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We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus’ generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(−)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5′ hammerhead and 3′ ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(−)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(−)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(−)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative‐strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize‐infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full‐length infectious clone was established using agrobacterium‐mediated delivery of full‐length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA‐directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST‐LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV‐GFP. Complementary DNA (cDNA) clones of MMV‐wild type (WT) and MMV‐GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV‐WT and MMV‐GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross‐kingdom studies of virus replication in plant and insect hosts. The maize mosaic virus infectious clone enables stable and robust expression of heterologous genes in maize and planthoppers, and is a versatile tool for cross‐kingdom studies of virus replication in plant and insect hosts.

Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants. The recombinant NCMV‐RFP virus was rescued in barley plants and insect vectors to reveal cellular superinfection exclusion of two cytorhabdoviruses, NCMV‐RFP and BYSMV‐GFP, in barley.

Fowl adenovirus serotype 4 (FAdV-4) is a non-enveloped double-stranded DNA virus with a 43–45 kb genome. This characteristic makes it a promising viral vector for expressing other antigens in developing multi-valent or multi-series vaccines in the poultry industry. To create an easy-to-use reverse genetics system for manipulating FAdV-4 genomic DNA, a full-length infectious clone of FAdV-4 was constructed using lambda Red-mediated recombination in Escherichia coli DH10B. Viable viruses were successfully rescued after the transfection of linearised infectious clones into LMH cells. The rescued viruses showed the same cytopathic effect and growth kinetics as wild-type FAdV-4 viruses. Based on the FAdV-4 infectious clone, the hexon coding sequence of the high-pathogenicity FAdV-4 was replaced by that of the nonpathogenic FAdV-4 using lambda Red-mediated recombination combined with rpsL counter selection without leaving extra sequences after engineering. The rescued recombinant virus was highly attenuated and showed low pathogenicity to 21-day-old SPF chickens. Hereto, the easy-to-use reverse genetics system for FAdV-4 was successfully established. With this platform, the genomic DNA of FAdV-4 can be manipulated and purified in DH10B, making it quicker and easier to generate a recombinant FAdV-4 virus to develop multi-valent/multi-series vaccines.

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.

Rabies is a lethal zoonotic disease caused by lyssaviruses, such as rabies virus (RABV), that results in nearly 100% mortality once clinical symptoms appear. There are no curable drugs available yet. RABV contains five structural proteins that play an important role in viral replication, transcription, infection, and immune escape mechanisms. In the past decade, progress has been made in research on the pathogenicity of RABV, which plays an important role in the creation of new recombinant RABV vaccines by reverse genetic manipulation. Here, we review the latest advances on the interaction between RABV proteins in the infected host and the applied development of rabies vaccines by using a fully operational RABV reverse genetics system. This article provides a background for more in-depth research on the pathogenic mechanism of RABV and the development of therapeutic drugs and new biologics.

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome.

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.