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Tomato yellow leaf curl China virus (TYLCCNV) is a major agricultural pathogen and primary model for circular single‐stranded DNA (cssDNA) virus studies. The infectious clones of TYLCCNV and other cssDNA viruses are usually constructed as two tandem copies of the small viral genomes in Agrobacterium‐ mediated T‐DNA vectors. However, during our experiment, we observed that successive cultivation of the agrobacterial infectious clone of TYLCCNV led to a reduction or complete loss of its virulence in host plants. Further analysis revealed that the instability of this infectious clone is analogous to the mechanism of viral genome release from the dimeric infectious clones of cssDNA viruses during rolling circle replication, with key contributing factors being the activity of the viral Replication protein (Rep) and the replication origins. Unlike the infectious clones of RNA viruses, which often utilize introns to disrupt toxic protein coding sequences in order to achieve stabilization, the infectious clones of DNA viruses are unable to remove introns before releasing viral genomes. To address this challenge, we developed a micro‐homology mediated end joining (MMEJ)‐based system that disrupts the Rep coding sequences with an I‐ Sce I site flanked by microhomologous regions, stabilizing the infectious clone in prokaryotic cells. Then the transiently co‐expressed I‐ Sce I enzyme seamlessly removes the introduced I‐ Sce I site through MMEJ repair in plant hosts, resulting in efficient release of functional TYLCCNV viral genomes in planta . Theoretically, this approach can be applied to the construction of stable infectious clones for all plant DNA viruses.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus in the Flaviviridae, which is classified into two different lineages Asian and African. The outbreak of ZIKV Asian lineage isolates in 2015–2016 is associated with the increase in cases with prenatal microcephaly and Guillain–Barré syndrome, and has sparked attention throughout the world. Genome sequence alignment and the analysis of Asian and African lineage isolates indicate that amino acid changes, particular in positively charged amino acid substitutions in the pr region of prM protein might involve a phenotypic change that links with the global outbreak of ZIKV Asian-lineage. The study generated and characterized the virological properties of wild type and mutants of single-round infectious particles (SRIPs) and infectious clones (i.c.s) of ZIKV Asian-lineage Natal RGN strain, and then identified the function of amino acid substitutions at the positions 139 [Asn139→Ser139 (N139S)] and 143 [Glu143→Lys143 (E143K)] in ZIKV polyproteins (located within the pr region of prM protein) in the infectivity and cytopathogenicity. The E143K SRIP and i.c. of Natal RGN strain exhibited relatively higher levels of cytopathic effect, EGFP reporter, viral RNA and protein synthesis, and virus yield in three types of human cell lines, TE617, SF268 and HMC3, compared to wild type (WT), N139S SRIPs and i.c.s, which displayed more efficiency in replication kinetics. Additionally, E143K Natal RGN i.c. had greater activities of virus attachment and entry, yielded higher titers of intracellular and extracellular virions, and assembled the E proteins near to the plasma membrane in infected cells than the other i.c.s. The results indicate that the positively charged amino acid residue Lys143, a conserved residue in the pr region of prM of ZIKV African lineages, plays a crucial role in viral replication kinetics, including viral attachment, entry, assembly and egress. Thus, the negatively charged amino acid residue Glu143 within the pr region of prM leads to an alteration of the phenotypes, in particular, a lower replication efficiency of ZIKV Asian-lineage isolates with the attenuation of infectivity and cytopathicity.

To study the host range of Rose rosette virus (RRV), we employed crude sap inoculum extracted from RRV-infected roses and the RRV infectious clone. We inoculated plants from the families Solanaceae, Cucurbitaceae, Leguminosae, Malvaceae, Amaranthaceae, and Brassicaceae. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect RRV in the inoculated plants throughout their growth stages. Interestingly, RRV was detected in the newly developed leaves of tomato, pepper, tobacco, cucumber, squash, zucchini, pumpkin, pea, peanut, soybean, spinach, okra, and Chenopodium spp. The speed of upward advancement of RRV within infected plants was variable between plants as it took two to three weeks for some plant species and up to five weeks in other plant species to emerge in the newest leaves. No severe symptoms were detected on most of the inoculated plants. Chenopodium spp., spinach, cucumber and Nicotiana rustica exhibited either chlorotic or necrotic lesions with variable shapes and patterns on the systemically infected leaves. Double membrane-bound particles of 80–120 nm in diameter were detected by transmission electron microscopy in the infected tissues of cucumber, pepper, and N. benthamiana plants. This finding infers the validity of mechanical inoculation for RRV on a wide range of plants that would serve as potential natural reservoirs.

A picornavirus strain of Duck/FC22/China/2017 (FC22) with an unclear taxonomic status was isolated in our laboratory in 2017. To study the biological properties of the virus, an infectious clone of the FC22 strain was successfully rescued by the infectious subgenomic amplicon method. The FC22 gene was amplified in two segments and fused with CMV and HDR after the addition of marker sites. The fusion fragment was transfected into LMH cells and passaged, and the obtained virus was named rFC22 and identified by PCR, IFA and electron microscopy. The biological characteristics of strain rFC22 were studied by inoculation with LMH cells, and the results showed that the TCID 50 of rFC22 was essentially the same as that of its parent strain FC22 (1 × 10 6.6 TCID 50 /mL) and that the growth curve of rFC22 was consistent with that of its parent strain FC22. The results indicated no significant difference in the biological characteristics between the rescued strain rFC22 and its parent strain FC22. In summary, our findings suggest that a duck-derived picornavirus was successfully rescued by ISA and the method was more convenient and more successful than alternative approaches.

Summary Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.

Infectious cloning of plant viruses is a powerful tool for studying the reverse genetic manipulation of viral genes in virus–host plant interactions, contributing to a deeper understanding of the life history and pathogenesis of viruses. Yet, most of the infectious clones of RNA virus constructed in E. coli are unstable and toxic. Therefore, we modified the binary vector pCass4-Rz and constructed the ternary shuttle vector pCA4Y. The pCA4Y vector has a higher copy number in the E. coli than the conventional pCB301 vector, can obtain a high concentration of plasmid, and is economical and practical, so it is suitable for the construction of plant virus infectious clones in basic laboratories. The constructed vector can be directly extracted from yeast and transformed into Agrobacterium tumefaciens to avoid toxicity in E. coli. Taking advantage of the pCA4Y vector, we established a detailed large and multiple DNA HR-based cloning method in yeast using endogenous recombinase. We successfully constructed the Agrobacterium-based infectious cDNA clone of ReMV. This study provides a new choice for the construction of infectious viral clones.

Squash leaf curl China virus (SLCCNV) belongs to the species Begomovirus cucurbitachinaense in the genus Begomovirus and can infect some Cucurbitaceae crops except for watermelon (Citrullus lanatus). In this study, watermelon plants showing symptoms typical to begomovirus infection in field were observed in Zhejiang Province of China, and SLCCNV presence was identified through PCR and next-generation sequencing (NGS). The pairwise sequence identity of the DNA-A genome shows that SLCCNV watermelon isolate belongs to the SLCCNV/CN strain and shares 96% nucleotide identity with the previously sequenced SLCCNV/CN. An infectious clone of SLCCNV watermelon isolate was constructed using the tandem repeat fragment method. Through agrobacterium-mediated inoculation, the clone could induce systemic infection with typical symptoms in watermelon, melon (Cucumis melo), squash (Cucurbita pepo), pumpkin (Cucurbita maxima), wax gourd (Benicasa hispida), cucumber (Cucumis sativus), and N. benthamiana. It was further demonstrated that the progeny virions derived from the cloned watermelon isolate could be transmitted by whitefly rather than the sap. To the best of our knowledge, this is the first report of a natural infection of SLCCNV on watermelon in China, and the first complete report on the molecular characteristics and pathogenicity of watermelon-infecting SLCCNV in the world.

Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, has become more serious and poses a potential threat to animal safety and public health. Given the lack of antivirals and vaccines against GETV, further development of tools, including reverse genetics techniques, is crucial for combating this pathogen. Herein, we describe the design and construction of a DNA-launched infectious clone for GETV. The full-length genome of the GETV HuN1 strain, flanked by the cytomegalovirus immediate-early (CMV) promoter sequence at the 5' end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3' end, was packaged in a bacterial artificial chromosome vector to establish the GETV infectious clone pBR322-GETV-HuN1. In parallel, recombinant reporter viruses carrying the reporter gene EGFP between the E1 gene and the 3' UTR were constructed on the basis of the established CMV-driven cDNA clone. Both in vivo and in vitro experiments have shown that the rescued recombinant virus (rGETV-HuN1 and rGETV-EGFP) possesses viral biological activity similar to that of the parental virus. In summary, this study develops a concise and efficient GETV infectious cDNA clone and a recombinant virus carrying an EGFP reporter gene. The availability of GETV infectious clones will facilitate further studies on understanding the molecular mechanisms of GETV biology, virulence determinants, molecular pathogenesis, vaccine development and virus‒host interactions.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.