In Vivo Dynamics of Reporter Flaviviridae Viruses

Abstract

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals. IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

Development of a High-Throughput Serum Neutralization Test Using Recombinant Pestiviruses Possessing a Small Reporter Tag

“…In our previous studies, recombinant Flaviviridae viruses possessing a small reporter HiBiT tag were developed as tools to understand the viral life cycle and pathogenesis and to provide a robust platform for the development of novel antivirals [30,31]. For those studies, two recombinant pestiviruses, vCSFV GPE − /HiBiT and vBVDV-1 NCP7/HiBiT, were established from the recombinant full-length cDNA of CSFV GPE − and BVDV-1 NCP7 strains, respectively.…”

“…For those studies, two recombinant pestiviruses, vCSFV GPE − /HiBiT and vBVDV-1 NCP7/HiBiT, were established from the recombinant full-length cDNA of CSFV GPE − and BVDV-1 NCP7 strains, respectively. To establish a novel SNT using pestiviruses possessing a small reporter tag, the recombinant full-length cDNA of the BDV FNK2012-1 strain, BDV subgenotype-1 (BDV-1) isolated from domestic pigs in Japan [22] was newly established and a BDV recombinant was generated by inserting HiBiT between C and E rns (vBDV FNK/HiBiT, Figure 1A), since the N-termini of E rns of CSFV and BVDV tolerate the exogenous tag [27,30,31]. To compare the growth of recombinant vBDV FNK/HiBiT with the wild-type BDV, swine kidney line-L (SK-L) cells were inoculated with 200 50% tissue-culture infective doses (TCID 50 ) of either virus.…”

“…Since the first recombinant pestivirus carrying a marker gene was constructed [25], various reporter genes have been applied for pestivirus research [26][27][28]. Recently, to reduce the risk of undesirable effects following the insertion of a large foreign gene into a viral genome, NanoLuc binary technology (NanoBiT) [29] was employed for the construction of recombinant CSFV and BVDV-1 carrying a small reporter gene [30,31]. NanoBiT is a split reporter protein consisting of two subunits: a high-affinity…”

“…In the present study, a recombinant BDV carrying the HiBiT gene was newly constructed and applied for SNT, together with previously established recombinant CSFV and BVDV-1 possessing HiBiT [30,31], to reduce the time and effort required to detect specific neutralizing antibodies. This new SNT method using these recombinant viruses showed the same sensitivity and specificity as conventional SNTs.…”

Real Time Analysis of Bovine Viral Diarrhea Virus (BVDV) Infection and Its Dependence on Bovine CD46

“…Members of this genus are pathogens of cloven-hoofed animals, including the highly economically relevant pathogens bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). In recent years, genetically labelled BVDV and CSFV clones have been constructed based on N-terminal fusion of luciferase or fluorophores to either E rns or E2 [10][11][12][13]. For a BVDV E2-fluorophore fusion protein, visualization of purified particles in fluorescence microscopy based on the specific fluorescence signal has been reported [13].…”