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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-β, 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12250-021-00418-3.

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 EThe CSFV EIndirect ELISA was established based on recombinant CSFV EThe newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

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., Author summary Coronavirus disease 2019 (COVID-19) is currently destroying both lives and economies. However, patients infected with severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) usually present heterogeneous and complicated progressions, such as different incubation periods (short and long), symptoms (asymptomatic and symptomatic) and severity (mild-moderate and severe). Currently, various clinical data and experimental data are available from different countries, which has great significance for integrating different types of data to comprehensively understand the diverse disease progression in COVID-19 patients and guide individual treatment strategies. Here, we developed a multi-scale computational model to describe the dynamical process of patients infected with SARS-CoV-2, including intracellular viral dynamics, multicellular infection process, and immune responses. By combining data integration, stochastic simulation and quantitative analysis based on the multi-scale mathematical model, we addressed an important question regarding how IFN response and T cell exhaustion quantitatively affect heterogeneous progression in patients with respect to incubation periods, symptoms and severity. Furthermore, the efficacy of various therapeutic strategies for treating COVID-19 patients with different severity degrees was evaluated and validated. The computational framework in this study can also be extended to explore the dynamical process of other coronavirus infections.

Highlights • A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. • The detection limit of mRT-PCR was in agreement with the singleplex RT-PCR. • The mRT-PCR were successfully used to detect clinical specimens. • The mRT-PCR developed is a useful tool for clinical diagnosis of single virus and/or mixed infections in swine., A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1 × 103 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine.

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.

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for the virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between the positively charged patch on protease module and NS3 function. The surface patch was composed of four basic residues R50, K74 and K94 in NS3 protease domain and H24 in the structurally integrated cofactor NS4A PCS . Single residue or simultaneous four-residue substitution in the patch to alanine or aspartic acid hardly affect ATPase activity. However, the single R50, K94 or H24 residue or simultaneous four-residue substitution resulted in the apparent changes of the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, the single K94 residue or simultaneous four-residue substitution (Qua_A or Qua_D) impaired the infectious virus production. Furthermore, the replication efficiency of the CSFV variants was partially correlated to the helicase activity of NS3 in vitro . Our results suggest that the conserved positively charged patch on the NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

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.

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.

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.

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 β, 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.

Japanese encephalitis virus (JEV) infection induces brain tissue disease characterized by neuron death. however, little is known about the underlying mechanism. Using RNA sequencing, we profiled global mRNA expression changes in response to in vitro and in vivo JEV infection. Integration analysis of in vitro and in vivo mRNA transcriptome revealed that JEV infection regulated apoptosis-related Foxo signaling pathway. Foxo expression was reduced by JEV infection in vitro and in vivo. Knockdown of Foxo promoted apoptosis, while its overexpression reduced apoptosis in JEV-infected Neuro-2a cells. JEV infection in Neuro-2a cells decreased the expression of Foxo downstream genes including pro-apoptotic protein Bim, anti-apoptotic protein Bcl-6 and p21. Overexpression of anti-apoptotic proteins Bcl-6 and p21 repressed JEV-induced apoptosis. These findings suggest that Foxo primarily exerts an anti-apoptotic function via Bcl-6 and p21 in JEV-infected Neuro-2a cells. A STAT3 binding site was identified in the promoter region of Foxo by TFBIND software and confirmed by ChIP and reporter assays. JEV infection reduced STAT3 expression as well as its binding at the Foxo promoter compared to mock infection in Neuro-2a cells. Moreover, STAT3 knockdown reduced Foxo promoter activity and Foxo expression. Therefore, JEV reduced Foxo expression, at least in part, by downregulating STAT3. Taken together, we found that JEV induced cell apoptosis by inhibiting STAT3-Foxo-Bcl-6/p21 pathway, which provides a novel insight into JEV-caused encephalitis.

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.

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.

Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated the thermostable recombinant NDV (rNDV) expressing the different forms of hemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) H5N1 based on the full-length cDNA clone of thermostable TS09-C strain. The recombinant thermostable Newcastle disease viruses, rTS-HA, rTS-HA1 and rTS-tPAs/HA1, expressed the HA, HA1 or modified HA1 protein with the tissue plasminogen activator signal sequence (tPAs), respectively. The rNDVs displayed similar thermostability, growth kinetics and pathogenicity compared with the parental TS09-C virus. The tPAs facilitated the expression and secretion of HA1 protein in cells infected with rNDV. Animal studies demonstrated that immunization with rNDVs elicited effective H5N1- and NDV-specific antibody responses and conferred immune protection against lethal H5N1 and NDV challenges in chickens and mice. Importantly, vaccination of rTS-tPAs/HA1 resulted in enhanced protective immunity in chickens and mice. Our study thus provides a novel thermostable NDV-vectored vaccine candidate expressing a soluble form of a heterologous viral protein, which will greatly aid the poultry industry in developing countries.

Respiratory syncytial virus (RSV) infection causes severe disease in the lower respiratory tract of infants and young children. Currently, no licensed vaccine is available. In this study, we generated the chimeric virus-like particles (tHBc/FE1E2, tHBc/FE1E2/M282-90 and tHBc/FE1E2/M282-90/tG VLPs) containing multiple antigenic peptides of RSV proteins based on a truncated hepatitis B virus core carrier (tHBc). We investigated the immune protection against RSV infection induced by these VLPs in a mouse model. Immunization with the VLPs elicited RSV-specific IgG and neutralizing antibody production and conferred protection against RSV infection in vivo. Compared with UV-RSV or tHBc/FE1E2/M282-90/tG VLPs, the tHBc/FE1E2 and tHBc/FE1E2/M282-90 VLPs induced significantly decreased Th2 cytokines (IL-4, IL-5) and increased Th1 cytokines (IFN-γ, TNF-α, IL-2) as well as increased IgG2a/IgG1 ratios. tHBc/FE1E2 and tHBc/FE1E2/M282-90 VLPs also elicited an increased regulatory T (Treg) cell frequency and IL-10 secretion in the lungs of vaccinated mice, thereby relieving pulmonary pathology upon subsequent RSV infection. Our results demonstrate that the VLPs containing antigenic peptides of F protein combined with a CTL epitope of M2 may represent a promising RSV subunit vaccine candidate.

Salmonella Pullorum is a pathogen specific to birds that can cause Pullorum disease in young chickens and lead to considerable economic losses in the poultry industry. During transmission and infection, S. Pullorum will encounter various environmental stresses and host defenses. The stringent response is an important adaptation response induced by (p)ppGpp, and in Salmonella, (p)ppGpp is synthesized by two (p)ppGpp synthetases, RelA and SpoT. To investigate the role of (p)ppGpp synthetases in the adaptation and pathogenicity of S. Pullorum, a (p)ppGpp synthetases mutant (ΔrelAΔspoT) was constructed, and its physiological phenotypes and pathogenicity, as well as transcription profiling, were compared with the parent strain. The ΔrelAΔspoT mutant showed decreased ability to form biofilms, and reduced resistance to acidic, alkaline, high osmolarity and H2O2 conditions. The internalization of the ΔrelAΔspoT mutant into host cells in vitro and its lethality and colonization abilities within young chickens were also significantly reduced. RNA sequencing showed that the (p)ppGpp synthetases did not only affect the classic stringent response, such as inhibition of DNA replication and protein synthesis, but also controlled the expression of many virulence factors, in particular, the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs), and adhesion factors. These results suggest that the (p)ppGpp synthetases are required for the pathogenicity of S. Pullorum by affecting its stress response and the expression of the virulence factors.

To investigate the feasibility and efficacy of a virus-like particle (VLP) vaccine composed of the conserved antigenic epitopes of respiratory syncytial virus (RSV), the chimeric RSV VLPs HBcΔ-tG and HBcΔ-tG/M282-90 were generated based on the truncated hepatitis B virus core protein (HBcΔ). HBcΔ-tG consisted of HBcΔ, the conserved region (aa 144-204) of the RSV G protein. HBcΔ-tG was combined with a single peptide (aa 82-90) of the M2 protein to generate HBcΔ-tG/M282-90. Immunization of mice with the HBcΔ-tG or HBcΔ-tG/M282-90 VLPs elicited RSV-specific IgG and neutralizing antibody production and conferred protection against RSV infection. Compared with HBcΔ-tG, HBcΔ-tG/M282-90 induced decreased Th2 cytokine production (IL-4 and IL-5), increased Th1 cytokine response (IFN-γ, TNF-α, and IL-2), and increased ratios of IgG2a/IgG1 antibodies, thereby relieving pulmonary pathology upon subsequent RSV infection. Our results demonstrated that chimeric HBcΔ-tG/M282-90 VLPs represented an effective RSV subunit vaccine candidate.

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.

Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV.

Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2 containing the E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain Shimen (vSM) was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To investigate the molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant (vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of vSM/CE2-p11 were observed. Based on reverse genetic manipulation of the chimeric cDNA clone pSM/CE2, the mutated viruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were rescued. The data from infection of pigs demonstrated that the M979K amino acid substitution was responsible for pathogenicity. Studies in vitro indicated that T745I and M979K increased infectious virus production and replication. Our results indicated that two residues located at sites 745 and 979 within E2 play a key role in determining the replication in vitro and pathogenicity in vivo of chimeric CSFV vSM/CE2.

Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication.

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.

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-Å 2 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.

The classical swine fever virus (CSFV) nonstructural protein p7 is crucial for virus production, yet precisely how the p7 modulates this process is unclear. In this study, we first identified the interactions of p7 with E2 and NS2. The key binding regions of both p7 and NS2 mapped to the first transmembrane (TM1) domain of two proteins. Three amino acid substitutions in the TM1 region of p7 (p7TDI18/19/20AAA, p7EVV21/22/23AAA and p7YFY25/26/30AAA) impaired infectious virus production and reduced the interaction of p7 with the NS2 protein. The E2p7 processing and mature p7, but not the E2p7 precursor, are essential for infectious virus production. Bicistronic mutants (pSM/E2/IRES) with single substitutions at residues 1 to 9 of p7 exhibited a significantly increased infectious CSFV titer compared to their counterparts in the context of pSM. Viral genomic RNA copies of the mutants exhibited similar levels compared with the wt CSFV. Our results demonstrated that CSFV p7 and its precursor E2p7 modulate viral protein interactions and infectious virus production without influencing viral RNA replication.

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand-foot-and-mouth disease (HFMD). To investigate novel combined vaccines to prevent EV71 and CA16 infection, we constructed chimeric virus-like particles (tHBc/SPA or tHBc/SP VLPs) displaying conserved epitopes of EV71 (aa 208-222 of VP1 and aa 248-263 of VP2) and CA16 (aa271-285 of VP1) using a truncated hepatitis B virus core carrier (tHBc). Immunization with the chimeric VLPs induced epitope- or virus-specific IgG and neutralization antibodies against EV71 and CA16 in the mice. Compared with inactivated EV71, the chimeric VLPs induced significantly increased Th1 cytokine (IFN-γ, IL-2) production and decreased Th2 cytokine (IL-4, IL-10) responses. Neonatal mice born to dams immunized with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection. Co-expression of the conserved human MHC class I CD4+ T cell epitope (aa248-263 of VP2) did not improve the antiviral immunity of the chimeric VLP vaccine in mice. Our results demonstrate that experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.

A multiplex reverse transcription-PCR (mRT-PCR) assay was developed for the rapid detection of influenza A viruses. The assay simultaneously differentiated H1, H3, H5 and H9 hemagglutinin subtypes in a single reaction mixture. Five sets of specific primers targeted to the M, H1, H3, H5 and H9 genes were used in this assay. The amplified products were visualized by agarose gel electrophoresis. The sizes of the PCR amplified fragments were 612 bp for H1, 187 bp for H3, 338 bp for H5, 289 bp for H9 and 239 bp for M. The detection limit of the viral RNA template was 1 ng for the H1, H3 and H5 subtypes and 0.1 ng for the H9 subtype. Nonspecific product bands from RNAs of other viral pathogens were not amplified. The sensitivity analysis demonstrated that the mRT-PCR assay is as sensitive as conventional RT-PCR and 10 times less sensitive than SYBR Green real-time RT-PCR. In conclusion, the mRT-PCR assay developed in this study was able to type influenza A viruses and simultaneously differentiate H1, H3, H5 and H9 subtypes in both human and avian clinical specimens, and thus, the mRT-PCR assay could be a rapid, convenient and relatively inexpensive molecular diagnostic tool for large-scale screening of clinical samples.

We report here the complete genome sequence of virulent fowl adenovirus serotype 4 strain HB1510, isolated from a diseased chicken with hydropericardium-hepatitis syndrome in Hubei, China, in 2015. The viral genome is 43,721 bp long, and sequence analysis showed an 11-amino-acid deletion in open reading frame 29 (ORF29).

p13 gene was first described in Leucania separata multinuclear polyhedrosis virus (Ls-p13) several years ago, but the function of P13 protein has not been experimentally investigated to date. In this article, we indicated that the expression of p13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (Ha-p13) was regulated by both early and late promoter. Luciferase assay demonstrated that the activity of Ha-p13 promoter with hr4 enhancer was more than 100 times in heterologous Sf9 cells than that in nature host Hz-AM1 cells. Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocal microscopic analysis showed that both mainly located in the cytoplasm membrane at 48 h. Results of RNA interference indicated that Ha-p13 was a killing-associated gene for host insects H. armigera. The AcMNPV acquired the mentioned killing activity and markedly accelerate the killing rate when expressing Ls-p13. In conclusion, p13 is a killing associated gene in both homologous and heterologous nucleopolyhedrovirus. [BMB Reports 2012; 45(12): 730-735]

A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

To evaluate the effects of the Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF) on immune responses induced by DNA vaccines, mice were immunized with the eukaryotic expression plasmid pcDNA/E2 encoding classical swine fever virus (CSFV) E2 alone or in combination with the TRIF genetic adjuvant. Immune responses were examined in immunized mice. Our data demonstrates that co-delivery of the DNA vaccine pcDNA/E2 with the TRIF adjuvant augmented specific humoral and cellular immune responses in a mouse model. Vaccination of pigs confirmed that the pcDNA/E2 in combination with TRIF conferred total protection against lethal challenge with highly virulent CSFV. We conclude that TRIF enhances the effects of the DNA vaccine against CSFV infection and could be used as a potential genetic adjuvant for DNA vaccines in large animal species.

Classical swine fever virus (CSFV) nonstructural protein 3 (NS3) is believed to possess three enzyme activities that are likely to be essential for virus replication: a serine protease located in the N-terminus and NTPase as well as helicase activities located in the C-terminus. In this report, we expressed NS3 helicase domain (NS3h) in E. coli and characterized its helicase activity. The NS3h helicase activity was dependent on the presence of NTP and divalent cations, with a preference for ATP and Mn 2+ , and required the substrates possessing a 3′ un-base-paired region on the RNA template strand. The NS3h helicase activity was proportional to increasing lengths of the 3′ un-base-paired regions up to 16 nucleotides of the RNA substrates. We also investigated the modulation of NS3 NTPase/helicase activities by NS3 protease domain and NS5B, an RNA-dependent RNA polymerase (RdRp). Our data showed that the NS3 protease domain enhanced the helicase activity of NS3 but had no effect on its NTPase activity. For the truncated NS3 (helicase domain, NS3h), both NTPase and helicase activities were up-regulated by NS5B. However, for the full-length NS3 (NS3FL), the NTPase activity, but not the helicase activity, was stimulated by NS5B. Maltose-binding protein (MBP) pull-down as well as enzyme-linked immunosorbent assays confirmed the specific interaction between NS3 and NS5B.

Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease, due to their decreased dependence on cold chain for transport and storage. However, the genetic basis underlying the NDV thermostability is poorly understood. In this study, we generated chimeric viruses by exchanging viral genes between the thermostable TS09-C strain and thermolabile LaSota strain using reverse genetics technology. Evaluations of these chimeric NDVs demonstrated that the thermostability of NDV was dependent on the origin of HN protein. Chimeras bearing the HN protein derived from thermostable virus exhibited a thermostable phenotype and vice versa. Both hemagglutinin and neuraminidase activities of viruses bearing the TS09-C HN protein were more thermostable than those containing LaSota HN protein. Furthermore, the newly developed thermostable virus rLS-T-HN, encoding the TS09-C HN protein in LaSota backbone, induced significantly higher antibody response than the TS09-C virus and conferred complete protection against virulent NDV challenge. Taken together, the data suggest that the HN protein of NDV is a crucial determinant of thermostability and the HN gene from a thermostable NDV could be engineered into a thermolabile NDV vaccine strain for developing novel thermostable NDV vaccine.

Enterovirus 71 (EV71) belonging to the Picornaviridae family is considered the most frequently detected causative agent in hand-foot-and-mouth disease (HFMD) and is a serious threat to public health in the Asia-Pacific region. There are currently no approved vaccines or effective drugs for EV71. In this study, using recombinant vesicular stomatitis virus (rVSV) expressing viral VP1 protein (mVP1) of EV71 as a control, we generated two types of rVSVs that can form EV71 virus-like particles (VLPs). First, we co-infected two rVSVs singly expressing P1 (mP1) and 3CD (m3CD) of EV71. Second, we inserted P1 and 3CD into one VSV backbone to generate an rVSV expressing P1 and 3CD together (mP1-3CD). When P1 and 3CD were expressed in the cells either co-infected with mP1 and m3CD (mP1/m3CD) or infected with mP1-3CD, P1 was cleaved by 3CD and produced VP1, VP3, and VP0 to form VLPs. Furthermore, mice immunized with mP1/m3CD or mP1-3CD showed higher humoral and cellular immunity responses than mice immunized with mVP1. Finally, the rVSVs expressing the EV71 proteins were evaluated in mice to determine their potential to protect against a lethal EV71 virus challenge, and among all the rVSVs, the mP1-3CD was shown to be the most promising vaccine candidate for EV71 protection.

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.

The nonstructural protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK(232)T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA(354) for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K(m) and k(cat)) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.