A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTRN-S1NCOE to obtain vC/SM3'UTRN-tPAsS1NCOE and vC/SM3'UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-ß induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-ß induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus.

BACKGROUND: Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. METHODS: The CSFV Erns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). RESULTS: Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. CONCLUSION: The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Data-driven multi-scale mathematical modeling of SARS-CoV-2 infection reveals heterogeneity among COVID-19 patients.

Patients with coronavirus disease 2019 (COVID-19) often exhibit diverse disease progressions associated with various infectious ability, symptoms, and clinical treatments. To systematically and thoroughly understand the heterogeneous progression of COVID-19, we developed a multi-scale computational model to quantitatively understand the heterogeneous progression of COVID-19 patients infected with severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2). The model consists of intracellular viral dynamics, multicellular infection process, and immune responses, and was formulated using a combination of differential equations and stochastic modeling. By integrating multi-source clinical data with model analysis, we quantified individual heterogeneity using two indexes, i.e., the ratio of infected cells and incubation period. Specifically, our simulations revealed that increasing the host antiviral state or virus induced type I interferon (IFN) production rate can prolong the incubation period and postpone the transition from asymptomatic to symptomatic outcomes. We further identified the threshold dynamics of T cell exhaustion in the transition between mild-moderate and severe symptoms, and that patients with severe symptoms exhibited a lack of naïve T cells at a late stage. In addition, we quantified the efficacy of treating COVID-19 patients and investigated the effects of various therapeutic strategies. Simulations results suggested that single antiviral therapy is sufficient for moderate patients, while combination therapies and prevention of T cell exhaustion are needed for severe patients. These results highlight the critical roles of IFN and T cell responses in regulating the stage transition during COVID-19 progression. Our study reveals a quantitative relationship underpinning the heterogeneity of transition stage during COVID-19 progression and can provide a potential guidance for personalized therapy in COVID-19 patients.

Hepatitis B Virus Core Particles Containing a Conserved Region of the G Protein Combined with Interleukin-35 Protected Mice against Respiratory Syncytial Virus Infection without Vaccine-Enhanced Immunopathology.

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in infants and young children. The vaccine-enhanced disease (VED) has greatly hindered the development of an RSV vaccine. Currently, there are no licensed vaccines for RSV. In this study, immunization of mice with hepatitis B virus core particles containing a conserved region of the G protein (HBc-tG) combined with interleukin-35 (IL-35) elicited a Th1-biased response and a high frequency of regulatory T (Treg) cells and increased the levels of IL-10, transforming growth factor ß, and IL-35 production. Importantly, immunization with HBc-tG together with IL-35 protected mice against RSV infection without vaccine-enhanced immunopathology. To explore the mechanism of how IL-35 reduces lung inflammation at the gene expression level, transcription profiles were obtained from lung tissues of immunized mice after RSV infection by the Illumina sequencing technique and further analyzed by a systems biology method. In total, 2,644 differentially expressed genes (DEGs) were identified. Twelve high-influence modules (HIMs) were selected from these DEGs on the basis of the protein-protein interaction network. A detailed analysis of HIM10, involved in the immune response network, revealed that Il10 plays a key role in regulating the host response. The selected DEGs were consistently confirmed by quantitative real-time PCR (qRT-PCR). Our results demonstrate that IL-35 inhibits vaccine-enhanced immunopathology after RSV infection and has potential for development in novel therapeutic and prophylactic strategies.IMPORTANCE In the past few decades, respiratory syncytial virus (RSV) has still been a major health concern worldwide. The vaccine-enhance disease (VED) has hindered RSV vaccine development. A truncated hepatitis B virus core protein vaccine containing the conserved region (amino acids 144 to 204) of the RSV G protein (HBc-tG) had previously been shown to induce effective immune responses and confer protection against RSV infection in mice but to also lead to VED. In this study, we investigated the effect of IL-35 on the host response and immunopathology following RSV infection in vaccinated mice. Our results indicate that HBc-tG together with IL-35 elicited a balanced immune response and protected mice against RSV infection without vaccine-enhanced immunopathology. Applying a systems biology method, we identified Il10 to be the key regulator in reducing the excessive lung inflammation. Our study provides new insight into the function of IL-35 and its regulatory mechanism of VED at the network level.

A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production.

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

Y-Box-Binding Protein 3 (YBX3) Restricts Influenza A Virus by Interacting with Viral Ribonucleoprotein Complex and Imparing its Function.

The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.

Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway.

Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

Characterization of Salmonella spp. isolated from chickens in Central China.

BACKGROUND: Salmonella is an important zoonotic pathogen, and chickens are one of its main hosts. Every year, Salmonella infections pose a serious threat to the poultry industry in developing countries, especially China. In this study, a total of 84 Salmonella isolates recovered from sick and healthy-looking chickens in central China were characterized by serotyping, MLST-based strain typing, presence of potential virulence factors, and antimicrobial resistance profiles. RESULT: Data showed that the main serotypes of Salmonella isolates in central China were Salmonella enterica serovar Gallinarum biovar Pullorum, Salmonella enterica serovar Gallinarum biovar Gallinarum, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, and among them, S. Pullorum was the dominant type in both sick and healthy-looking chickens, accounting for 43.9 and 46.5%, respectively, while S. Enteritidis was only found in healthy-looking chickens. All isolates exhibited higher resistance rates to ampicillin (97.6%), tetracycline (58.3%) and colistin (51.2%), and among these isolates, 49.5% were resistant to more than three drugs in different combinations. S. Enteritidis was the most severe multidrug-resistant serotype, which showed higher resistance rates to colistin, meropenem and ciprofloxacin. Multilocus sequence typing (MLST) revealed that S. Gallinarum and S. Enteritidis isolates were clustered in clade 1, which belonged to two and one STs, respectively. All S. Typhimurium isolates were clustered in clade 3, and belonged to three STs. However, S. Pullorum were distributed in three clades, which belonged to 7 STs. Twenty-seven virulence-associated genes were detected, and expected cdtB, which was absent in all the isolates, the other 26 genes were conserved in the closely related Salmonella serogroup D (S. Enteritidis, S. Pullorum, and S. Gallinarum). CONCLUSION: Salmonella serogroup D was the major subgroup, and S. Pullorum was the most common type in sick and healthy-looking chickens in central China. Drug resistance assays showed serious multiple antimicrobial resistances, and S. Enteritidis was the most severe drug-resistant serotype. MLST showed that there was correlation between serotypes and genotypes in most Salmonella isolates, except S. Pullorum, which showed complicated genetic diversity firstly. These results provide important epidemiological information for us to control Salmonella in chickens.

Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

Induction of USP25 by viral infection promotes innate antiviral responses by mediating the stabilization of TRAF3 and TRAF6.

Host pathogen-recognition receptors detect nucleic acid from invading viruses and initiate a series of signaling pathways that lead to the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we found that a viral infection-induced deubiquitinase (DUB), ubiquitin-specific protease 25 (USP25) was required for host defense against RNA and DNA viruses. The activation of transcription factors IRF3 and NF-κB was impaired and the production of type I IFNs and proinflammatory cytokines was inhibited in Usp25-/- cells compared with the wild-type counterparts after RNA or DNA viruses infection. Consistently, USP25 deficient mice were more susceptible to H5N1 or HSV-1 infection compared with the wild-type mice. USP25 was associated with TRAF3 and TRAF6 after infection by RNA or DNA viruses and protected virus-induced proteasome-dependent or independent degradation of TRAF3 and TRAF6, respectively. Moreover, reconstitution of TRAF3 and TRAF6 into Usp25-/- MEFs restored virus-triggered production of type I IFNs and proinflammatory cytokines. Our findings thus reveal a previously uncovered positive feedback regulation of innate immune responses against RNA and DNA viruses by USP25.

Synergistic roles of the E2 glycoprotein and 3' untranslated region in the increased genomic stability of chimeric classical swine fever virus with attenuated phenotypes.

The E2 glycoprotein and 3' untranslated region (UTR) of classical swine fever virus (CSFV) are virulence determinants. To investigate the synergistic roles of E2 and 3'UTR for pathogenicity and genomic stability, a series of chimeric CSFVs were constructed by replacing the E2 gene and/or 3'UTR of virulent CSFV strain Shimen with the corresponding sequence of the lapinized 'Chinese' strain (C-strain) using a reverse genetic approach. The in vitro growth characterization and in vivo pathogenicity of the chimeric CSFVs were investigated. Our results demonstrated that the E2 glycoprotein mediates virus cell-to-cell spread and viral particle release and that the 3'UTR regulates viral RNA replication. The CSFV E2 and 3'UTR synergistically modulate infectious virus production, viral genomic stability in vitro, and attenuation in swine. This work contributes to our understanding of the structure and function of the CSFV genome and virus pathogenicity and will be useful for the development of a novel CSF vaccine.

Development of a novel single-step reverse genetics system for the generation of classical swine fever virus.

We describe an alternative reverse genetics system for generating classical swine fever virus (CSFV) based on swine RNA polymerase I promoter (pSPI)-mediated vRNA transcription. The recombinant plasmid pSPTI/SM harboring a full-length CSFV Shimen strain cDNA, flanked by a swine RNA polymerase I (pol I) promoter sequence at the 5' end and a murine pol I terminator sequence at the 3' end, was constructed. When the plasmid pSPTI/SM was introduced into PK-15 cells by transfection, an infectious CSFV with termini identical to those of the parental virus was generated directly. CSFV rescued from this reverse genetics system exhibited similar growth kinetics and plaque formation compared with the parental CSFV. When the novel reverse genetics system was used to generate the CSFV vaccine C-strain, infectious virus was detected in the supernatant of PK-15 cells transfected with the recombinant plasmid pSPTI/C. This novel reverse genetics system is a simple and efficient tool for the investigation of the structure and function of the viral genome, for molecular pathogenicity studies, and for the development of genetically engineered vaccines for CSFV.

Japanese encephalitis virus induces apoptosis by the IRE1/JNK pathway of ER stress response in BHK-21 cells.

The role of endoplasmic reticulum (ER) stress in Japanese encephalitis is largely unknown. In this study, we found that Japanese encephalitis virus (JEV) strain SA14-14-2 regulates the expression of glucose-regulated protein 78 (GRP78), transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and splicing of X-box-binding protein 1 (XBP1) mRNA in BHK-21 cells. SA14-14-2-induced cytopathic effect and decrease in viability were also observed. Moreover, the inositol-requiring enzyme 1 (IRE1) inhibitor 3,5-dibromosalicylaldehyde and JNK inhibitor SP600125 increased cell viability and reduced cell apoptosis but did not alter virus replication in SA14-14-2-infected BHK-21 cells. These results, for the first time, demonstrate that JEV induces apoptosis by the IRE1/JNK pathway of ER stress response.

Xuanfei Formula inhibited RSV infection by normalizing the SREBP2-mediated cholesterol synthesis process.

Background and aims: Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children and the elderly, often progressing to pneumonia and severe sequelae. However, there are currently no feasible and cost-effective interventions with proven efficacy for children, making medications with anti-RSV activity urgently needed. Traditional Chinese medicine has shown promising therapeutic efficacy in alleviating viral infection symptoms. Therefore, we aimed to develop effective strategies for RSV treatment based on traditional Chinese medicine. Methods and results: The infection status was assessed in BALB/c mice with or without Xuanfei Formula (XFF) treatment over a one-week period using H&E staining, cytokine assays and RSV titer testing after RSV challenge. Remarkably, on the first day of XFF intervention, both the pro-inflammation cytokine levels in the serum and RSV-N gene copies in the lung of mice were plummeted, compared to the RSV-infected group. This implied that XFF might possess the immune-independent anti-RSV capability. To elucidate the underlying mechanism, we employed transcriptome analysis followed by k-means analysis. The reversal effects of XFF against RSV primarily focused on the processes of innate and adaptive immunity. Additionally, we found that XFF administration corrected the disordered fatty acid and cholesterol metabolism processes during RSV infection. Lipidomics profiling indicated consistent cholesterol abundance with transcriptional changes but not fatty acids. Cholesterol synthesis-related genes mRNA levels and cholesterol synthesis intermediates detection supported XFF's repression upon cholesterol biosynthesis. Aberrantly increased cholesterol production has been reported as necessary for RSV infection. To mimic that, we observed lovastatin treatment inhibited RSV replication and pro-inflammation cytokine expression in vitro. Transcription factor prediction of differentially expressed genes (DEGs) involved in cholesterol synthesis implicated SREBP2. Through network pharmacology, stigmasterol and ß-sitosterol were identified as the effective active ingredients within the XFF, with the help of further molecular docking and mass spectrum detection. In vitro experiments demonstrated ß-sitosterol and stigmasterol reinforced the bonding between SREBP cleavage-activation protein (SCAP) and insulin-induced gene proteins (INSIGs) to inhibit SREBP2 cleavage maturation and consequent RSV infection. Conclusion: Xuanfei Formula (XFF) exhibits excellent anti-RSV efficacy by inhibiting SREBP2-mediated cholesterol synthesis to reduce RSV replication and ameliorate inflammation in the lung of infected mice.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Role of the conserved E2 residue G259 in classical swine fever virus production and replication.

The E2 glycoprotein of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle. The chimeric live attenuated C strain with the E2 substitution of bovine viral diarrhea virus (BVDV) is a promising marker vaccine candidate. In this study, the recombinant chimeric CSFV/bE2 cDNA clone harboring heterologous E2 (bE2) of BVDV was constructed by genetic approaches. Recombinant infectious virus rCSFV/bE2 (P11) was recovered by 11 serial passages of transfected PK15 cells. Viral genome sequencing showed that a glutamic acid to glycine mutation (E260G) at position 260 of the bE2 was observed in rCSFV/bE2 P11. Alignment of amino acid sequences displayed that the glycine was one of three conserved residues in pestivirus E2. When the glutamic acid to glycine substitution (E260G) was introduced into chimeric CSFV/bE2 cDNA clone, the high-titer infectious rCSFV/bE2E260G was rescued. The glycine to glutamic acid substitution at corresponding position in CSFV E2 resulted in significantly decreased rCSFV/E2G259E production. We further identified that the conserved E2 residue G259 played a critical role in the release and binding activity of CSFV and that the E2 residues G259 and V111 modulated synergistically infectious virus production and replication.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis.

The 3'untranslated region (3'UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3'UTR and vC/b3'UTR, with 3'UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3'UTR and vC/b3'UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3'UTR or vC/b3'UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3'UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

Dynamic Host Immune and Transcriptomic Responses to Respiratory Syncytial Virus Infection in a Vaccination-Challenge Mouse Model.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children. Inactivated RSV vaccine was developed in the late 1960's, but the vaccine-enhanced disease (VED) occurred to vaccinated infants upon subsequent natural RSV infection. The excessive inflammatory immunopathology in the lungs might be involved in the VED, but the underlying mechanisms remain not fully understood. In this study, we utilized UV-inactivated RSV in the prime/boost approach followed by RSV challenge in BALB/c mice to mimic RSV VED. The dynamic virus load, cytokines, histology and transcriptome profiles in lung tissues of mice were investigated from day 1 to day 6 post-infection. Compared to PBS-treated mice, UV-RSV vaccination leads to a Th2 type inflammatory response characterized by enhanced histopathology, reduced Treg cells and increased IL4+CD4 T cells in the lung. Enhanced production of several Th2 type cytokines (IL-4, IL-5, IL-10) and TGF-ß,  reduction of IL-6 and IL-17 were observed in UV-RSV vaccinated mice. A total of 5582 differentially expressed (DE) genes between PBS-treated or vaccinated mice and naïve mice were identified by RNA-Seq. Eleven conserved high-influential modules (HMs) were recognized, majorly grouped into regulatory networks related to cell cycle and cell metabolism, signal transduction, immune and inflammatory responses. At an early time post-infection, the vaccinated mice showed obvious decreased expression patterns of DE genes in 11 HMs compared to PBS-treated mice. The extracellular matrix (HM5) and immune responses (HM8) revealed tremendous differences in expression and regulation characteristics of transcripts between PBS-treated and vaccinated mice at both early and late time points. The highly connected genes in HM5 and HM8 networks were further validated by RT-qPCR. These findings reveal the relationship between RSV VED and immune responses, which could benefit the development of novel RSV vaccines.

Chimeric enterovirus 71 virus-like particle displaying conserved coxsackievirus A16 epitopes elicits potent immune responses and protects mice against lethal EV71 and CA16 infection.

Hand-foot-and-mouth disease (HFMD) is an infectious disease of infants and young children frequently caused by the enterovirus A species, mainly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, we prepared the EV71 virus-like particle (EV71-VLP) and its chimeras using recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under CMV-IE promoter) proteins in Sf9 cells. EV71-VLP chimera ChiEV71(1E)-VLP or ChiEV71(4E)-VLP displayed single CA16 PEP71 epitope in VP1 or four conserved CA16 neutralizing epitopes (PEP71 in VP1, aa136-150 in VP2, aa176-190 in VP3 and aa48-62 in VP4) by substitution of the corresponding regions of EV71 structure proteins, respectively. In mice, EV71-VLP and its chimeras elicited similar EV71-specific IgG and neutralizing antibody (NAb) titers compared to inactivated EV71. Expectedly, vaccination of ChiEV71(1E)-VLP or ChiEV71(4E)-VLP resulted in significantly increased CA16-specific IgG and NAb production and improved cross-protection against CA16 infection compared to EV71-VLP. Interestingly, the VLPs induced potent cellular immune responses and significantly decreased Th2 type (IL-4 and IL-10) cytokines secretion in the splenocytes of immunized mice compared to inactivated EV71 or inactivated CA16. Neonatal mice born to dams immunized with the chimeric VLPs or neonatal mice passively transferred with sera of immunized mice were completely protected from lethal EV71 challenge and partially protected from lethal CA16 infection. Our study provides a novel bivalent or multivalent vaccine strategy to prevent EV71 and related-enterovirus infections.