Rotaviruses (RVs) are considered to be one of the most common causes of viral gastroenteritis in young children and infants worldwide. Before recent developments, studies on rotavirus biology have suffered from the lack of an effective reverse genetics (RG) system to generate recombinant rotaviruses and study the precise roles of the viral proteins in the context of RV infection. Lately a fully-tractable plasmid-only based RG system for rescuing recombinant rotaviruses has been developed leading to a breakthrough in the RV field. Since then, the reproducibility and improvements of this technology have led to the generation of several recombinant rotaviruses with modifications on different gene segments, which has allowed the manipulation of viral genes to characterise the precise roles of viral proteins during RV replication cycle or to encode exogenous proteins for different purposes. This review will recapitulate the different RG approaches developed so far, highlighting any similarities, differences and limitations of the systems as well as the gene segments involved. The review will further summarise the latest recombinant rotaviruses generated using the plasmid-only based RG system showing the enormous potentials of this technique to shed light on the still unanswered questions in rotavirus biology.
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| http://dx.doi.org/10.1016/j.virusres.2021.198576 | DOI Listing |
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An improved reverse genetics system for rotavirus vaccine strain LLR using five plasmid vectors.
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National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID), NHC Key Laboratory for Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Beijing 100052, PR China.
Species A rotaviruses (RVs), which belong to the family and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.
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Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses.
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