A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

Generation and Efficacy of Two Chimeric Viruses Derived from GPE- Vaccine Strain as Classical Swine Fever Vaccine Candidates.

A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020.

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.