A reverse genetics system for human rotavirus G2P[4].

Rotaviruses (RVs) are an important cause of acute gastroenteritis in young children. Recently, versatile plasmid-based reverse genetics systems were developed for several human RV genotypes; however, these systems have not been developed for all commonly circulating human RV genotypes. In this study, we established a reverse genetics system for G2P[4] human RV strain HN126. Nucleotide sequence analysis, including that of the terminal ends of the viral double-stranded RNA genome, revealed that HN126 possessed a DS-1-like genotype constellation. Eleven plasmids, each encoding 11 gene segments of the RV genome, and expression plasmids encoding vaccinia virus RNA capping enzyme (D1R and D12L), Nelson Bay orthoreovirus FAST, and NSP2 and NSP5 of HN126, were transfected into BHK-T7 cells, and recombinant strain HN126 was generated. Using HN126 or simian RV strain SA11 as backbone viruses, reassortant RVs carrying the outer and intermediate capsid proteins (VP4, VP7 and VP6) of HN126 and/or SA11 (in various combinations) were generated. Viral replication analysis of the single, double and triple reassortant viruses suggested that homologous combination of the VP4 and VP7 proteins contributed to efficient virus infectivity and interaction between other viral or cellular proteins. Further studies of reassortant viruses between simian and other human RV strains will contribute to developing an appropriate model for human RV research, as well as suitable backbone viruses for generation of recombinant vaccine candidates.

Novel apoptosis suppressor Apsup from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus precludes apoptosis by preventing proteolytic processing of initiator caspase Dronc.

We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells.

Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Degradation of rRNA in BM-N cells from the silkworm Bombyx mori during abortive infection with heterologous nucleopolyhedroviruses.

Cell lines derived from the silkworm, Bombyx mori, are only permissive for B. mori nucleopolyhedrovirus (NPV), with other NPVs generally resulting in abortive infection. Here, we demonstrate that rRNA of B. mori BM-N cells undergoes rapid degradation through site-specific cleavage upon infection with NPVs from Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua (SeMNPV) and Spodoptera litura (SpltMNPV). No significant decreases in cellular RNA were observed in Ld652Y, Se301, Sf9, SpIm and S2 cells infected with AcMNPV or HycuMNPV, indicating the response is unique to BM-N cells. A transient expression assay using a cosmid library of the HycuMNPV genome demonstrated that HycuMNPV P143 is responsible for rRNA degradation, which was also detected in BM-N cells transfected with plasmids expressing the P143 proteins from AcMNPV, SeMNPV and SpltMNPV. These results indicate that B. mori evolved to acquire a unique antiviral immune mechanism that is activated by P143 proteins from heterologous NPVs.

Cloning and functional characterization of the Lymantria dispar initiator caspase dronc.

Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.

The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells.

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

NISES-AnPe-428 cell line derived from the Chinese oak silkworm Antheraea pernyi is permissive for multiple nucleopolyhedrovirus species from insects of four different families.

The cell line NISES-AnPe-428 (AnPe), derived from the Chinese oak silkworm Antheraea pernyi, was characterized for its permissiveness and productivity for six different nucleopolyhedrovirus (NPV) species. These NPVs included homologous Antheraea pernyi NPV (AnpeNPV) and heterologous Autographa californica multiple NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Spodoptera exigua MNPV (SeMNPV), and Lymantria dispar MNPV (LdMNPV), representing viruses that had been isolated from insect species belonging to five different families (Saturniidae, Noctuidae, Bombycidae, Arctiidae, and Lymantriidae). We found that AnPe cells supported productive replication of AnpeNPV, AcMNPV, BmNPV, HycuMNPV, and SeMNPV to varying degrees. Upon infection with SeMNPV, a subset of AnPe cell population in the culture underwent apoptosis, while remaining cells produced limited amounts of progeny virions and polyhedra. AnPe cells were refractory to LdMNPV infection and failed to support replication of viral DNA, indicating that viral replication was restricted at or prior to the step of viral DNA replication. These results indicated that AnPe cells have the potential to provide excellent systems for studying the molecular mechanisms underlying cellular permissiveness for NPV replication and host-range determination of NPVs.

Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection.

High-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.

Identification of the minimal AcMNPV P143 protein region responsible for triggering apoptosis and rRNA degradation of Bombyx mori cells.

Bombyx mori cells induce antiviral responses including global protein synthesis shutdown, rRNA degradation, and apoptosis upon infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We previously demonstrated that five and six amino acid residues located at positions between 514 and 599 of AcMNPV P143 (Ac-P143) protein are important for induction of apoptosis and rRNA degradation, respectively. However, it remains unexplored whether other residues of Ac-P143 protein also participate in antiviral immune responses. Here, we conducted transient expression analysis using a number of Ac-P143 protein deletion and truncation mutants and found that some of the N-terminal 413 residues (amino acids 1-413), besides previously identified residues between amino acids 514 and 599, are indispensable, whereas C-terminal 622 residues (amino acids 600-1221) are dispensable, for Ac-P143 protein to induce apoptosis or rRNA degradation. In addition, we found that the N-terminal 413 sequence (amino acids 1-413) of Ac-P143 protein can be substituted with corresponding BmNPV P143 (Bm-P143) protein sequence. Further analysis demonstrated that mutant Ac-P143 protein consisting of 275 residues (amino acids 325-599), but not 274 residues (amino acids 326-599) lacking glutamine residue at position 325 (Q325), is sufficient for triggering apoptosis and rRNA degradation of B. mori cells. These 275 residues are located outside the region of DNA helicase motifs of Ac-P143 protein, indicating that induction of apoptosis or rRNA degradation occurs independently of viral DNA replication-related function of the Ac-P143 protein. Moreover, Ac-P143(325-599/Q325A) and Ac-P143(1-599/Q325A) proteins harboring Q325A substitution retain the ability to induce apoptosis and rRNA degradation in B. mori cells. These findings suggest that the Ac-P143 protein needs minimal sequence length starting from the Q325 residue that contains a specific effector domain to induce apoptosis and rRNA degradation.

Antiviral immune responses of Bombyx mori cells during abortive infection with Autographa californica multiple nucleopolyhedrovirus.

Lepidopteran cells rely on multiple antiviral responses to defend against baculovirus infections, including apoptosis, global protein synthesis shutdown, and rRNA degradation. Here, we characterized apoptosis and rRNA degradation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Bombyx mori cells, a system resulting in abortive infection, in relation to viral DNA replication and viral late gene expression. RNAi-mediated silencing of viral DNA replication-related genes prevented apoptosis, but not rRNA degradation, in B. mori cells infected with p35-deficient AcMNPV. Additionally, AcMNPV, but not B. mori nucleopolyhedrovirus (BmNPV), drastically reduced B. mori cellular iap1 transcript levels and p35-deficient AcMNPV induced more prominent apoptosis than did p35-deficient BmNPV. These results, together with previous results that global protein synthesis shutdown follows viral DNA replication, demonstrate that rRNA degradation is the primary antiviral response that abolishes productive AcMNPV infection of B. mori cells. Our results also demonstrate that B. mori cells induce apoptosis to a different extent depending on NPV species.

Bombyx mori homolog of tumor suppressor p53 is involved in apoptosis-mediated antiviral immunity of B. mori cells infected with nucleopolyhedrovirus.

Apoptosis is important in antiviral immunity and affects viral multiplication and pathogenesis. Here, we showed that Bombyx mori cells transiently expressing B. mori homolog of the tumor suppressor p53 (Bm-p53) protein underwent apoptosis accompanied by elevated caspase-3-like protease activity and processing of B. mori Dronc (Bm-Dronc). RNAi-mediated silencing of bm-p53 expression, which significantly diminished accumulation of bm-p53 transcript and Bm-p53 protein, prevented apoptosis of B. mori cells infected with a recombinant B. mori nucleopolyhedrovirus defective in the anti-apoptotic p35 gene (vBmΔp35) and abolished the activation of caspase-3-like protease and processing of Bm-Dronc. Apoptosis in vBmΔp35-infected B. mori cells is associated with viral DNA replication, suggesting involvement of the DNA damage response. The Bm-p53 pro-apoptotic function is also found in Spodoptera frugiperda and Lymantria dispar cells. These results indicate that apoptosis induction in vBmΔp35-infected B. mori cells is a Bm-p53-mediated process promoted by the commencement of viral DNA replication.

P143 proteins from heterologous nucleopolyhedroviruses induce apoptosis in BM-N cells derived from the silkworm Bombyx mori.

We previously demonstrated that ribosomal RNA (rRNA) of Bombyx mori BM-N cells is rapidly degraded upon infection with heterologous nucleopolyhedroviruses (NPVs), including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV and S. litura MNPV, and that this response is triggered by viral P143 proteins. The transient expression of P143 proteins from heterologous NPVs was also shown to induce apoptosis and caspase-3-like protease activation in BM-N cells. In the present study, we conducted a transient expression assay using BM-N cells expressing mutant AcMNPV P143 (Ac-P143) proteins and demonstrated that five amino acid residues cooperatively participate in Ac-P143 protein-triggered apoptosis of BM-N cells. Notably, these five residues were previously shown to be required for triggering rRNA degradation in BM-N cells. As rRNA degradation in BM-N cells does not result from apoptosis, the present results suggest that Ac-P143-triggered rRNA degradation is the upstream signal for apoptosis induction in BM-N cells. We further showed that P143 protein-triggered apoptosis does not occur in S. frugiperda Sf9 or Lymantria dispar Ld652Y cells, indicating that apoptosis induction by heterologous P143 proteins is a BM-N cell-specific response. In addition, the observed induction of apoptosis in BM-N cells was found to be mediated by activation of the initiator caspase Bm-Dronc. Taken together, these results suggest that BM-N cells evolved a unique antiviral system that recognizes heterologous NPV P143 proteins to induce rRNA degradation and caspase-dependent apoptosis.

HCF-1 encoded by baculovirus AcMNPV is required for productive nucleopolyhedrovirus infection of non-permissive Tn368 cells.

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in both Spodoptera frugiperda Sf21 and Trichoplusia ni Tn368 cells, whereas AcMNPV defective in hcf-1 (host cell-factor 1) gene productively infects only Sf21 cells, indicating that HCF-1 is indispensable for the AcMNPV productive infection of Tn368 cells. Here, we demonstrated that HCF-1 protein transiently expressed in Tn368 cells promotes the DNA synthesis of Hyphantria cunea MNPV (HycuMNPV), Orygia pseudotsugata MNPV and Bombyx mori NPV, which are normally unable to replicate in Tn368 cells. We also demonstrated that a recombinant HycuMNPV harboring the hcf-1 gene successfully replicates in Tn368 cells, generating substantial yields of progeny viruses and polyhedra. These results indicate that HCF-1 encoded by AcMNPV is an essential viral factor for productive NPV infection of Tn368 cells. Taken together with the previous findings on HRF-1 (host range factor 1), the present results provide strong evidence that viral genes acquired through horizontal gene transfer play an important role in baculovirus evolution, serving to expand the host range of baculoviruses.

Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori.

Recent advances in genome-wide surveys have revealed a number of lepidopteran insect homologs of mammalian and Drosophila genes that are responsible for apoptosis regulation. However, the underlying molecular mechanisms for apoptosis regulation in lepidopteran insect cells remain poorly understood. In the present study, we demonstrated that the transfection of Bombyx mori BM-N cells with dsRNA against the B. mori cellular iap1 gene (cbm-iap1) induces severe apoptosis that is accompanied by an increase of caspase-3-like protease activity. In these apoptotic cells, the cleaved form of the endogenous initiator caspase Dronc (Bm-Dronc) was detected, indicating that cBm-IAP1 protein depletion by RNAi silencing resulted in the activation of Bm-Dronc. In transient expression assays in BM-N cells, cBm-IAP1 suppressed the apoptosis triggered by Bm-Dronc overexpression and depressed the elevation of caspase-3-like protease activity, but also increased the cleaved form of Bm-Dronc protein. cBm-IAP1 also suppressed the caspase-3-like protease activity stimulated by Bm-caspase-1 overexpression. Co-immunoprecipitation experiments demonstrated that cBm-IAP1 strongly interacts with Bm-Dronc, but only has weak affinity for Bm-caspase-1. Transient expression analyses showed that truncated cBm-IAP1 proteins defective in the BIR1, BIR2 or RING domain were unable to suppress Bm-Dronc-induced apoptosis. In addition, BM-N cells expressing truncated cBm-IAP1 proteins underwent apoptosis, suggesting that intact cBm-IAP1, which has anti-apoptotic activity, was replaced or displaced by the overexpressed truncated cBm-IAP1 proteins, which are incapable of interfering with the apoptotic caspase cascade. Taken together, the present results demonstrate that cBm-IAP1 is a vital negative regulator of apoptosis in BM-N cells and functions by preventing the activation and/or activity of Bm-Dronc and Bm-caspase-1.

Identification of amino acid residues of AcMNPV P143 protein involved in rRNA degradation and restricted viral replication in BM-N cells from the silkworm Bombyx mori.

We previously demonstrated that rRNA undergoes rapid and extensive degradation in Bombyx mori BM-N cells upon infection with AcMNPV, which is triggered by AcMNPV P143 (Ac-P143) protein. Here, we showed that six amino acid residues of Ac-P143 protein, distributing between positions 514 and 599, are involved in rRNA degradation in BM-N cells. The six residues are highly conserved among P143 proteins from AcMNPV, HycuMNPV, SeMNPV and SpltMNPV, which trigger rRNA degradation in BM-N cells upon infection, but are only partially conserved in Bm-P143 protein, which does not induce rRNA degradation in BM-N cells. We also demonstrated that substitution of only two selected residues (N565S/L578F) of Bm-P143 protein with the corresponding Ac-P143 protein residues generates a mutant Bm-P143 protein that is capable of triggering rRNA degradation in BM-N cells. These results indicate that BmNPV evolved a unique P143 protein to evade the antiviral response and allow replication in B. mori cells.

Baculovirus genes modulating intracellular innate antiviral immunity of lepidopteran insect cells.

Innate immunity is essential for insects to survive infectious pathogens. In baculovirus-infected lepidopteran cells, apoptosis and global protein synthesis shutdown are major mechanisms of intracellular innate immunity that inhibit viral replication. In contrast, baculoviruses have evolved diverse genes and mechanisms to counter the antiviral immunity activated in infected cells. In this review, we summarize the current knowledge of the cellular antiviral pathways and the baculovirus genes that modulate antiviral immunity. The studies highlighted illustrate a high degree of diversity in both the cellular responses against viral infections and viral responses against intracellular antiviral immunity, providing an important basis of further studies in this field.