Transcriptomic Analysis of PDCoV-Infected HIEC-6 Cells and Enrichment Pathways PI3K-Akt and P38 MAPK.

Porcine Deltacoronavirus (PDCoV) is a newly identified coronavirus that causes severe intestinal lesions in piglets. However, the understanding of how PDCoV interacts with human hosts is limited. In this study, we aimed to investigate the interactions between PDCoV and human intestinal cells (HIEC-6) by analyzing the transcriptome at different time points post-infection (12 h, 24 h, 48 h). Differential gene analysis revealed a total of 3560, 5193, and 4147 differentially expressed genes (DEGs) at 12 h, 24 h, and 48 h, respectively. The common genes among the DEGs at all three time points were enriched in biological processes related to cytokine production, extracellular matrix, and cytokine activity. KEGG pathway analysis showed enrichment of genes involved in the p53 signaling pathway, PI3K-Akt signaling pathway, and TNF signaling pathway. Further analysis of highly expressed genes among the DEGs identified significant changes in the expression levels of BUB1, DDIT4, ATF3, GBP2, and IRF1. Comparison of transcriptome data at 24 h with other time points revealed 298 DEGs out of a total of 6276 genes. KEGG analysis of these DEGs showed significant enrichment of pathways related to viral infection, specifically the PI3K-Akt and P38 MAPK pathways. Furthermore, the genes EFNA1 and KITLG, which are associated with viral infection, were found in both enriched pathways, suggesting their potential as therapeutic or preventive targets for PDCoV infection. The enhancement of PDCoV infection in HIEC-6 was observed upon inhibition of the PI3K-Akt and P38 MAPK signaling pathways using sophoridine. Overall, these findings contribute to our understanding of the molecular mechanisms underlying PDCoV infection in HIEC-6 cells and provide insights for developing preventive and therapeutic strategies against PDCoV infection.

[Advances in nanobody screening technology].

Nanobody (Nb) is a novel type of antibody discovered in the serum of Camelidae. It is characterized by its small size, high specificity, stability, and ease of preparation. Nanobodies exhibit the ability to identify hidden epitopes and have diverse applications across various fields. This review aims to introduce three key stages in the screening and optimization of nanobodies, including nanobody library construction, in vitro surface display, and affinity maturation. We provide a brief description of preparation and characteristics of natural, immunological, and semi-synthetic/synthetic libraries. Additionally, we systematically explain eight in vitro display methods, including phage display, yeast display, bacterial display, ribosome display/mRNA display, and eukaryotic cell display. Furthermore, we discuss the application of yeast two-hybrid system high-throughput sequencing and mass spectrometry identification. A thorough analysis of their advantages and limitations is presented in this protocols. Finally, we summarize the platforms for in vitro or computer-aided affinity maturation techniques aimed at enhancing the functional stability of nanobodies. Consequently, this review provides a comprehensive approach to the integrated utilization of various technologies for the rapid development of stable, reliable, and specific nanobody-based drugs or diagnostic agents.

Effectiveness of an online management platform (Joint Cloud) versus standard process for patients undergoing total knee arthroplasty: study protocol for a prospective randomised controlled trial.

INTRODUCTION: Osteoarthritis (OA) is one of the main causes of mobility impairment in the elderly worldwide. Therefore, total knee arthroplasty (TKA) is often performed and is one of the most successful surgery and has resulted in substantial quality-of-life gains for people with end-stage arthritis. There is still room for improvement in the standard treatment process in the preoperative, intraoperative and postoperative period of TKA. Telerehabilitation has the potential to become a positive alternative to face-to-face rehabilitation nowadays. But it remains unclear how well telemedicine interventions cover the entire surgical pathway (preoperation, intraoperation, postoperation). This study aims to explore the effectiveness of Joint Cloud (JC, an online management platform) compared with existing standard process in regulating functional recovery, pain management, muscle strength changes and other health-related outcomes in patients undergoing total knee arthroplasty preoperation, intraoperation and postoperation. METHODS AND ANALYSIS: A randomised controlled trial was designed to compare the online management platform (JC) with standard process (SP) in patients undergoing TKA. A total of 186 TKA patients will be randomly assigned to the intervention (n=93) or control (n=93) group. Patients in the intervention group will receive access to the 'JC' mini-program. This mini-program provides popular science information (eg, information about OA and TKA), functional exercise information and communication channels. Patients evaluate their condition and functional level through standardised digital questionnaires. The control group of patients will not accept any functions of this mini-program. The primary outcome is knee functional recovery, and the secondary outcomes are pain management, isometric knee extensor muscle strength, patient satisfaction and cost-benefit analysis. Assessments will be performed 1 month and 3 days before surgery (T0) and 1 month and 3 months after surgery. Data analysis will be performed according to the intent-to-treat (ITT) principle. Repeated measures of linear mixed models and parametric and non-parametric testing will be used for statistical analysis. ETHICS AND DISSEMINATION: The study was reviewed and approved by the Tianjin Hospital Medical Ethics Review Committee on 10 February 2023 (2022YLS155). Test data are considered highly sensitive but are available upon request. The findings will be disseminated in peer-reviewed publications. TRIAL REGISTRATION NUMBER: ChiCTR2300068486.

Nanobodies against porcine CD163 as PRRSV broad inhibitor.

PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a major swine pathogen causing economic losses. To the date, effective broad PRRSV inhibitory strategies have not been available in practice yet. Targeting the key viral receptor CD163 to block PRRSV entry has emerged as an alternative approach beside vaccines for PRRSV inhibition. As an effective therapeutic tool, nanoantibodies (Nbs) have been widely used in antiviral research. In this study, a phage display VHH library was constructed for the selection of Nbs against porcine CD163 scavenger receptor cysteine-rich 5-9 domain (SRCR5-9). After five rounds of bio-panning and indirect ELISA, seven CD163-specific Nbs (Nb1-Nb7) were identified. All obtained Nbs displayed strong affinity to CD163 receptor and excellent antiviral activity. In particular, Nb2 exhibited significant broad inhibitory effects on variable PRRSV lineages and downregulated virus-related NF-κB signaling. Further studies suggested that Nbs mainly exerted antiviral functions by interfering with virus attachment stage, and also decreased the transcription of CD163. The conformational epitopes recognized by Nbs were localized in the SRCR5 domain of CD163, a crucial region in PRRSV infection. Overall, our findings provide a novel insight into the biofunction of CD163 in antiviral infection and the development of broad-spectrum strategies against PRRSV.

Genomic characteristics of an avipoxvirus 282E4 strain.

Avipoxvirus 282E4 strain was extensively applied into recombinant vaccine vector to prevent other infectious diseases. However, little information on the genomic background, functional and genetic evolutionary of the isolate 282E4 strain was clarified. The results showed that the linear genome of avipoxvirus 282E4 was 308,826 bp, containing 313 open reading frames (ORFs) and 12 new predicted ORFs. The 282E4 strain appears to encode two novel thymidine kinase proteins and two TGF-beta-like proteins that may be associated with the suppression of the host's antiviral response. Avipoxvirus 282E4 also encodes 57 ankyrin repeat proteins and 5 variola B22R-like proteins, which composed 7% of the avipoxvirus 282E4 genome. GO and KEGG analysis further revealed that 12 ORFs participate in viral transcription process, 7 ORFs may function during DNA repair, replication and biological synthesis, and ORF 208 is involved in the process of virus life cycle. Interestingly, phylogenetic analysis based on concatenated sequences p4b and DNA polymerase of avipoxviruses gene demonstrates that avipoxvirus 282E4 strain is divergent from known FWPV isolates and is similar to shearwater poxvirus (SWPV-1) that belongs to the CNPV-like virus. Sequencing avipoxvirus 282E4 is a significant step to judge the genetic position of avipoxviruses within the larger Poxviridae phylogenetic tree and provide a new insight into the genetic background of avipoxvirus 282E4 and interspecies transmission of poxviruses, meanwhile, explanation of gene function provides theoretical foundation for vaccine design with 282E4 strain as skeleton.

A micro-sized vaccine based on recombinant Lactiplantibacillus plantarum fights against SARS-CoV-2 infection via intranasal immunization.

COVID-19 has globally spread to burden the medical system. Even with a massive vaccination, a mucosal vaccine offering more comprehensive and convenient protection is imminent. Here, a micro-sized vaccine based on recombinant Lactiplantibacillus plantarum (rLP) displaying spike or receptor-binding domain (RBD) was characterized as microparticles, and its safety and protective effects against SARS-CoV-2 were evaluated. We found a 66.7% mortality reduction and 100% protection with rLP against SARS-CoV-2 in a mouse model. The histological analysis showed decreased hemorrhage symptoms and increased leukocyte infiltration in the lung. Especially, rLP:RBD significantly decreased pulmonary viral loads. For the first time, our study provides a Lactiplantibacillus plantarum-vectored vaccine to prevent COVID-19 progress and transmission via intranasal vaccination.

Mining Potential Drug Targets for Osteoporosis Based on CeRNA Network.

OBJECTIVE: To identify key pathological hub genes, micro RNAs (miRNAs), and circular RNAs (circRNAs) of osteoporosis (OP) and construct their ceRNA network in an effort to explore the potential biomarkers and drug targets for OP therapy. METHODS: GSE7158, GSE201543, and GSE161361 microarray datasets were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified by comparing OP patients with healthy controls and hub genes were screened by machine learning algorithms. Target miRNAs and circRNAs were predicted by FunRich and circbank, then ceRNA network were constructed by Cytoscape. Pathways affecting OP were identified by functional enrichment analysis. The hub genes were verified by receiver operating characteristic (ROC) curve and real time quantitative PCR (RT-qPCR). Potential drug molecules related to OP were predicted by DSigDB database and molecular docking was analyzed by autodock vina software. RESULTS: A total of 179 DEGs were identified. By combining three machine learning algorithms, BAG2, MME, SLC14A1, and TRIM44 were identified as hub genes. Three OP-associated target miRNAs and 362 target circRNAs were predicted to establish ceRNA network. The ROC curves showed that these four hub genes had good diagnostic performance and their differential expression was statistically significant in OP animal model. Benzo[a]pyrene was predicted which could successfully bind to protein receptors related to the hub genes and it was served as the potential drug molecules. CONCLUSION: An mRNA-miRNA-circRNA network is reported, which provides new ideas for exploring the pathogenesis of OP. Benzo[a]pyrene, as potential drug molecules for OP, may provide guidance for the clinical treatment.

The "LLQY" Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S protein synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2' sites are very important, like the S2' cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.

Ferroptosis in viral infection: the unexplored possibility.

Virus-induced cell death has long been thought of as a double-edged sword in the inhibition or exacerbation of viral infections. The vital role of iron, an essential element for various enzymes in the maintenance of cellular physiology and efficient viral replication, places it at the crossroads and makes it a micronutrient of competition between the viruses and the host. Viruses can interrupt iron uptake and the antioxidant response system, while others can utilize iron transporter proteins as receptors. Interestingly, the unavailability of iron facilitates certain viral infections and causes cell death characterized by lipid peroxide accumulation and malfunction of the antioxidant system. In this review, we discuss how iron uptake, regulation and metabolism, including the redistribution of iron in the host defense system during viral infection, can induce ferroptosis. Fenton reactions, a central characteristic of ferroptosis, are caused by the increased iron content in the cell. Therefore, viral infections that increase cellular iron content or intestinal iron absorption are likely to cause ferroptosis. In addition, we discuss the hijacking of the iron regulatoy pathway and the antioxidant response, both of which are typical in viral infections. Understanding the potential signaling mechanisms of ferroptosis in viral infections will aid in the development of new therapeutic agents.

Mucosal IgA response elicited by intranasal immunization of Lactobacillus plantarum expressing surface-displayed RBD protein of SARS-CoV-2.

Coronavirus Disease 2019 (COVID-19) caused by a novel betacoronavirus SARS-CoV-2 has been an ongoing global pandemic. Several vaccines have been developed to control the COVID-19, but the potential effectiveness of the mucosal vaccine remains to be documented. In this study, we constructed a recombinant L. plantarum LP18:RBD expressing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein via the surface anchoring route. The amount of the RBD protein was maximally expressed under the culture condition with 200 ng/mL of inducer at 33 °C for 6 h. Further, we evaluated the immune response in mice via the intranasal administration of LP18:RBD. The results showed that the LP18:RBD significantly elicited RBD-specific mucosal IgA antibodies in respiratory tract and intestinal tract. The percentages of CD3 + CD4+ T cells in spleens of mice administrated with the LP18:RBD were also significantly increased. This indicated that LP18:RBD could induce a humoral immune response at the mucosa, and it could be used as a mucosal vaccine candidate against the SARS-CoV-2 infection. We provided the first experimental evidence that the recombinant L. plantarum LP18:RBD could initiate immune response in vivo, which implies that the mucosal immunization using recombinant LAB system could be a promising vaccination strategy to prevent the COVID-19 pandemic.

Association between matrix metalloproteinase-1 (MMP-1) protein level and the risk of rheumatoid arthritis and osteoarthritis: a meta-analysis

Recent publications have investigated the potential role of the protein level of matrix metalloproteinase-1 (MMP-1) in the susceptibility to rheumatoid arthritis (RA) and osteoarthritis (OA). However, no unanimous conclusion was obtained. Therefore, we carried out a meta-analysis to explore the association between MMP-1 expression and these two clinical disorders. After database searching and screening, we enrolled a total of eighteen articles for the pooled analysis. We observed a significant association between RA cases and controls in the whole population [SMD (standard mean difference)=1.01, P=0.017]. There were similar positive results in the subgroup analysis of "population-based control" (SMD=1.50, P=0.032) and "synovial fluid" (SMD=1.32, P=0.049). In addition, we observed an increased risk in OA cases, compared with controls, in the overall analysis (SMD=0.47, P=0.004) and subsequent subgroup analysis of "knee OA" (SMD=0.86, P<0.001), "Asian/China" (SMD=0.76, P=0.003), "cartilage-Asian/China" (SMD=1.21, P<0.001), and "synovial fluid-Asian/China" (SMD=0.73, P=0.004). In summary, a high protein level of MMP-1 in synovial fluid may be associated with the susceptibility to RA, and the high MMP-1 level in the cartilage tissue or synovial fluid may be related to the pathogenesis of knee OA in the Chinese population. This should be confirmed by larger sample sizes.

Association between matrix metalloproteinase-1 (MMP-1) protein level and the risk of rheumatoid arthritis and osteoarthritis: a meta-analysis.

Recent publications have investigated the potential role of the protein level of matrix metalloproteinase-1 (MMP-1) in the susceptibility to rheumatoid arthritis (RA) and osteoarthritis (OA). However, no unanimous conclusion was obtained. Therefore, we carried out a meta-analysis to explore the association between MMP-1 expression and these two clinical disorders. After database searching and screening, we enrolled a total of eighteen articles for the pooled analysis. We observed a significant association between RA cases and controls in the whole population [SMD (standard mean difference)=1.01, P=0.017]. There were similar positive results in the subgroup analysis of "population-based control" (SMD=1.50, P=0.032) and "synovial fluid" (SMD=1.32, P=0.049). In addition, we observed an increased risk in OA cases, compared with controls, in the overall analysis (SMD=0.47, P=0.004) and subsequent subgroup analysis of "knee OA" (SMD=0.86, P<0.001), "Asian/China" (SMD=0.76, P=0.003), "cartilage-Asian/China" (SMD=1.21, P<0.001), and "synovial fluid-Asian/China" (SMD=0.73, P=0.004). In summary, a high protein level of MMP-1 in synovial fluid may be associated with the susceptibility to RA, and the high MMP-1 level in the cartilage tissue or synovial fluid may be related to the pathogenesis of knee OA in the Chinese population. This should be confirmed by larger sample sizes.

A recombinant Lactobacillus plantarum strain expressing the spike protein of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6-10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection.

mRNA Vaccines Encoding the HA Protein of Influenza A H1N1 Virus Delivered by Cationic Lipid Nanoparticles Induce Protective Immune Responses in Mice.

The design of the mRNA vaccine involves the selection of in vitro transcription (IVT) systems and nonviral delivery vectors. This study aimed to verify the effect of 5' and 3' untranslated region (UTR) sequences on the translation efficiency of mRNA. Three modes of IVT-mRNA systems (IVT-mRNA-n1/n2/n3) with diverse UTRs were constructed, and EGFP (enhanced green fluorescent protein) and HA (hemagglutinin) gene of H3N2 influenza virus were introduced into each of them. The results showed that the mode of 5' and 3' UTRs originating from human ß-globulin was better than the mode of UTRs from human α-globulin, and the n3 mode was the best. mEGFP-n3, mH3HA-n3, and mLuciferease-n3 were prepared to compare the effect of cationic lipid nanoparticle (LNP) with that of mannose-conjugated LNP (LNP-Man) on the efficiency of gene delivery. The results showed that the effect of LNP-Man was better than that of LNP both in vitro and in vivo. Choosing appropriate ligands might help in vaccine design. After selecting the IVT-mRNA-n3 system and delivery vectors, mRNA vaccines were constructed against the H1N1 influenza virus, and C57BL/6 mice were immunized through intranasal administration. The results showed that mRNA vaccines could elicit both humoral and cellular immune responses and completely protect mice from the tenfold LD50 H1N1 influenza virus challenge.

Construction and optimization of Lactobacillus plantarum expression system expressing glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes serious reproductive failure and respiratory disease in pigs. Although numerous vaccines were developed against the virus, licensed vaccines showed limited efficacy. Here, we describe the construction and optimization of Lactobacillus plantarum expression system of PRRSV GP5 gene. The wild-type truncated GP5 or codon-optimized truncated GP5 was linked with endogenous signal peptide and target peptides (DCpep or Mpep) at 5' and 3' end of the gene, respectively. Then, the fragments were cloned into the L. plantarum expression plasmid pSIP411 and expressed under the induction of SppIP. As a result, PRRSV GP5 genes with optimized codons have higher expressions than that of the GP5 genes with wild-type codons, indicating codons optimization is an effective way to enhance the expression of an exogenous gene in L. plantarum. Further analysis showed that the codon-optimized GP5 with endogenous signal peptide can be effectively displayed on the surface of the L. plantarum, and the GP5 harboring target peptide Mpep displayed the highest antigenicity than the others. The highest production of PRRSV GP5 was obtained under the following conditions: L. plantarum harboring the plasmid pSIP-1320-O5MH are induced with 200 ng/mL SppIP at 33 °C for 7 h.

Construction, expression and antiviral activity analysis of recombinant adenovirus expressing human IFITM3 in vitro.

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits the replication of multiple pathogenic viruses by blocking their entry. In this study, we constructed a shuttle plasmid, harboring human IFITM3. Thereafter, recombinant adenovirus rAd5-IFITM3 was obtained by co-transfection of the linearized viral backbone vector pAd5 and the shuttle plasmid. The results showed that human IFITM3 did not affect the assembly and morphogenesis of progeny adenovirus. Human IFITM3 can be expressed in both A549 and MDCK cells in a time dependent manner. Furthermore, cells infected with rAd5-IFITM3 at a multiplicity of infection (MOI) of 100 for 24 h were challenged with avian influenza virus (AIV) H5N1 at an MOI of 1 for 6, 12 and 24 h. Rates of H5N1 infection in rAd5-IFITM3 cells were significantly decreased at 24 h post-infection (hpi), in a time dependent manner, compared with that of wild type wtAd5-infected cells. The expressions of viral genes were significantly inhibited at transcriptional and translational levels at 6 and 12 hpi. These results suggest that IFITM3 can suppress H5N1 replication in the early stage of the infection, which may be used as a promise agent against H5N1 infection in vivo.

Metagenomic Analysis of Flaviviridae in Mosquito Viromes Isolated From Yunnan Province in China Reveals Genes From Dengue and Zika Viruses.

More than 6,000 mosquitoes of six species from six sites were collected and tested for their virome using metagenomics sequencing and bioinformatic analysis. The identified viral sequences belonged to more than 50 viral families. The results were verified by PCR of selected viruses in all mosquitoes, followed by phylogenetic analysis. In the present study, we identified the partial dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) sequences in mosquitoes. Metagenomic analysis and the PCR amplification revealed three DENV sequences, one of which encodes a partial envelope protein. Two ZIKV sequences both encoding partial nonstructural protein 3 and one JEV sequence encoding the complete envelope protein were identified. There was variability in the viral titers of the newly isolated virus JEV-China/YN2016-1 of different passage viruses. The newly identified Zika virus gene from ZIKV-China/YN2016-1 was an Asian genotype and shared the highest nucleotide sequence identity (97.1%) with a ZIKV sequence from Thailand isolated in 2004. Phylogenetic analysis of ZIKV-China/YN2016-1 and ZIKV-China/YN2016-2 with known Flavivirus genes indicated that ZIKV has propagated in Yunnan province, China.

Metagenomic Sequencing From Mosquitoes in China Reveals a Variety of Insect and Human Viruses.

We collected 8,700 mosquitoes in three sites in China, which belonged to seven species. Their viromes were tested using metagenomic sequencing and bioinformatic analysis. The abundant viral sequences were detected and annotated belonging to more than 50 viral taxonomic families. The results were verified by PCR, followed by phylogenetic analysis. In the present study, we identified partial viral genes of dengue virus (DENV), a novel circovirus (CCV), densovirus (DNV), Japanese encephalitis virus (JEV), and Wuhan mosquito virus (WMV) in mosquitoes. Metagenomic analysis and PCR amplification revealed three DENV sequences, which were as homologous to the NS3 gene of DENV from Singapore isolated in 2005, with at least 91% nucleotide (nt) identity. Seven fragments of JEV encoding structural proteins were identified belonging to genotype I. They all shared high homology with structural protein genes of JEV isolated from Laos in 2009. The production of infectious virus particles of the newly isolated virus YunnanJEV2017-4 increased after passage from the BHK-21 cell line to the Vero cell line. Novel circovirus-related genes were identified and as being related to an unnamed gene of a mosquito circovirus (MCCV) sequence from the USA isolated in 2011, with at least 41% nt identity: this distant relationship suggests that the parent virus might belong to a novel circovirus genus. Additionally, numerous known viruses and some unknown viruses were also detected in mosquitoes from Yunnan province, China, which will be tested for propagation.

SIV-Specific Antibodies are Elicited by a Recombinant Fowlpox Virus Co-expressing SIV Gag and envT.

Given the failures of past HIV-1 vaccine clinical trials, potential HIV-1 vaccine candidates should be rigorously screened in preclinical models including simian immunodeficiency virus (SIV) primate models and small animal models. In this study, we tested the immunogenicity of a recombinant fowlpox virus (rFPV) expressing the SIV gag and SIV envT (rFPVsg-se) proteins in BALB/c mice, to establish a foundation for further development. rFPVsg-se was constructed through homologous recombination techniques and purified through plaque screening assays using enhanced green fluorescent protein as the reporter gene. The integration, transcription, and translation of the SIV genes were measured by PCR (genomic DNA), RT-PCR (RNA), Western-blot, respectively. The levels of SIV-specific antibodies were assessed by ELISA following a single immunization (n = 18/group) or a prime-boost strategy (n = 24/group) with rFPVsg-se and compared to FPV and PBS controls. Residual virus was measured in distant organs following immunization using PCR. SIV-specific IgG titers against gag and gp120 were detected following single vaccination and the prime-boost. As expected the titers were higher following the prime-boost approach. The levels of Gag- and gp120-specific antibodies were significantly higher than controls (p < 0.01) 14 days after the booster immunization. Residual rFPVSg-Se was detected in the muscle at the site of injection, but not in distant organs, from day 1-7 post immunization. In summary, rFPVsg-se induced high levels of SIV-specific antibodies suggesting it may be a viable candidate for further development.

The Host Restriction Factor Interferon-Inducible Transmembrane Protein 3 Inhibits Vaccinia Virus Infection.

Interferons (IFNs) establish dynamic host defense mechanisms by inducing various IFN-stimulated genes that encodes many antiviral innate immune effectors. IFN-inducible transmembrane (IFITM) proteins have been identified as intrinsic antiviral effectors, which block the entry of a broad spectrum of enveloped RNA viruses by interrupting virus-endosomal fusion. However, antiviral activity of IFITM proteins against mammalian DNA virus has not been demonstrated till date. Here, we sought to investigate the antiviral activities and mechanisms of interferon-inducible transmembrane protein 3 (IFITM3) protein against poxvirus infection. Analysis of expression kinetics of cell endogenous IFITM3 protein indicated that vaccinia virus (VACV) infection suppressed its translation, which was independent of IRF3 phosphorylation triggered by VACV. Although silencing of endogenous IFITM proteins did not affect their baseline antiviral effects in the cell, it has reduced the IFN-α-mediated inhibition of VACV infection, and also modulated VACV-induced cell death. Moreover, we discovered that overexpression of IFITM3 significantly restricted VACV infection, replication and proliferation mainly by interfering with virus entry processes prior to the virus nucleocapsid entry into the cytoplasm. Interestingly, IFITM3 overexpression showed an impact on virus binding. Furthermore, IFITM3 interfered with the cytosolic entry of virus through low pH-dependent fashion. Taken together, our findings provide the first evidence of exogenously expressed IFITM3 protein restricting infection of an enveloped DNA virus, thus expanding their antiviral spectrum. This study further explores the complex mechanism and provides novel insights into the interaction between virus infection and host defense.