Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX.

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Efficacy of the marker vaccine rAdV-SFV-E2 against classical swine fever in the presence of maternally derived antibodies to rAdV-SFV-E2 or C-strain.

Classical swine fever (CSF) is an economically important disease caused by Classical swine fever virus (CSFV). In order to eradicate CSF, many marker vaccines that allow differentiation of infected from vaccinated animals (DIVA) have been developed. In our previous studies, a DIVA CSF vaccine rAdV-SFV-E2 has been demonstrated to completely protect pigs against lethal CSFV challenge. In the context of risk assessments for an emergency vaccination scenario, the question has been raised whether preexisting maternally derived antibodies (MDAs) interfere with the efficacy of the vaccine. In this study, six groups of piglets (n=5), with or without anti-C-strain or anti-rAdV-SFV-E2 MDAs, were immunized twice with 106 TCID50 rAdV-SFV-E2 and challenged with the CSFV Shimen strain. Clinical signs, CSFV-specific antibodies, viremia and pathological and histopathological changes were monitored. The results showed that the vaccinated piglets, either with or without MDAs directed against C-strain (about 67% blocking rate) or rAdV-SFV-E2 (about 50% blocking rate) were completely protected; however, the mock-vaccinated piglets displayed severe CSF-typical clinical symptoms, viremia, pathological/histopathological changes and deaths (5/5). These findings demonstrate that the MDAs to either rAdV-SFV-E2 or C-strain do not interfere with the efficacy of rAdV-SFV-E2, which highlights the great potential of the vaccine for control and eradication of CSF.

Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines.

Piglets with maternally derived antibodies from sows immunized with rAdV-SFV-E2 were completely protected against lethal CSFV challenge.

Classical swine fever (CSF) is an economically important infectious disease of pigs caused by Classical swine fever virus (CSFV). To facilitate the eradication of CSF in endemic areas, a marker vaccine enabling differentiation of infected from vaccinated animals (DIVA) is urgently needed. Previously, we have demonstrated that the DIVA vaccine rAdV-SFV-E2, an adenovirus-vectored Semliki Forest virus replicon expressing the E2 glycoprotein of CSFV, induces complete protection from lethal CSFV challenge. The aim of this study was to investigate whether maternally derived antibodies (MDAs) from sows immunized with rAdV-SFV-E2 can effectively protect piglets against lethal CSFV challenge. Three groups of five-week-old piglets (n=4), with or without MDAs, were challenged with the highly virulent CSFV Shimen strain. Clinical signs, CSFV-specific antibodies, viremia and pathological and histopathological changes were monitored. The results showed that the piglets with MDAs from the sow immunized with rAdV-SFV-E2 were protected clinically, virologically and pathologically, while the piglets with undetectable MDAs from the rAdV-SFV-E2-immunized sow were partially protected (2/4 survival), in contrast with the piglets from the non-vaccinated sow, which displayed CSF-typical clinical signs, viremia, deaths (4/4) and pathological/histopathological lesions. These results indicate that MDAs from the sow immunized with rAdV-SFV-E2 are able to confer full passive immunity to newborn piglets.

Safety and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs.

Classical swine fever (CSF) and pseudorabies (PR) are both major infectious diseases of pigs, causing enormous economic losses to the swine industry in many countries. A marker vaccine that enables differentiation of infected from vaccinated animals (DIVA) is highly desirable for control and eradication of these two diseases in endemic areas. Since late 2011, PR outbreaks have been frequently reported in many Bartha-K61-vaccinated pig farms in China. It has been demonstrated that a pseudorabies virus (PRV) variant with altered antigenicity and increased pathogenicity was responsible for the outbreaks. Previously, we showed that rPRVTJ-delgE/gI/TK, a gE/gI/TK-deleted PRV variant, was safe for susceptible animals and provided a complete protection against lethal PRV variant challenge, indicating that rPRVTJ-delgE/gI/TK can be used as an attractive vaccine vector. To develop a safe bivalent vaccine against CSF and PR, we generated a recombinant virus rPRVTJ-delgE/gI/TK-E2 expressing the E2 protein of classical swine fever virus (CSFV) based on rPRVTJ-delgE/gI/TK and evaluated its safety and immunogenicity in pigs. The results indicated that pigs (n=5) immunized with rPRVTJ-delgE/gI/TK-E2 of different doses did not exhibit clinical signs or viral shedding following immunization, the immunized pigs produced anti-PRV or anti-CSFV neutralizing antibodies and the pigs immunized with 10(6) or 10(5) TCID50 rPRVTJ-delgE/gI/TK-E2 were completely protected against the lethal challenge with either CSFV Shimen strain or variant PRV TJ strain. These findings suggest that rPRVTJ-delgE/gI/TK-E2 is a promising bivalent DIVA vaccine candidate against CSFV and PRV coinfections.

Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity.

UNLABELLED: Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE: Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.

Pathogenicity and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant in susceptible animals.

A pseudorabies virus (PRV) variant with enhanced pathogenicity has emerged in many vaccinated swine herds in China since 2011. rPRVTJ-delgE/gI, a previously described gE/gI-deleted PRV based on the PRV variant TJ strain, has been shown to be avirulent to pigs yet virulent to sheep. To ensure desirable biosafety, we further deleted the thymidine kinase (TK) gene of rPRVTJ-delgE/gI to generate a gE/gI/TK-deleted mutant rPRVTJ-delgE/gI/TK, and evaluated its pathogenicity and immunogenicity in susceptible animals. Groups of mice (n=5), sheep (n=3), and pigs (n=4) were inoculated with different doses of rPRVTJ-delgE/gI/TK or rPRVTJ-delgE/gI, and clinical signs, viral shedding, pathological changes, and serum antibodies were examined following inoculation. The results showed that rPRVTJ-delgE/gI/TK displayed higher safety than rPRVTJ-delgE/gI for mice (10(3)-10(6) TCID50) and sheep (10(5) TCID50), and pigs inoculated with rPRVTJ-delgE/gI/TK (10(5) TCID50) induced PRV-specific antibodies and protection against lethal PRV challenge comparable to those immunized with rPRVTJ-delgE/gI. In conclusion, rPRVTJ-delgE/gI/TK has the potential to be developed as a vaccine for controlling the currently prevalent PR in China.

Dose-dependent pathogenicity of a pseudorabies virus variant in pigs inoculated via intranasal route.

Pseudorabies (PR) or Aujeszky's disease (AD), caused by pseudorabies virus (PRV), is an economically important viral disease in many countries. The modified live vaccine Bartha-K61 strain has played an important role in the control of PR in many countries including China. Since late 2011, however, increasing PR outbreaks caused by an emerging PRV variant have been reported in Bartha-K61-vaccinated swine population on many farms in China. Previously, we showed that the PRV variant TJ strain exhibited enhanced pathogenicity in pigs inoculated via intramuscular route. To develop an animal infection model for accurate evaluation of novel vaccines against the emergent PRV variant, we evaluated the pathogenicity of the PRV TJ strain of different doses in pigs infected via intranasal route. Groups (n=5) of 7-week-old healthy pigs were inoculated intranasally with 10(3), 10(4), 10(5), or 10(6) TCID50 (median tissue culture infective dose) PRV TJ strain. Clinical signs, rectal temperature, virus shedding, pathological changes, and seroconversion were monitored. The results showed that the PRV TJ strain induced varied morbidity and mortality (0/5 to 5/5), clinical signs, and tissue lesions, increasingly correlated with the infection doses, and the median lethal dose (LD50) of the virus was determined to be 10(4.5) TCID50. Together, this study demonstrates the dose-dependent pathogenicity of the PRV variant via the intranasal route of infection, which provides an ideal animal infection model for evaluation of novel vaccines against the emerging PRV variant.