reverse genetics
Vaccines and Antiviral AgentsReverse Genetics Approach for Developing Rotavirus Vaccine Candidates Carrying VP4 and VP7 Genes Cloned from Clinical Isolates of Human RotavirusAlthough vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral...
Genome Replication and Regulation of Viral Gene Expression | SpotlightThe Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established...
Virus-Cell Interactions | SpotlightA Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus ReplicationAfter entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for...
Vaccines and Antiviral AgentsDevelopment of Reverse Genetics for the Prototype New World Mammarenavirus Tacaribe VirusTo date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide...
Genome Replication and Regulation of Viral Gene Expression | SpotlightExpression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart ElementRotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous...
Genome Replication and Regulation of Viral Gene ExpressionAn Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like RotavirusesGroup A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV...
Genome Replication and Regulation of Viral Gene ExpressionEstablishment of a Reverse Genetics System for Influenza D VirusThe bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies...
Vaccines and Antiviral AgentsRapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein ActivityImmunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-...
Genome Replication and Regulation of Viral Gene ExpressionRecombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral FactoriesThe rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully...
Genome Replication and Regulation of Viral Gene ExpressionGeneration of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics SystemPrevious studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have...