can infect Cucumis sativus L. and Bidens pilosa L. However, except mild leaf deformation exhibited on the PpCMV-inoculated C. moschata plants, no obvious symptom were observed in these plants including P. yunnanensis. A total of 179 field P. yunnanensis leaf samples from four counties in 2020–2021, and all 640 P. yunnanensis plants from a whole study plot of Lijiang in 2024, were tested using RT-PCR and specific primers, the results showed that PpCMV is a potential preponderant species in some regions, and ParPV-5 has the possible transmission from the original site to other regions.

Construction and Characterization of a Full-Length Infectious cDNA Clone of a Strain of Watermelon Mosaic Virus Isolated from Melon.

Watermelon mosaic virus (WMV), a member of the genus Potyvirus, causes serious economic losses in cucurbit crops. For molecular biological studies of viruses, it is necessary to construct an infectious clone that can facilitate gene functional analysis. In this study, we constructed an infectious cDNA clone of WMV genomic RNA by Gibson assembly and evaluated its virulence and symptoms on a variety of host plants. A WMV isolate (WMV-IS-me2) collected from melon in Iksan, Jeonbuk Province, Korea in 2015 caused mosaic symptoms on leaves. Four overlapping PCR fragments of the full-length genome of this isolate and the pJL89 binary vector with overhanging ends of 40 bp were amplified by reverse transcription PCR and joined in a single isothermal reaction. The complete nucleotide sequence of the infectious cDNA clone of WMV was determined and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. Following agro-infiltration, an infectious clone (pWMV-M, KACC95145P) systemically induced mosaic symptoms on watermelon (Citrullus lanatus), zucchini (Cucurbita pepo), melon (Cucumis melo), and Nicotiana benthamiana plants. This infectious clone may be useful for screening cucurbit varieties resistant to WMV, as well as for studying viral gene function.

Construction of a full-length infectious cDNA clone of zucchini tigre mosaic virus infecting snake gourd and genetic diversity analysis based on complete genome sequences of ZTMV isolates.

Zucchini tigre mosaic virus (ZTMV) is a positive-sense single-stranded RNA virus belonging to the genus Potyvirus. In this study, a full-length infectious cDNA clone of a ZTMV strain infecting snake gourd (Trichosanthes cucumerina var. anguina L.) was constructed and shown to infect snake gourd, chieh-qua, zucchini, ridge gourd, and bitter melon. The complete genome sequence of ZTMV-FS7 (PP291701) showed the highest nucleotide sequence similarity to ZTMV-TW (86.2% identity). Genetic diversity analysis of 12 ZTMV isolates showed that the P1 gene had the highest variability. Selection pressure analysis indicated that all of the ZTMV genes were under negative selection. However, some sites, particularly within the P1 gene, were under positive selection.

Development and characterization of reverse genetics systems of feline infectious peritonitis virus for antiviral research

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome.

Generation of an infectious cDNA clone for NADC30-like PRRSV.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly significant infectious disease that poses a substantial threat to the global pig industry. In recent years, the NADC30-like strain has gradually emerged as prevalent in China, causing a profound impact on the country's pig farming industry. Therefore, it is important to conduct an in-depth study on the characteristics and gene functions of the NADC30-like strain. An infectious cDNA clone is an indispensable tool for investigating the functions of viral genes. In this current study, we successfully isolated a NADC30-like strain and constructed its full-length infectious cDNA clone. The utilization of this clone will facilitate our investigation into the viral replication, pathogenesis, and immune response associated with the PRRSV NADC30-like strain.

Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain

The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures. Currently, the mechanism underlying its attenuation phenotype remain largely unknown. In this study, we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques. The recovered H2 strain (H2ic) from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production, similar to the parental H2 strain. Additionally, H2ic did not cause disease in Ifnar1−/− C57 mice, consistent with the H2 strain. To explore the cell-adaptive mutations of the H2 strain, chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone. The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic. Other chimeric viruses containing the 2B, 2C, or 3A proteins from H2w failed to be recovered. Furthermore, there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses. These results demonstrate that adaptive mutations in the 2B, 2C, and 3A proteins are essential for efficient replication of the H2 strain in cell cultures. Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice. Together, this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.

A potyvirus provides an efficient viral vector for gene expression and functional studies in Asteraceae plants.

Plant virus-derived vectors are rapid and cost-effective for protein expression and gene functional studies in plants, particularly for species that are difficult to genetically transform. However, few efficient viral vectors are available for functional studies in Asteraceae plants. Here, we identified a potyvirus named zinnia mild mottle virus (ZiMMV) from common zinnia (Zinnia elegans Jacq.) through next-generation sequencing. Using a yeast homologous recombination strategy, we established a full-length infectious cDNA clone of ZiMMV under the control of the cauliflower mosaic virus 35S promoter. Furthermore, we developed an efficient expression vector based on ZiMMV for the persistent and abundant expression of foreign proteins in the leaf, stem, root, and flower tissues with mild symptoms during viral infection in common zinnia. We showed that the ZiMMV-based vector can express ZeMYB9, which encodes a transcript factor inducing dark red speckles in leaves and flowers. Additionally, the expression of a gibberellic acid (GA) biosynthesis gene from the ZiMMV vector substantially accelerated plant height growth, offering a rapid and cost-effective method. In summary, our work provides a powerful tool for gene expression, functional studies, and genetic improvement of horticultural traits in Asteraceae plant hosts.

Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

Complete genome sequencing and construction of full-length infectious cDNA clone of papaya ringspot virus-HYD isolate and its efficient in planta expression.

Papaya ringspot virus (PRSV) is a devastating Potyvirus that causes papaya ringspot disease in Carica papaya plantations globally. In this study, the complete genome sequence of a PRSV isolate from Shankarpalli, Telangana, India, was reported and designated as PRSV-HYD (KP743981.1). The genome is a single-stranded positive-sense RNA comprising 10,341 nucleotides. Phylogenetic analysis revealed that PRSV-HYD is closely related to PRSV Pune (Aundh) isolate with 92 and 95% nucleotide and amino acid sequence identity, respectively. To develop infectious cDNA (icDNA), the complete nucleotide sequence of PRSV-HYD was cloned between the right and left borders in the binary vector pCB301 using BglII and XmaI restriction sites. Cauliflower mosaic virus (CaMV) double promoter (35S) was fused at the 5'-end and Avocado sunblotch viroid (ASBVd) ribozyme (RZ) sequence was fused to the 3' end to generate an authentic 3' viral end in the transcribed mRNAs. The icDNA generated was mobilized into the Agrobacterium tumefaciens EHA 105, and the agrobacterial cultures were infiltrated into the natural host C. papaya and a non-host Nicotiana benthamiana plants; both did not show any symptoms. In RT-PCR analysis of RNAs isolated from N. benthamiana, we could detect viral genes as early as 3 days and continued up to 28 days post infiltration. Alternatively, virion particles were purified from agroinfiltrated N. benthamiana plants and introduced into C. papaya by mechanical inoculation as well as by pinprick method. In both cases, we could see visible systemic symptoms similar to that of wild type by 40 days. Additionally, we studied the expression patterns of the genes related to plant defense, transcription factors (TFs), and developmental aspects from both C. papaya and N. benthamiana.

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, Citrullus 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.

Cell type dependent stability and virulence of a recombinant SARS-CoV-2, and engineering of a propagation deficient RNA replicon to analyze virus RNA synthesis

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu-1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV-2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening.

Evaluation of packaging capacity at the genomic 2C/3A junction region in Porcine enterovirus G

Porcine enterovirus G (EV-G) is endogenous to most pig farming countries worldwide. Reports that a papain-like protease (PLP) gene has been naturally inserted into the 2C/3A junction region of the EV-G genome, has increased the potential public health threats from this virus. We constructed a full-length infectious cDNA clone of EV-G, CH/17GXQZ/2017, in order to determine the packaging capacity at the 2C/3A insertion site. Subsequently, recombinants viruses containing the coding tags, GFP, iLOV and His at the 2C/3A junction region, were synthesized. The infectious virus was successfully rescued only with the insertion of the His-tag, which displayed similar virological and molecular properties to its parental strain. This study determined the packaging capacity of the 2C/3A insertion site, and it provides a practical tool for studying the functions and pathogenic mechanisms of EV-G in pigs.

Molecular identification and development of an infectious cDNA clone of Trichosanthes kirilowii-infecting cucurbit mild mosaic virus

Trichosanthes kirilowii has been mainly grown for use in traditional Chinese medicine. In this study, cucurbit mild mosaic virus (CuMMV) belonging to the genus Fabavirus was identified from T. kirilowii plants. CuMMV possesses a segmented, bipartite linear single-stranded RNA genome composed of RNA1 and RNA2. Sequence analysis showed that each genomic segment shares the highest sequence similarity with those of CuMMV isolated from pumpkin. A full-length infectious cDNA clone of CuMMV was further constructed and was found to induce typical symptoms in T. kirilowii, Cucumis sativus, C. melo, Citrullus lanatus, and Cucurbita pepo. The sap inoculum derived from the infectious cDNA clone of CuMMV could be mechanically transmitted and reproduce similar symptoms in the tested plants. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of CuMMV, which will provide a useful tool in understanding CuMMV-encoded proteins and plant-CuMMV interactions.

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

Citrus yellow mottle-associated virus (CiYMaV) belonging to the subgenus Mandarivirus within the genus Potexvirus, was first identified in 2018 from Pakistan (CiYMaV-PK), where it is endemic in several regions. Here, three full-length cDNA clones (pCiYMaV-FL-1, pCiYMaV-FL-18, and pCiYMaV-FL-22) corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo (Citrus grandis) seedlings using the vacuum infiltration method. All the inoculated plants developed severe vein yellowing, leaf mottling, and dwarfing symptoms by 40 days post-infiltration (dpi). The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100% infection rates of pCiYMaV-FL at 60 dpi. Despite there being no observed difference among the three clones in the severity of symptom, pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins. Moreover, pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them. Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration. Taken together, the results indicate that Koch's postulates were fulfilled for CiYMaV in citrus plants. This is the first report of full-length infectious cDNA clones of CiYMaV, and thus, the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus.

The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production.

Construction of coxsackievirus B5 viruses with luciferase reporters and their applications in vitro and in vivo

Coxsackievirus belongs to the Picornaviridae family and is one of the major pathogens that cause hand, foot and mouth disease (HFMD) in infants and children with potential serious complications and even deaths. The pathogenesis of this virus is not fully elucidated and no vaccine or antiviral drug has been approved. In this study, a full-length infectious cDNA clone of coxsackievirus B5 virus was assembled and the recombinant virus displayed similar growth kinetics and ability to cause cytopathic effects as the parental virus. Luciferase reporter was then incorporated to generate both full-length and subgenomic replicon (SGR) reporter viruses. The full-length reporter virus is suitable for high-throughput antiviral screening, while the SGR is a useful tool to study viral-host interactions. More importantly, the full-length reporter virus has also been shown to infect the suckling mouse model and the reporter gene could be detected using an in vivo imaging system, thus providing a powerful tool to track viruses in vivo. In summary, we have generated coxsackievirus B5 reporter viruses and provided unique tools for studying virus-host interactions in vitro and in vivo as well as for high-throughput screenings (HTS) to identify novel antivirals.

Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP.

Human adenovirus 55 (HAdV-55) has recently caused outbreaks of acute respiratory disease (ARD), posing a significant public threat to civilians and military trainees. Efforts to develop antiviral inhibitors and quantify neutralizing antibodies require an experimental system to rapidly monitor viral infections, which can be achieved through the use of a plasmid that can produce an infectious virus. Here, we used a bacteria-mediated recombination approach to construct a full-length infectious cDNA clone, pAd55-FL, containing the whole genome of HadV-55. Then, the green fluorescent protein expression cassette was assembled into pAd55-FL to replace the E3 region to obtain a recombinant plasmid of pAd55-dE3-EGFP. The rescued recombinant virus rAdv55-dE3-EGFP is genetically stable and replicates similarly to the wild-type virus in cell culture. The virus rAdv55-dE3-EGFP can be used to quantify neutralizing antibody activity in sera samples, producing results in concordance with the cytopathic effect (CPE)-based microneutralization assay. Using an rAdv55-dE3-EGFP infection of A549 cells, we showed that the assay could be used for antiviral screening. Our findings suggest that the rAdv55-dE3-EGFP-based high-throughput assay provides a reliable tool for rapid neutralization testing and antiviral screening for HAdV-55.

Molecular characteristics and pathogenicity of infectious cDNA clones of the Chinese isolates of pea enation mosaic virus 1 and pea enation mosaic virus 2

The pea enation mosaic disease (PEMD) causes significant yield losses worldwide. PEMD is caused by two taxonomically unrelated but symbiotic viruses, pea enation mosaic virus 1 (PEMV-1) and pea enation mosaic virus 2 (PEMV-2). In this study, the complete genomes of four isolates of PEMV-1 and PEMV-2 from Yunnan Province of China were determined and analyzed. The four isolates of PEMV-1 or PEMV-2 shared ≥ 98.7% nucleotide sequence identities at whole genome level. The four Chinese PEMV-1 isolates were most closely related to PEMV-1-Ramsey-1 with nucleotide sequence identities of 97.8–98.0%, while the four Chinese PEMV-2 isolates were most closely related to PEMV-2-DSMZ PV-0088, with nucleotide sequence identities of 94.3–94.5%. The two full-length infectious cDNA clones of the Yunnan-Dali isolates of the two viruses (PEMV-1-YDL and PEMV-2-YDL) were constructed. Co-inoculation of PEMV-1-YDL and PEMV-2-YDL in pea seedlings caused systemic infection, with typical enation and mosaic symptoms on new leaves after inoculation, while the inoculated Nicotiana benthamiana plants showed curling symptoms. Inoculation of pea seedlings with either PEMV-1-YDL or PEMV-2-YDL did not cause obvious symptoms on the new leaves. It has been known that PEMV-2 helps the systemic infection of PEMV-1. However, our results showed that PEMV-1-YDL alone could cause a systemic infection. The pathogenicity of the two infectious clones was tested on different pea cultivars, and some cultivars were PEMD-tolerant. This is the first report of the infectious clones constructed from the Chinese isolates of PEMV-1 and PEMV-2. The current approach will facilitate determination of the synergistic relationship between PEMV-1 and PEMV-2 and their interactions with the host plant by reverse genetic analysis.

Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Construction of Full-Length Infectious cDNA Clones of Citrus Mosaic Virus RNA1 and RNA2 and Infection of Citrus Seedlings by Agrobacterium-Mediated Vacuum-Infiltration

The development of full-length infectious cDNA clones for plant RNA viruses is important for studying their molecular biological characteristics, functional genomics, pathogenesis, and vectorization applications. Citrus mosaic virus (CiMV), a member of the genus Sadwavirus, is of economic importance to the citrus industry and comprises a bipartite, positive-sense, single-stranded RNA genome encapsidated in icosahedral virions. In the present study, full-length cDNA clones of CiMV RNA1 and RNA2 were constructed based on a ternary yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pTY, using transformation-associated recombination (TAR) strategy. Infectivity of cDNA clones of CiMV RNA1 and RNA2 was examined in multiple citrus varieties via Agrobacterium-mediated vacuum-infiltration (AVI) through symptom observation, RT-PCR, and virion detection with an electron microscope. Furthermore, the genome-sized RT-PCR fragments of RNA1 and RNA2 were obtained from symptomatic Jinchengyou (Citrus grandis) plants infected by the cloned virus (CiMV211). In addition, CiMV211 produced typical symptoms of wild-type CiMV in cowpea (Vigna angularis) plants inoculated by Agrobacterium-mediated injection. This is the first report of infectious cDNA clones of CiMV, which may lay the foundation for research on the pathogenesis and vectorization of the virus.