The first successful recovery of recombinant rotaviruses (RVs) derived entirely from cloned cDNAs in a Japanese study will both improve understanding of RV pathobiology and promote the development of new vaccines and therapeutics.

RVs are double-stranded RNA viruses, which are the most common cause of severe gastroenteritis and diarrhea in infants and children. Two RV vaccines are available in the U.S., Rotarix (Glaxosmithkline plc) and Rotateq (Merck & Co. Inc.), with several other vaccines also being available elsewhere.

Such attenuated vaccines can prevent up to 95 percent of severe diarrheal infections in developed areas. While those vaccines are recommended by WHO for inclusion in national vaccine programs, as they apparently reduce the risk of diarrhea-related death in young children, they are suboptimal in developing countries.

"Although these vaccines provide protection against RV infections causing vomiting and severe diarrhea in infants and children, there are concerns about their efficacy, safety and cost," said principal study investigator Takeshi Kobayashi, an associate professor in the Department of Virology, Research Institute for Microbial Diseases, Osaka University.

"Both of the currently available attenuated RV vaccines were made using the classic approach of 'forward genetics,'" Kobayashi told BioWorld Today. Forward genetics is generally a time-consuming and somewhat random process since "induced viral genome mutations are not controllable."

In contrast, "reverse genetics involves creating targeted mutations in known viral genes, in order to determine the phenotype," he noted. "With the reverse genetics system, we can rapidly create desired gene modifications for appropriate antigenicity and attenuated mutations for producing tailor-made vaccine strains."

Although a plasmid-based reverse genetics system has been developed for all major groups of animal RNA viruses, despite extensive research efforts, the generation of an RV entirely from cloned cDNAs has not been achieved. That lack of a reliable reverse genetics platform to generate infectious RVs entirely from cloned cDNAs has limited the study of those important viruses in general, hampering efforts to develop the next generation of more effective RV vaccines in particular.

The Osaka University research team has now developed the first truly plasmid-based reverse genetics system, which is free from helper viruses and independent of any selection for RV, they reported in the Jan.30, 2017, early online edition of Proceedings of the National Academy of Sciences.

Kobayashi explained that helper virus-dependent reverse genetics systems require a helper virus and an appropriate selection system is required to eliminate those. "These systems can manipulate only a single gene segment, which is very laborious. Our new plasmid-based system offers the potential for studies of all the RV gene segments, without needing a helper virus and selection system."

He said that while there are already "partial reverse genetics systems," in which a single RV gene segment could be incorporated into helper RVs, "we have generated, for the first time, recombinant virus entirely from cloned cDNAs."

That is a significant development, as an "entirely plasmid-based reverse genetics system to engineer viable viruses that contain a specific sequence modification, provides a powerful approach for studying viral replication and pathogenesis, and developing new vaccines and viral vectors," Kobayashi said.

"While viral genomic RNAs cannot be manipulated directly, it is relatively easy to introduce mutations into viral cDNAs using basic DNA monocular cloning techniques. Then, using those modified plasmids, we can generate gene-modified recombinant RVs."

The researchers then used this newly developed reverse genetics system to obtain valuable new insights into the process by which RV nonstructural protein 1 (NSP1) disrupts host innate immune responses.

"NSP1 mutants incapable of blocking interferon signaling and apoptosis pathways may be attractive candidates for development of new attenuated RV vaccines," said Kobayashi.

Moreover, by insertion into the NSP1 gene segment, they recovered recombinant "reporter" RVs that encoded split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. Such fluorescent or chemiluminescent signaling reporter viruses are useful for basic research, as the presence of viruses can be seen in live imaging.

"We are planning to use reporter viruses for screening antiviral drugs for efficacy against RV. Further, our results using reporter viruses should reveal the potential for exploitation of RV as a gene-transduction vector with useful applications in developing new oral and mucosal vaccine vectors."

In the future, the researchers will use the new reverse genetics system "to attempt to generate functional mutants in all RV gene segments to understand the function of each segment in viral replication and pathogenesis," said Kobayashi.

"For example, by using reverse genetics and animal models, we would like to discover how RV infection induces more severe symptoms, including RV-related encephalopathy and intussusception. We will also use reverse genetics to design safer and more effective attenuated vaccine strains."