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Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

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.

Arabidopsis (Arabidopsis thaliana) exhibits natural genetic variation in drought response, including varying levels of proline (Pro) accumulation under low water potential. As Pro accumulation is potentially important for stress tolerance and cellular redox control, we conducted a genome-wide association (GWAS) study of low water potential-induced Pro accumulation using a panel of natural accessions and publicly available single-nucleotide polymorphism (SNP) data sets. Candidate genomic regions were prioritized for subsequent study using metrics considering both the strength and spatial clustering of the association signal. These analyses found many candidate regions likely containing gene(s) influencing Pro accumulation. Reverse genetic analysis of several candidates identified new Pro effector genes, including thioredoxins and several genes encoding Universal Stress Protein A domain proteins. These new Pro effector genes further link Pro accumulation to cellular redox and energy status. Additional new Pro effector genes found include the mitochondrial protease LON1, ribosomal protein RPL24A, protein phosphatase 2A subunit A3, a MADS box protein, and a nucleoside triphosphate hydrolase. Several of these new Pro effector genes were from regions with multiple SNPs, each having moderate association with Pro accumulation. This pattern supports the use of summary approaches that incorporate clusters of SNP associations in addition to consideration of individual SNP probability values. Further GWASguided reverse genetics promises to find additional effectors of Pro accumulation. The combination of GWAS and reverse genetics to efficiently identify new effector genes may be especially applicable for traits difficult to analyze by other genetic screening methods.

Recently developed plasmid-based reverse genetics systems for rotavirus A (RVA) enable rapid engineering of reassortants carrying human RVA antigens. However, complete genome segment sequences are required for successful generation of such reassortants, and sequencing of the untranslated regions (UTRs) of field strains is often not accomplished. To address this problem, we established a system that permits the generation of reassortants using only the open reading frame (ORF) nucleotide sequence information. Plasmids containing the VP7-ORF nucleotide sequence of six human RVA field strains (genotypes G2, G5, G8, G9, G12 and G29) derived from GenBank and flanked by the UTR sequences of simian RVA strain SA11 were constructed. Using these plasmids, four VP7 (G2, G5, G9 and G12) reassortants in an SA11 backbone were successfully generated. In contrast, the G8 and G29 reassortants were not viable. BLASTp search of the G8 and G29 sequences revealed an unusual amino acid substitution in each sequence, which was not present in related field strains. Site-directed reversion of the corresponding C656T mutation in G8 led to effective rescue of reassortant virus. However, reverting the G84C mutation in G29 did not result in replicating virus. The results suggest that most human RVA VP7 UTRs can be substituted with simian RVA UTRs. However, generation of reassortants might be impeded by potential sequencing errors or intrinsic reassortment limitations. The established system could help to broaden the antigenic repertoire for rapid engineering of potential novel RVA vaccine strains. Key Points • Generation of diverse rotavirus vaccine strains is impeded by missing UTR sequences. • UTRs from SA11 can be used instead of missing UTR sequences from field strains. • Human RVA reassortants of genotypes G2, G5, G8, G9, G12 were successfully rescued.

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines.

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.

Medicago truncatula is one of the model species for legume studies. In an effort to develop legume genetics resources, > 21 700 Tnt1 retrotransposon insertion lines have been generated. To facilitate fast-growing needs in functional genomics, two reverse genetics approaches have been established: web-based database searching and PCR-based reverse screening. More than 840 genes have been reverse screened using the PCR-based approach over the past 6 yr to identify mutants in these genes. Overall, c. 84% (705 genes) success rate was achieved in identifying mutants with at least one Tnt1 insertion, of which c. 50% (358 genes) had three or more alleles. To demonstrate the utility of the two reverse genetics platforms, two mutant alleles were isolated for each of the two floral homeotic MADS-box genes, MtPISTILATA and MtAGAMOUS. Molecular and genetic analyses indicate that Tnt1 insertions in exons of both genes are responsible for the defects in floral organ development. In summary, we have developed two efficient reverse genetics platforms to facilitate functional characterization of M. truncatula genes.

Background Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. Methods To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. Results The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. Conclusion This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.