Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

The 3' end of the coding region of senecavirus A contains a highly conserved sequence that potentially forms a stem-loop structure required for virus rescue.

Senecavirus A (SVA) can cause a vesicular disease in swine. It is a positive-strand RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Positive-strand RNA viruses possess positive-sense, single-stranded genomes whose untranslated regions (UTRs) have been reported to contain cis-acting RNA elements. In the present study, a total of 100 SVA isolates were comparatively analyzed at the genome level. A highly conserved fragment (HCF) was found to be located in the 3D sequence and to be close to the 3' UTR. The HCF was computationally predicted to form a stem-loop structure. Eight synonymous mutations can individually disrupt the formation of a single base pair within the stem region. We found that SVA itself was able to tolerate each of these mutations alone, as evidenced by the ability to rescue all eight single-site mutants from their individual cDNA clones, and all of them were genetically stable during serial passaging. However, the replication-competent SVA could not be rescued from another cDNA clone containing all eight mutations. The failure to recover SVA might be attributed to disruption of the predicted stem-loop structure, whereas introduction of a wild-type HCF into the cDNA clone with eight mutations still had no effect on virus recovery. These results suggest that the putative stem-loop structure at the 3' end of the 3D sequence is a cis-acting RNA element that is required for SVA growth.

Rescue of recombinant canine distemper virus that expresses S1 subunit of SARS-CoV-2 spike protein in vitro.

The coronavirus disease 2019 (COVID-19), as an unprecedented pandemic, has rapidly spread around the globe. Its etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus in the family Coronaviridae. The viral S1 subunit has been demonstrated to have a powerful potential in inducing protective immune responses in vivo. Since April 2020, farmed minks were frequently reported to be infected with the SARS-CoV-2 in different countries. Unfortunately, there has been no available veterinary vaccine as yet. In this study, we used reverse genetics to rescue a recombinant canine distemper virus (CDV) that could express the SARS-CoV-2 S1 subunit in vitro. The S1 subunit sequence was demonstrated to be relatively stable in the genome of recombinant CDV during twenty serial viral passages in cells. However, due to introduction of the S1 subunit sequence into CDV genome, this recombinant CDV grew more slowly than the wild-type strain did. The genomic backbone of recombinant CDV was derived from a virulence-attenuating strain (QN strain). Therefore, if able to induce immune protections in minks from canine distemper and COVID-19 infections, this recombinant would be a potential vaccine candidate for veterinary use.

Molecular characteristics of a canine distemper virus isolated from mink (Neovison vison) in Shandong, China, 2013.

No abstract Keywords: canine distemper virus; H gene; phylogenetic analysis; amino-acid substitution; N-linked glycolysation; cysteine.

Molecular Characterization of African Swine Fever Virus, China, 2018.

On August 3, 2018, an outbreak of African swine fever in pigs was reported in China. We subjected a virus from an African swine fever-positive pig sample to phylogenetic analysis. This analysis showed that the causative strain belonged to the p72 genotype II and CD2v serogroup 8.

Major mutation events in structural genes of peste des petits ruminants virus through serial passages in vitro.

Peste des petits ruminants (PPR) is an highly contagious disease of small ruminants, and caused by peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus in the family Paramyxoviridae. The first outbreak of PPR in China was officially reported in July 2007, when a PPRV strain was successfully isolated from a sick goat in Tibet, followed by sequencing at a full-genome level (China/Tibet/Geg/07-30, GenBank: FJ905304.1). To date, this isolate has been virulently attenuated by more than 90 serial passages in Vero-Dog-SLAM cells at our laboratory. In this study, a total of nine strains by serial passages (namely the 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, and 90th passages) were chosen for sequencing of six structural genes in PPRV. The sequence analysis showed that mutation rates in all viral genes were relatively low, and only a few identical mutations within certain genes were stably maintained after an earlier passage, perhaps indicating a predominance of mutants after such a passage.

Comparative transcriptomics analysis on Senecavirus A-infected and non-infected cells.

Senecavirus A (SVA) is an emerging virus that causes the vesicular disease in pigs, clinically indistinguishable from other high consequence vesicular diseases. This virus belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-stranded RNA, approximately 7,300 nt in length, with a 3' poly(A) tail but without 5'-end capped structure. SVA can efficiently propagate in different cells, including some non-pig-derived cell lines. A wild-type SVA was previously rescued from its cDNA clone using reverse genetics in our laboratory. In the present study, the BSR-T7/5 cell line was inoculated with the passage-5 SVA. At 12 h post-inoculation, SVA-infected and non-infected cells were independently collected for the analysis on comparative transcriptomics. The results totally showed 628 differentially expressed genes, including 565 upregulated and 63 downregulated ones, suggesting that SVA infection significantly stimulated the transcription initiation in cells. GO and KEGG enrichment analyses demonstrated that SVA exerted multiple effects on immunity-related pathways in cells. Furthermore, the RNA sequencing data were subjected to other in-depth analyses, such as the single-nucleotide polymorphism, transcription factors, and protein-protein interactions. The present study, along with our previous proteomics and metabolomics researches, provides a multi-omics insight into the interaction between SVA and its hosts.

AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome.

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem-loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.

Structure and function of type IV IRES in picornaviruses: a systematic review.

The Picornaviridae is a family of icosahedral viruses with single-stranded, highly diverse positive-sense RNA genomes. Virions consist of a capsid, without envelope, surrounding a core of RNA genome. A typical genome of picornavirus harbors a well-conserved and highly structured RNA element known as the internal ribosome entry site (IRES), functionally essential for viral replication and protein translation. Based on differences in their structures and mechanisms of action, picornaviral IRESs have been categorized into five types: type I, II, III, IV, and V. Compared with the type IV IRES, the others not only are structurally complicated, but also involve multiple initiation factors for triggering protein translation. The type IV IRES, often referred to as hepatitis C virus (HCV)-like IRES due to its structural resemblance to the HCV IRES, exhibits a simpler and more compact structure than those of the other four. The increasing identification of picornaviruses with the type IV IRES suggests that this IRES type seems to reveal strong retention and adaptation in terms of viral evolution. Here, we systematically reviewed structural features and biological functions of the type IV IRES in picornaviruses. A comprehensive understanding of the roles of type IV IRESs will contribute to elucidating the replication mechanism and pathogenesis of picornaviruses.

Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins.

Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs.

Structural and nonstructural proteins of Senecavirus A: Recent research advances, and lessons learned from those of other picornaviruses.

Senecavirus A (SVA) is an emerging virus, causing vesicular disease in swine. SVA is a single-stranded, positive-sense RNA virus, which is the only member of the genus Senecavirus in the family Picornaviridae. SVA genome encodes 12 proteins: L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D. The VP1 to VP4 are structural proteins, and the others are nonstructural proteins. The replication of SVA in host cells is a complex process coordinated by an elaborate interplay between the structural and nonstructural proteins. Structural proteins are primarily involved in the invasion and assembly of virions. Nonstructural proteins modulate viral RNA translation and replication, and also take part in antagonizing the antiviral host response and in disrupting some cellular processes to allow virus replication. Here, we systematically reviewed the molecular functions of SVA structural and nonstructural proteins by reference to literatures of SVA itself and other picornaviruses.

Cells at early and late stages of infection with Senecavirus A: Comparative analysis of N6-methyladenosine modification on mRNAs.

Infection with Senecavirus A (SVA) causes differential phenotypes in cells. In this study, cells were inoculated with SVA for culture. At 12 and 72 h post infection, cells were independently harvested for high-throughput RNA sequencing, and further methylated RNA immunoprecipitation sequencing. The resultant data were comprehensively analyzed for mapping N6-methyladenosine (m6A)-modified profiles of SVA-infected cells. More importantly, m6A-modified regions were identified in the SVA genome. A dataset of m6A-modified mRNAs was generated for screening out differentially m6A-modified mRNAs, further subjected to a series of in-depth analyses. This study not only showed statistical differentiation of m6A-modified sites between two SVA-infected groups, but also demonstrated that SVA genome, as a positive-sense, single-stranded mRNA, itself could be modified through the m6A pattern. Out of the six samples of SVA mRNAs, only three were identified to be m6A-modified, implying that the epigenetic effect might not be a crucial driving force for SVA evolution.

A putative wild-type or wild-type-like hairpin structure is required within 3' untranslated region of Senecavirus A for virus replication.

The 3' untranslated region (UTR) of Senecavirus A (SVA) was predicted to harbor two hairpin structures, hairpin-I and -II. The former is composed of two internal loops, one terminal loop and three stem regions; the latter comprises one internal loop, one terminal loop and two stem regions. In this study, we constructed a total of nine SVA cDNA clones, which contained different point mutations within a stem-formed motif in the hairpin-I or -II, for rescuing replication-competent viruses. Only three mutants were successfully rescued and moreover genetically stable during at least five serial passages. Computer-aided prediction showed these three mutants bearing either a wild-type or a wild-type-like hairpin-I in their individual 3' UTRs. Neither wild-type nor wild-type-like hairpin-I could be computationally predicted to exist in 3' UTRs of the other six unviable "viruses". The results suggested that the wild-type or wild-type-like hairpin-I was necessary in the 3' UTR for SVA replication.

Identification of cis-acting replication element in VP2-encoding region of Senecavirus A genome.

Picornavirus possesses one positive-sense, single-stranded RNA genome, in which a cis-acting replication element (cre) is located. The cre is a stem-loop structure that harbors a conserved AAACA motif within its loop region. This motif functions as a template for adding two U residues to the viral VPg, therefore generating a VPg-pUpU that is required for viral RNA synthesis. Senecavirus A (SVA) is an emerging picornavirus. Its cre has not been identified as yet. In the present study, one putative cre containing a typical AAACA motif was computationally predicted to exist within the VP2-encoding sequence of SVA. To test the role of this putative cre, 22 SVA cDNA clones with different point mutations in their cre-formed sequences were constructed in an attempt to rescue replication-competent SVAs. A total of 11 viruses were rescued from their individual cDNA clones, implying that some mutated cres exerted lethal impacts on SVA replication. To eliminate these impacts, an intact cre was artificially inserted into those SVA cDNA clones without ability of recovering virus. The artificial cre was proven to be able of compensating for some, but not all, defects caused by mutated cres, leading to successful recovery of SVAs. These results indicated that the putative cre of SVA was functionally similar to those of other picornaviruses, perhaps involved in the uridylylation of VPg.

Translation of Senecavirus A polyprotein is initiated from the IRES-proximal initiation codon.

To clarify whether Senecavirus A (SVA) has the potential of alternative translation, an extra G residue was inserted into an SVA cDNA clone, resultantly generating an "AUGAUG" motif. The second AUG is the authentic SVA initiation codon, whereas the first AUG is a putative one. Subsequently, eighteen nucleotides were inserted one by one between AUG and AUG for reconstructing cDNA clones. The test of virus recovery showed that three replication-competent SVAs, whose AUG/AUG-flanked sequences were not multiples of three nucleotides, were successfully rescued from their individual cDNA clones. The wild-type SVA possesses a UUUUU motif within the polyprotein-encoding region. Sanger sequencing showed that these three replication-competent SVAs harbored one or two extra U residues in the UUUUU motif, implying that polyprotein translation was initiated from the putative AUG, and the authentic AUG would be inactivated. This is probably attributed to the lack of ribosome scanning along an SVA genome.

Only fourteen 3'-end poly(A)s sufficient for rescuing Senecavirus A from its cDNA clone, but inadequate to meet requirement of viral replication.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-strand RNA that has 5' and 3' untranslated regions. There is a poly(A) tail at the 3' end of viral genome. Although the number of poly(A)s is variable, the length of poly(A) tail generally has the minimum nucleotide limit for picornaviral replication. To identify a range limit of poly(A)s for SVA recovery, five SVA cDNA clones, separately containing 25, 20, 15, 10 and 5 poly(A)s, were constructed for rescuing viruses. Replication-competent SVAs could be rescued from the first three cDNA clones, implying the range limit of poly(A)s was (A)15 to (A)10. To recognize the precise limit, four extra cDNA clones, separately containing 14, 13, 12 and 11 poly(A)s, were constructed to rescue SVAs further. The replication-competent SVA was rescued only from the poly(A)14-containing plasmid, indicating that the precise limit was poly(A)14 at the 3' end of cDNA clone for SVA recovery. The rescued SVA was serially passaged in cells. The passage-5 and -10 progenies were independently subjected to the analysis of 3'-rapid amplification of cDNA ends. Both progenies showed their own poly(A) tails far more than 14 (A)s, implying extra (A)s added to the poly(A)14 sequence during viral passaging. It can be concluded that fourteen (A)s are sufficient for rescuing a replication-competent SVA from its cDNA clone, but inadequate for maintaining viral propagation in cells.

Recovery of Two Replication-Competent Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis with a high mortality rate in humans. Additionally, dogs are frequently reported to be infected with this disease. There has been no commercially available vaccine for humans and animals as yet. The SFTS is caused by Dabie bandavirus (DBV), formerly known as SFTS virus. The DBV is now classified into the genus Bandavirus in the family Phenuiviridae. DBV Gn and Gc can induce specific immune responses in vivo. In this study, we used reverse genetics technique to construct two recombinant canine distemper viruses (rCDVs), rCDV-Gn and -Gc, which could express Gn and Gc in vitro, respectively. Both of the recombinants, derived from a common parental CDV, were independently subjected to twenty serial passages in cells for Sanger sequencing. Neither point mutation nor fragment deletion was found in the Gn open reading frame (ORF), whereas the rCDV-Gc showed a nonsynonymous mutation (A157C) in the Gc ORF, correspondingly resulting in a mutation of amino acid (T53P) in the Gc. Growth curve of the rCDV-Gc almost coincided with that of a wild-type CDV, but exhibited a significant difference from that of the rCDV-Gn. Much research remains to be performed to demonstrate whether both recombinants are able of inducing specific immune responses in vivo.

Experimental evidence for occurrence of putative copy-choice recombination between two Senecavirus A genomes.

Senecavirus A (SVA), formerly known as Seneca Valley virus, belongs to the genus Senecavirus in the family Picornaviridae. SVA has a single-stranded, positive-sense RNA genome, which is actually an mRNA that initiates translation via its own internal ribosome entry site (IRES). The SVA IRES has been demonstrated to be the hepatitis C virus (HCV)-like IRES, containing eight stem-loop domains: domain (D)II, DIIIa, DIIIb, DIIIc, DIIId1, DIIId2, DIIIe and DIIIf. In this study, stem-forming motifs (SFMs) in the eight domains were independently subjected to site-directed mutagenesis (SDM) to construct eight SVA minigenomes for dual-luciferase reporter assay. The result suggested that except the DII, the other seven domains were closely evolved in the IRES activity. Subsequently, a full-length SVA cDNA clone tagged with a reporter gene was genetically modified to construct eight SFM-mutated ones, separately transfected into BSR-T7/5 cells in an attempt to rescue replication-competent SVAs. Nevertheless, no virus was successfully rescued from its own cDNA clone, implying each of the putative domains necessary in SVA IRES for viral replication. Further, we attempted to rescue replication-competent SVA via pairwise transfection of cDNA clones. Out of 28 combinations of co-transfection, four were demonstrated to be able to rescue replication-competent SVAs. Sanger sequencing showed that all four viruses had the wild-type IRES genotype, suggesting the occurrence of putative copy-choice recombination between two IRES-modifying genomes.

Genomic profile of eGFP-expressing canine distemper virus that undergoes serial plaque-to-plaque transfers.

Canine distemper virus (CDV) is classified into the genus Morbillivirus in the family Paramyxoviridae. This virus has a single-stranded genomic RNA with negative polarity. The wild-type CDV genome is generally composed of 15 690 nucleotides. We previously rescued an enhanced green fluorescence protein (eGFP)-tagged recombinant CDV (rCDV-eGFP) using reverse genetics. In this study, the rCDV-eGFP at passage-7 was subjected to 38 serial plaque-to-plaque transfers (or bottleneck passages) and two extra common passages in cells. In theory, the effect of Muller's ratchet may fix deleterious mutations in a single viral population after consecutive plaque-to-plaque transfers. In order to uncover a mutated landscape of the rCDV-eGFP under the circumstances of bottleneck passages, the passage-47 progeny was collected for the in-depth analysis via next-generation sequencing. The result revealed a total of nine single-nucleotide mutations (SNMs) in the viral antigenome. Out of them, SNMs at nt 1832, 5022, 5536, 5580, 5746, 6913 and 8803 were identified as total single-nucleotide substitution, i.e., 100% of mutation frequency. The result suggested no notable formation of viral quasispecies in the rCDV-eGFP population after consecutive plaque-to-plaque transfers.