Reverse genetics of parrot bornavirus 4 reveals a unique splicing of the glycoprotein gene that affects viral propagation.

Viruses can utilize host splicing machinery to enable the expression of multiple genes from a limited-sized genome. Orthobornaviruses use alternative splicing to regulate the expression level of viral proteins and achieve efficient viral replication in the nucleus. Although more than 20 orthobornaviruses have been identified belonging to eight different viral species, virus-specific splicing has not been demonstrated. Here, we demonstrate that the glycoprotein (G) transcript of parrot bornavirus 4 (PaBV-4; species Orthobornavirus alphapsittaciforme), a highly virulent virus in psittacines, undergoes mRNA splicing and expresses a soluble isoform termed sGP. Interestingly, the splicing donor for sGP is not conserved in other orthobornaviruses, including those belonging to the same orthobornavirus species, suggesting that this splicing has evolved as a PaBV-4-specific event. We have also shown that exogenous expression of sGP does not affect PaBV-4 replication or de novo virion infectivity. In this study, to investigate the role of sGP in viral replication, we established a reverse genetics system for PaBV-4 by using avian cell lines and generated a recombinant virus lacking the spliced mRNA for sGP. Using the recombinant viruses, we show that the replication of the sGP-deficient virus is significantly slower than that of the wild-type virus and that the exogenous expression of sGP cannot restore its propagation efficiency. These results suggest that autologous or controlled expression of sGP by splicing may be important for PaBV-4 propagation. The reverse genetics system for avian bornaviruses developed here will be a powerful tool for understanding the replication strategies and pathogenesis of avian orthobornaviruses. IMPORTANCE Parrot bornavirus 4 (PaBV-4) is the dominant cause of proventricular dilatation disease, a severe gastrointestinal and central nervous system disease among avian bornaviruses. In this study, we discovered that PaBV-4 expresses a soluble isoform of glycoprotein (G), called sGP, through alternative splicing of the G mRNA, which is unique to this virus. To understand the role of sGP in viral replication, we generated recombinant PaBV-4 lacking the newly identified splicing donor site for sGP using a reverse genetics system and found that its propagation was significantly slower than that of the wild-type virus, suggesting that sGP plays an essential role in PaBV-4 infection. Our results provide important insights not only into the replication strategy but also into the pathogenesis of PaBV-4, which is the most prevalent bornavirus in captive psittacines worldwide.

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity.

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low infectious viral particle production in cells and the lack of cytopathic effects, which are typically useful for screening. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of members of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the methionine at BoDV-1-G residue 307 increases the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell‒cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1.

Borna disease virus 1 (BoDV-1) is a non-segmented, negative-strand RNA virus that is characterized by persistent infection in the nucleus and low production of progeny virions. This feature impedes not only the harvesting of infectious viral particles from infected cells but also the rescue of high titres of recombinant BoDV-1 (rBoDV-1) by reverse genetics. Here, we demonstrate that exogenous expression of both matrix protein (M) and glycoprotein (G), which are constituents of the viral lipid envelope, significantly facilitates the formation of infectious particles and propagation of BoDV-1 without affecting its viral RNA synthesis. Furthermore, simultaneous transfection of M and G expression plasmids with N, P and L helper plasmids by reverse genetics drastically enhances the rescue efficiency of rBoDV-1. On the other hand, we also show that overexpression of M induces obvious cytotoxicity similar to that of other Mononegaviruses. Together with our recent report showing that excess expression of G induces aberrant accumulation of immature G, a potential stimulator of the host innate immune response, it is conceivable that BoDV-1 may suppress excess expression of M and G to reduce the cytopathic effect, thereby leading to maintenance of persistent infection. Our results contribute not only to the establishment of an efficient method to recover high-titre BoDV-1 but also to understanding the unique mechanism of persistent BoDV-1 infection.

The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity.

An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate an REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus. We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, conversely, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also to understanding of the molecular regulation of orthobornavirus polymerase activity. IMPORTANCE Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus, is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus, can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.

Optimal Expression of the Envelope Glycoprotein of Orthobornaviruses Determines the Production of Mature Virus Particles.

An RNA virus-based episomal vector (REVec) whose backbone is Borna disease virus 1 (BoDV-1) can provide long-term gene expression in transduced cells. To improve the transduction efficiency of REVec, we evaluated the role of the viral envelope glycoprotein (G) of the genus Orthobornavirus, including that of BoDV-1, in the production of infectious particles. By using G-pseudotype assay in which the lack of G in G-deficient REVec (ΔG-REVec) was compensated for expression of G, we found that excess expression of BoDV-1-G does not affect particle production itself but results in uncleaved and aberrant mature G expression in the cells, leading to the production of REVec particles with low transduction titers. We revealed that the expression of uncleaved G in the cells inhibits the incorporation of mature G and vgRNA into the particles. This feature of G was conserved among mammalian and avian orthobornaviruses; however, the cleavage efficacy of canary bornavirus 1 (CnBV-1)-G was exceptionally not impaired by its excess expression, which led to the production of the pseudotype ΔG-REVec with the highest titer. Chimeric G proteins between CnBV-1 and -2 revealed that the signal peptide of CnBV-1-G was responsible for the cleavage efficacy through the interaction with intracellular furin. We showed that CnBV-1 G leads to the development of pseudotyped REVec with high transduction efficiency and a high-titer recombinant REVec. Our study demonstrated that the restricted expression of orthobornavirus G contributes to the regulation of infectious particle production, the mechanism of which can improve the transduction efficiency of REVec.IMPORTANCE Most viruses causing persistent infection produce few infectious particles from the infected cells. Borna disease virus 1, a member of the genus Orthobornavirus, is an RNA virus that persistently infects the nucleus and has been applied to vectors for long-term gene expression. In this study, we showed that, common among orthobornaviruses, excessive G expression does not affect particle production itself but reduces the production of infectious particles with mature G and genomic RNA. This result suggested that limited G expression contributes to suppressing abnormal viral particle production. On the other hand, we found that canary bornavirus 1 has an exceptional G maturation mechanism and produces a high-titer virus. Our study will contribute to not only understanding the mechanism of infectious particle production but also improving the vector system of orthobornaviruses.

Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes.

Foot-and-mouth disease (FMD) is a host-restricted disease of cloven-hoofed animals, such as cattle and pigs. There are seven major serotypes of FMD virus that exhibit high antigenic variation, making vaccine strain selection difficult. However, there is an internal ribosomal entry site (IRES) element within the 5' untranslated region of the FMD virus (FMDV) RNA genome that is relatively conserved among FMDV serotypes and could be used as a pan-serotype target for disease interventions. To determine the potential for targeting the IRES as promising drug target, we designed a short interfering RNA (siRNA) targeting a relatively conserved region in the FMDV-IRES. The siRNA affected FMDV-IRES expression but not the expression of the encephalomyocarditis virus or hepatitis C virus IRES. To evaluate the effects of siRNA-mediated silencing, we established cell lines expressing a bicistronic luciferase reporter plasmid, which contained an FMDV-IRES element between the Renilla and firefly luciferase genes. The designed siRNA inhibited FMDV-IRES-mediated translation in a concentration-dependent manner. In order to sustain this inhibitory effect, we designed a short hairpin RNA (shRNA)-expressing lentiviral vector. The results showed that the lenti-shRNA vector significantly suppressed FMDV-IRES activity for up to 2 weeks in cell culture. Thus, our findings in this study provided a basis for the development of effective pan-serotype FMDV inhibitors.

Genetic characterization of an avian H4N6 influenza virus isolated from the Izumi plain, Japan.

An influenza A virus of H4N6 subtype was isolated from the Izumi plain, Japan, in 2013. Genetic analyses revealed that two viral genes (M and NS gene segments) of this isolate were genetically distinct from those of the H4N6 virus isolated from the same place in 2012. Furthermore, three viral genes (PB2, PB1 and M gene segments) of this isolate share high similarity with those of the North American isolates of 2014. These results suggest a high frequency of genetic reassortment of avian influenza viruses in Asian waterfowl and intercontinental movements of avian influenza viruses via migratory waterfowl.

IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals.

BACKGROUND: Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5' untranslated region (5'UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs). Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far. We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells. RESULTS: Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV. CONCLUSIONS: IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.

Full genome sequences of torque teno sus virus strains that coinfected a pig with postweaning multisystemic wasting syndrome in Japan: implications for genetic diversity.

We determined the complete genome sequences of torque teno sus viruses (TTSuVs) detected in pigs with postweaning multisystemic wasting syndrome (PMWS) and in healthy pigs in Japan. Unexpectedly, we found coinfection of a PMWS-affected pig in Japan with one strain of TTSuV1, five strains of TTSuV2, and one strain of PCV2. Full-genome sequencing of each of these strains, followed by phylogenetic analysis, revealed broad genetic diversity in the TTSuV2 strains infecting the PMWS-affected pig. These results suggest that the geographical bias in the available genetic information about TTSuVs has a limited impact on the evaluation of their genetic diversity.

Reverse Genetics and Artificial Replication Systems of Borna Disease Virus 1.

Borna disease virus 1 (BoDV-1) is a neurotropic RNA virus belonging to the family Bornaviridae within the order Mononegavirales. Whereas BoDV-1 causes neurological and behavioral disorders, called Borna disease (BD), in a wide range of mammals, its virulence in humans has been debated for several decades. However, a series of case reports in recent years have established the nature of BoDV-1 as a zoonotic pathogen that causes fatal encephalitis in humans. Although many virological properties of BoDV-1 have been revealed to date, the mechanism by which it causes fatal encephalitis in humans remains unclear. In addition, there are no effective vaccines or antiviral drugs that can be used in clinical practice. A reverse genetics approach to generating replication-competent recombinant viruses from full-length cDNA clones is a powerful tool that can be used to not only understand viral properties but also to develop vaccines and antiviral drugs. The rescue of recombinant BoDV-1 (rBoDV-1) was first reported in 2005. However, due to the slow nature of the replication of this virus, the rescue of high-titer rBoDV-1 required several months, limiting the use of this system. This review summarizes the history of the reverse genetics and artificial replication systems for orthobornaviruses and explores the recent progress in efforts to rescue rBoDV-1.