Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1.

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

Genome characteristics and the ODV proteome of a second distinct alphabaculovirus from Spodoptera litura.

BACKGROUND: Spodoptera litura is a harmful pest that feeds on more than 80 species of plants, and can be infected and killed by Spodoptera litura nucleopolyhedrovirus (SpltNPV). SpltNPV-C3 is a type C SpltNPV clone, that was observed and collected in Japan. Compared with type A or type B SpltNPVs, SpltNPV-C3 can cause the rapid mortality of S. litura larvae. METHODS: In this study, occlusion bodies (OBs) and occlusion-derived viruses (ODVs) of SpltNPV-C3 were purified, and OBs were observed by scanning electron microscopy (SEM). ODVs were observed under a transmission electron microscope (TEM). RESULTS: Both OBs and ODVs exhibit morphological characteristics typical of nucleopolyhedroviruses (NPVs).The genome of SpltNPV-C3 was sequenced and analyzed; the total length was 148,634 bp (GenBank accession 780,426,which was submitted as SpltNPV-II), with a G + C content of 45%. A total of 149 predicted ORFs were found. A phylogenetic tree of 90 baculoviruses was constructed based on core baculovirus genes. LC‒MS/MS was used to analyze the proteins of SpltNPV-C3; 34 proteins were found in the purified ODVs, 15 of which were core proteins. The structure of the complexes formed by per os infectivity factors 1, 2, 3 and 4 (PIF-1, PIF-2, PIF-3 and PIF-4) was predicted with the help of the AlphaFold multimer tool and predicted conserved sequences in PIF-3. SpltNPV-C3 is a valuable species because of its virulence, and the analysis of its genome and proteins in this research will be beneficial for pest control efforts.

Cloning, expression, and characteristic analysis of the novel ß-galactosidase from silkworm, Bombyx mori.

ß-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the ß-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect ß-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal-spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.

IFN-γ establishes interferon-stimulated gene-mediated antiviral state against Newcastle disease virus in chicken fibroblasts.

Newcastle disease virus (NDV) causes severe economic losses through severe morbidity and mortality and poses a significant threat to the global poultry industry. Significant efforts have been made to develop novel vaccines and therapeutics; however, the interaction of NDV with the host is not yet fully understood. Interferons (IFNs), an integral component of innate immune signaling, act as the first line of defense against invading viruses. Compared with the mammalian repertoire of IFNs, limited information is available on the antiviral potential of IFNs in chickens. Here, we expressed chicken IFN-γ (chIFN-γ) using a baculovirus expression vector system, characterized its antiviral potential against NDV, and determined its antiviral potential. Priming of chicken embryo fibroblasts with chIFN-γ elicited an antiviral environment in primary cells, which was mainly due to interferon-stimulated genes (ISGs). A genome-wide transcriptomics approach was used to elucidate the possible signaling pathways associated with IFN-γ-induced immune responses. RNA-sequencing (RNA-seq) data revealed significant induction of ISG-associated pathways, activated temporal expression of ISGs, antiviral mediators, and transcriptional regulators in a cascade of antiviral responses. Collectively, we found that IFN-γ significantly elicited an antiviral response against NDV infection. These data provide a foundation for chIFN-γ-mediated antiviral responses and underpin functional annotation of these important chIFN-γ-induced antiviral influencers.

Identification and Characterization of 30 K Protein Genes Found in Bombyx mori (Lepidoptera: Bombycidae) Transcriptome.

The 30 K proteins, the major group of hemolymph proteins in the silkworm, Bombyx mori (Lepidoptera: Bombycidae), are structurally related with molecular masses of ∼30 kDa and are involved in various physiological processes, e.g., energy storage, embryonic development, and immune responses. For this report, known 30 K protein gene sequences were used as Blastn queries against sequences in the B. mori transcriptome (SilkTransDB). Twenty-nine cDNAs (Bm30K-1-29) were retrieved, including four being previously unidentified in the Lipoprotein_11 family. The genomic structures of the 29 genes were analyzed and they were mapped to their corresponding chromosomes. Furthermore, phylogenetic analysis revealed that the 29 genes encode three types of 30 K proteins. The members increased in each type is mainly a result of gene duplication with the appearance of each type preceding the differentiation of each species included in the tree. Real-Time Quantitative Polymerase Chain Reaction (Q-PCR) confirmed that the genes could be expressed, and that the three types have different temporal expression patterns. Proteins from the hemolymph was separated by SDS-PAGE, and those with molecular mass of ∼30 kDa were isolated and identified by mass spectrometry sequencing in combination with searches of various databases containing B. mori 30K protein sequences. Of the 34 proteins identified, 13 are members of the 30 K protein family, with one that had not been found in the SilkTransDB, although it had been found in the B. mori genome. Taken together, our results indicate that the 30 K protein family contains many members with various functions. Other methods will be required to find more members of the family.

Genomic sequence analysis of Helicoverpa armigera nucleopolyhedrovirus isolated from Australia.

The complete genomic sequence of Helicoverpa armigera nucleopolyhedrovirus from Australia, HearNPV-Au, was determined and analyzed. The HearNPV-Au genome was 130,992 bp in size with a G+C content of 39 mol% and contained 134 predicted open reading frames (ORFs) consisting of more than 150 nucleotides. HearNPV-Au shared 94 ORFs with AcMNPV, HearSNPV-G4 and SeMNPV, and was most closely related to HearSNPV-G4. The nucleotide sequence identity between HearNPV-Au and HearSNPV-G4 genome was 99%. The major differences were found in homologous regions (hrs) and baculovirus repeat ORFs (bro) genes. Five hrs and two bro genes were identified in the HearNPV-Au genome. All of the 134 ORFs identified in HearNPV-Au were also found in HearSNPV-G4, except the homologue of ORF59 (bro) in HearSNPV-G4. The sequence data strongly suggested that HearNPV-Au and HearSNPV-G4 belong to the same virus species.

Genomic sequence analysis of Helicoverpa armigera nucleopolyhedrovirus isolated from Australia.

The complete genomic sequence of Helicoverpa armigera nucleopolyhedrovirus from Australia, HearNPV-Au, was determined and analyzed. The HearNPV-Au genome was 130,992 bp in size with a G + C content of 39 mol% and contained 134 predicted open reading frames (ORFs) consisting of more than 150 nucleotides. HearNPV-Au shared 94 ORFs with AcMNPV, HearSNPV-G4 and SeMNPV, and was most closely related to HearSNPV-G4. The nucleotide sequence identity between HearNPV-Au and HearSNPV-G4 genome was 99 %. The major differences were found in homologous regions (hrs) and baculovirus repeat ORFs (bro) genes. Five hrs and two bro genes were identified in the HearNPV-Au genome. All of the 134 ORFs identified in HearNPV-Au were also found in HearSNPV-G4, except the homologue of ORF59 (bro) in HearSNPV-G4. The sequence data strongly suggested that HearNPV-Au and HearSNPV-G4 belong to the same virus species.

A SARS-CoV-2 Nanobody Displayed on the Surface of Human Ferritin with High Neutralization Activity.

Purpose: COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology. Methods: The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody's expression level and neutralizing activity were then evaluated. Results: The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity. Conclusion: Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.

The host transcriptome change involved in the inhibitory effect of exogenous interferon-γ on Getah virus replication.

Introduction: Getah virus (GETV) has become a growing potential threat to the global livestock industry and public health. However, little is known about the viral pathogenesis and immune escape mechanisms, leading to ineffective control measures. Methods: In this study, the antiviral activity of exogenous interferons (IFNs) was assessed by using western blotting (WB), real-time quantitative PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The comparative transcriptomics among mock- and GETV-infected (MOI = 0.1) ST cells with or without IFN-γ was performed by RNA-seq, and then the transcriptome profiling of GETV-infected ST cells and key pathways and putative factors involved in inhibitory effect of IFN-γ on GETV replication were analyzed by bioinformatics methods and RT-qPCR. Results: The results showed that treatment with IFN-γ could suppress GETV replication, and the inhibitory effect lasted for at least 48 h, while the exogenous IFN-α/ω and IFN-λ3 treatments failed to inhibit the viral infection and early replication in vitro. Furthermore, the blueprint of virus-host interaction was plotted by RNA-seq and RT-qPCR, showing systemic activation of inflammatory, apoptotic, and antiviral pathways in response to GETV infection, indicating viral hijacking and inhibition of innate host immunity such as IFN-I/III responses. Last and most importantly, activation of the JAK-STAT signaling pathway and complement and coagulation cascades may be a primary driver for IFN-γ-mediated inhibition of GETV replication. Discussion: These findings revealed that GETV possessed the capability of viral immune escape and indicated that IFN-γ aided in the prevention and control of GETV, implying the potential molecular mechanism of suppression of GETV by IFN-γ, all of which warrant emphasis or further clarification.

Baculovirus-expressed self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induce protective immunity in mice.

Spike (S) protein, a homotrimeric glycoprotein, is the most important antigen target for SARS-CoV-2 vaccines. A complete simulation of the advanced structure of this homotrimer during subunit vaccine development is the most likely method to improve its immunoprotective effects. In this study, preparation strategies for the S protein receptor-binding domain, S1 region, and ectodomain trimer nanoparticles were designed using ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare three nanoparticle vaccines with high expression levels recorded in silkworms. The results in mice showed that the nanoparticle vaccine prepared using this strategy could induce immune responses when administered via both the subcutaneous administration and oral routes. Given the stability of these ferritin-based nanoparticle vaccines, an easy-to-use and low-cost oral immunization strategy can be employed in vaccine blind areas attributed to shortages of ultralow-temperature equipment and medical resources in underdeveloped areas. Oral vaccines are also promising candidates for limiting the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.

The Isolation and Characterization of a Novel Group III-Classified Getah Virus from a Commercial Modified Live Vaccine against PRRSV.

As an epizootic causative agent, the Getah virus (GETV) can cause moderate illness in horses, lethal disease in foxes, and reproductive disorders and fetal death in pigs. Due to the wide range of hosts and multiple routes of transmission, GETV has become a growing potential threat to the global livestock industry, and even to public health. More attention and research on GETV are urgently needed. In this study, we successfully isolated a novel GETV strain, named BJ0304, from a commercial live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) and determined its growth kinetics. Then, genetic and phylogenetic analyses were performed. The results revealed that BJ0304 was clustered into Group III, and it was most related to the GETV-V1 strain based on the complete genome sequence. Furthermore, the pathogenicity of the isolate was assessed and found to be a low virulent strain in mice relative to its closest homolog GETV-V1. Finally, mutation and glycosylation analysis showed that a unique mutation (171 T > I) at one amino acid of E2, which affected the glycosylation of E2, may be associated with viral pathogenicity. In summary, the general characteristic of a novel Group III-classified GETV-BJ0304 isolated from commercial live PRRSV vaccine was defined and then mutation/glycosylation-related potential virulence factor was discussed. This study highlights the complexity of GETV transmission routes in swine and the need for more surveillance on commercial animal vaccines, contributes to the understanding of genetic characterization of clinical isolates, provides possible virulence factors in favor of unveiling the viral pathogenesis, and eventually lays the foundation for the prevention and control of GETV.

Genomic sequencing and analyses of HearMNPV--a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera.

BACKGROUND: HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. METHODS: The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the "partial filling-in" method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. RESULTS: Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome. CONCLUSIONS: HearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.

A Renewed Appreciation of Helicoverpa armigera Nucleopolyhedrovirus BJ (Formerly Helicoverpa assulta Nucleopolyhedrovirus) with Whole Genome Sequencing.

Helicoverpa assulta is a pest that causes severe damage to tobacco, pepper and other cash crops. A local strain of HearNPV-BJ (formerly Helicoverpa assulta nucleopolyhedrovirus (HeasNPV-DJ0031)) was isolated from infected H. assulta larvae in Beijing, which had been regarded as a new kind of baculovirus in previous studies. Describing the biological characteristics of the strain, including its external morphology, internal structure and the pathological characteristics of the infection of various cell lines, can provide references for the identification and function of the virus. HearNPV-BJ virion was defined as a single-nucleocapsid nucleopolyhedrovirus by scanning electron microscopy. QB-Ha-E-5 (H. armigera) and BCIRL-Hz-AM1 (H. zea) cell lines were sensitive to HearNPV-BJ. Undoubtedly modern developed sequencing technology further facilitates the increasing understanding of various strains. The whole genome sequence of the HearNPV-BJ was sequenced and analyzed. The HearNPV-BJ isolate genome was 129, 800 bp nucleotides in length with a G + C content of 38.87% and contained 128 open reading frames (ORFs) encoding predicted proteins of 50 or over 50 amino acids, 67 ORFs in the forward orientation and 61 ORFs in the reverse orientation, respectively. The genome shared 99% sequence identity with Helicoverpa armigera nucleopolyhedrovirus C1 strain (HearNPV-C1), and 103 ORFs had very high homology with published HearNPV sequences. Two bro genes and three hrs were found to be dispersed along the HearNPV-BJ genome. Three of the highest homologs, ORFs with HearNPV, were smaller due to the earlier appearance of the stop codon with unknown functions. P6.9 of HearNPV-BJ, a structural protein, is distinctly different from that of Autographa californica nucleopolyhedrovirus (AcMNPV); its homology with the corresponding gene in HearNPV-C1 was 93.58%. HearNPV-BJ contains 38 core genes identified in other baculoviruses, and phylogenetic analysis indicates HearNPV-BJ belongs to Alphabaculovirus Group II, same as HearNPV-C1. The resulting data provide a better understanding of virion structure, gene function and character of infection. By supplementing the whole-genome sequencing data and Kimura-2 model index, there is more evidence to indicate that HearNPV-BJ may be a variant of Helicoverpa armigera nucleopolyhedrovirus, which also deepens our understanding of the virus species demarcation criteria.

Transcriptome analysis of pre-immune state induced by interferon gamma inhibiting the replication of H9N2 avian influenza viruses in chicken embryo fibroblasts.

Interferon (IFN), a critical antiviral cytokine produced by pathogens-induced cells, plays an important role in host innate immune system. In this study, to investigate the inhibition effect of IFN on avian influenza virus (AIV), Chicken Embryo Fibroblasts (CEFs) was infected by H9N2 AIV. The pre-immune state and transcriptome analysis have been observed and performed. The result showed chicken interferon gamma (chIFN-γ) have the most inhibitory effect on H9N2 virus among three types of chicken interferons (chIFNs). Inhibition of chIFN-γ on H9N2 virus was verified by indirect immunofluorescence, RT-qPCR and western blot. The possible signaling pathways induced by chIFN-γ with or without virus were analyzed by transcriptome. The transcriptome data were compared among H9N2-infected, chIFN-γ-treated, chIFN-γ + H9N2-treated, and Control groups. In summary, RNA-sequencing (RNA-seq) data suggested that H9N2 virus infection resulted in corresponding response of certain defensive, inflammatory and metabolism pathways to the virus replication in CEFs. Furthermore, while CEFs were treated with chIFN-γ, many immune-related signaling pathways in cells are affected and altered. Antiviral genes involved in these immune pathways such as interferon regulatory factors, chemokines, interferon-stimulated genes (ISGs) and transcription factors were significantly up-regulated, and showed significant antiviral responses. Compared with virus infected CEFs alone, pretreatment with IFN induced the expression of antiviral genes and activated related antiviral pathways, inhibited the viral replication as result. Our study provided functional annotations for antiviral genes and the basis for studying the mechanism of chIFN-γ mediated response against H9N2 AIV.

Expression and Evaluation of a Novel PPRV Nanoparticle Antigen Based on Ferritin Self-Assembling Technology.

Peste des Petits Ruminants (PPR) is a highly pathogenic disease that is classified as a World Organization for Animal Health (OIE)-listed disease. PPRV mainly infects small ruminants such as goats and sheep. In view of the global and high pathogenicity of PPRV, in this study, we proposed a novel nanoparticle vaccine strategy based on ferritin (Fe) self-assembly technology. Using Helicobacter pylori (H. pylori) ferritin as an antigen delivery vector, a PPRV hemagglutinin (H) protein was fused with ferritin and then expressed and purified in both Escherichia coli (E. coli) and silkworm baculovirus expression systems. Subsequently, the nanoparticle antigens' expression level, immunogenicity and protective immune response were evaluated. Our results showed that the PPRV hemagglutinin-ferritin (H-Fe) protein was self-assembled in silkworms, while it was difficult to observe the correctly folded nanoparticle in E. coli. Meanwhile, the expression level of the H-Fe protein was higher than that of the H protein alone. Furthermore, the immunogenicity and protective immune response of H-Fe nanoparticle antigens expressed by silkworms were improved compared with the H antigen alone. Particularly, the protective immune response of H-Fe antigens expressed in E. coli did not change, as opposed to the H antigen, which was probably due to the incomplete nanoparticle structure in E. coli. This study indicated that the use of ferritin nanoparticles as antigen delivery carriers could increase the expression of antigen proteins and improve the immunogenicity and immune effect of antigens.

Alternative splicing of the antitrypsin gene in the silkworm, Bombyx mori.

Alternative splicing plays an important role in expanding protein diversity. In the present study, different splice variants of the antitrypsin gene (sw-AT) in the silkworm were identified by bioinformatics analyses using expressed sequence tags and genomic information. Four splice variants were obtained by RT-PCR with suitably designed primers, confirmed by sequencing, and designated as sw-AT-1, sw-AT-2, sw-AT-3, and sw-AT-4. The sw-AT gene contains 10 exons and nine introns. The splice variants differ in exon 9, with sw-AT-1, sw-AT-2, and sw-AT-3 using different versions of the exon, namely exon 9a, 9b, and 9c, respectively. In sw-AT-4, exon 9 consists of the combination of exons 9b and 9c. The expression patterns of the four isoforms in different tissues, at different developmental stages, and under different stress conditions (temperature, starvation, and mycotic infection) were characterized and quantified. The sw-AT isoforms showed tissue-specific expression patterns, with sw-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. The four isoforms were predominantly expressed in the fat body, body wall, and testes of larvae, and exhibited similar expression profiles during development of the fat body. Among the stress treatments, low temperature had the greatest effect on isoform expression, and expression was also upregulated with mycotic infection.

Genome sequence and organization of the Mythimna (formerly Pseudaletia) unipuncta granulovirus Hawaiian strain.

Purified occlusion bodies (OBs) of Mythimna (formerly Pseudaletia) unipuncta (the true armyworm) granulovirus Hawaiian strain (MyunGV-A) were observed, showing typical GV morphological characteristics under scanning and transmission electron microscopy (EM). The genome of MyunGV-A was completely sequenced and analysed. The genome is 176,677 bp in size, with a G+C content of 39.79%. It contains 183 open reading frames (ORFs) encoding 50 or more amino acids with minimal overlap. Comparison of MyunGV-A with TnGV, XcGV, and HearGV genomes revealed extensive sequence similarity and collinearity, and the four genomes contain the same nine homologous regions (hrs) with conserved structures and locations. Three unique genes, 12 baculovirus repeated ORF (bro), 2 helicase, and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome, in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. In addition, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV (99% amino acid identity) according to a phylogenetic tree based on the combined amino acid sequences of 38 core gene contents.

[Cloning and expression of ORF146 gene of Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII)].

OBJECTIVE: To study the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) ORF146 gene. METHODS: The primers were designed according to the sequence of SpltMNPVII genome. The promoter of ORF146 was amplified by PCR. The promoter activities and the time course of mRNA transcription were analyzed. The fragment of the ORF146 gene was then cloned into the vector of pET28a(+) and expressed. The polyclonal antibody was prepared by using the purified fusion protein. Titration determination of anti-ORF146 antibody was evaluated by ELISA. RESULT: Nucleotide sequence analysis demonstrated that this gene has a 1383 bp ORF, encoding 460 amino acids with a predicted molecular weight of 50.4 kDa. Analysis of both promoter activities and the time course of mRNA transcription of the ORF146 gene showed that ORF146 was transcripted in early stage as well as in late stage. The transcription began at 2 h post infection (hpi) and reached two peaks at 8 and 18 hpi and then the transcription level was slightly decreased from 24 hpi. pET-28a-ORF146 fusion protein expressed in prokaryotic and purified polyclonal antibody was with good specificity, with the titer above 1:3200. CONCLUSION: SpltMNPV II ORF146 gene is a composition structure protein, which was expressed at both early and late stage. ORF146 might be involved in viral DNA replication. The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.

Interaction with the Receptor SLAM and Baculovirus Surface Display of Peste des petits ruminants Virus Hemagglutinin.

Peste des petits ruminants (PPR) is an acute, highly infectious, and highly pathogenic disease, which mainly damages small ruminants such as goats and sheep. Hemagglutinin protein (H), the main antigenic protein of peste des petits ruminants virus (PPRV), has been a hot spot in the research of genetic engineering vaccine for PPRV. In this study, the silkworm baculovirus surface display technology is combined with the transmembrane structure of the silkworm baculovirus envelope protein GP64 and different characteristics of the promoters to display four kinds of fusion proteins, which contain Pph-H, Pph-HJ, Pie1-H, and Pie1-HJ. The fusion proteins displayed on baculovirus surface have been detected by western blotting, cell surface immunofluorescence, and immunogold electron microscopy. In addition, the dominant form of PPR H displayed on baculovirus surface has been determined which is fusion protein mediated by Pph containing the hemagglutinin protein and full-length GP64, Pph-H. Furthermore, by comparing the fluorescence intensity of binding of hemagglutinin protein and signaling lymphocyte activation molecules (SLAM) in Vero-SLAM cells by immunocytochemistry, Pph-H can be combined with the receptor protein of PPRV, SLAM. It provides technical support for displaying the different structure of hemagglutinin and exploring the key sites of hemagglutinin and SLAM binding. Meanwhile, it is important for exploring the pathogenesis and immune mechanism of PPRV.

A construction strategy for a baculovirus-silkworm multigene expression system and its application for coexpression of type I and type II interferons.

The Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus expression system (BES) is a eukaryotic expression system. It possesses great capability for post-translation modification in expression of foreign proteins. With the counterselection cassette rpsL-neo and phage λ-Red recombinase, the defective-rescue BmNPV BES reBmBac can be employed for efficient heterologous multigene coexpression at different gene sites in one baculovirus genome. In the present study, a recombinant baculovirus, reBm-Cαγ, carrying two types of chicken interferon (IFN) genes (chIFN-α and chIFN-γ) was constructed using the reBmBac system. The chIFN-α and chIFN-γ genes were inserted into the same baculovirus genome at the polyhedron and p10 gene sites, respectively. The recombinant baculovirus was capable of coexpressing both chIFN-α and chIFN-γ. The expression levels of the two types of IFN in the coexpression product were exponentially high, at approximately 1.7 and 2.5 times higher, respectively, than those in the corresponding single-expression products. The increase in expression level corresponds to replacement of the nonessential p10 gene in the reBm-Cαγ recombinant baculovirus. This coexpression of recombinant chicken IFNs showed superior antiviral activity.