The construction and immunogenicity analyses of a recombinant pseudorabies virus with Senecavirus A VP3 protein co-expression.

Senecavirus A (SVA)-associated porcine idiopathic vesicular disease (PIVD) and Pseudorabies (PR) are highly contagious swine disease that pose a significant threat to the global pig industry. In the absence of an effective commercial vaccine, outbreaks caused by SVA have occurred in many parts of the world. In this study, the PRV variant strain PRV-XJ was used as the parental strain to construct a recombinant PRV strain with the TK/gE/gI proteins deletion and the VP3 protein co-expression, named rPRV-XJ-ΔTK/gE/gI-VP3. The results revealed that PRV is a suitable viral live vector for VP3 protein expressing. As a vaccine, rPRV-XJ-ΔTK/gE/gI-VP3 is safe for mice, vaccination with it did not cause any clinical symptoms of PRV. Intranasal immunization with rPRV-XJ-ΔTK/gE/gI-VP3 induced strong cellular immune response and high levels of specific antibody against VP3 and gB and neutralizing antibodies against both PRV and SVA in mice. It provided 100% protection to mice against the challenge of virulent strain PRV-XJ, and alleviated the pathological lesion of heart and liver tissue in SVA infected mice. rPRV-XJ-ΔTK/gE/gI-VP3 appears to be a promising vaccine candidate against PRV and SVA for the control of the PRV variant and SVA.

Pathogenicity and immunogenicity of a quadruple gene-deleted pseudorabies virus variant as a vaccine candidate.

Since late 2011, the PRV variants have emerged in China, characterized by the increased virulence. The traditional attenuated vaccines have proven insufficient in providing complete protection, resulting in substantial economic losses to swine industry. In this study, a vaccine candidate strain, ZJ01-ΔgI/gE/TK/UL21, carrying the quadruple gene deletion was derived from the previously generated three gene-deleted virus ZJ01-ΔgI/gE/TK. As anticipated, piglets inoculated with ZJ01-ΔgI/gE/TK/UL21 exhibited normal body temperatures and showed no viral shedding, consistent with the observations from piglets treated with ZJ01-ΔgI/gE/TK. Importantly, a significant higher level of interferon induction was observed among piglets in the ZJ01-ΔgI/gE/TK/UL21 group compared to those in the ZJ01-ΔgI/gE/TK group. Upon challenge with the PRV variant ZJ01, piglets immunized with ZJ01-ΔgI/gE/TK/UL21 exhibited reduced viral shedding compared to the ZJ01-ΔgI/gE/TK group. Furthermore, piglets vaccinated with ZJ01-ΔgI/gE/TK/UL21 exhibited minimal pathological lesions in brain tissues, similar to those in the ZJ01-ΔgI/gE/TK group. These results underscore the potential of ZJ01-ΔgI/gE/TK/UL21 as a promising vaccine for controlling PRV infection.

Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

A self-assembled nanoparticle vaccine based on pseudorabies virus glycoprotein D induces potent protective immunity against pseudorabies virus infection.

Pseudorabies virus (PRV) mainly causes pseudorabies (PR) or Aujeszky's disease in pigs and can infect humans, raising public health concerns about zoonotic and interspecies transmission of PR. With the emergence of PRV variants in 2011, the classic attenuated PRV vaccine strains have failed to protect many swine herds against PR. Herein, we developed a self-assembled nanoparticle vaccine that induces potent protective immunity against PRV infection. PRV glycoprotein D (gD) was expressed using the baculovirus expression system and further presented on the lumazine synthase (LS) 60-meric protein scaffolds via the SpyTag003/SpyCatcher003 covalent coupling system. In mouse and piglet models, LSgD nanoparticles emulsified with the ISA 201VG adjuvant elicited robust humoral and cellular immune responses. Furthermore, LSgD nanoparticles provided effective protection against PRV infection and eliminated pathological symptoms in the brain and lungs. Collectively, the gD-based nanoparticle vaccine design appears to be a promising candidate for potent protection against PRV infection.

Isolation and identification of two novel pseudorabies viruses with natural recombination or TK gene deletion in China.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years due to outbreaks of emergent pseudorabies. However, there is limited information about the evolution and pathogenicity of emergent PRV field strains in China. In this study, two PRV field strains were isolated from an intensive pig farm with suspected PRV infection. These were named the GXLB-2015 and GXGG-2016 strains and their growth characteristics together with their genome sequences and pathogenicity were determined. Nucleotide homology and phylogenetic analysis revealed the GXLB-2015 stain was relatively close to the foreign PRV isolated strains with respect to the whole genome sequence. However, it formed an independent branch between the foreign PRV isolates and the previous PRV variants isolated in China. Further recombination and genetic evolution analysis showed that the GXLB-2015 strain was a natural recombinant between the Bartha strain and PRV variants. The GXGG-2016 strain was highly homologous with the Chinese classical strains, but it has a natural deletion of 69 aa in the thymidine kinase (TK) gene. Pathogenicity analysis showed that, the GXLB-2015 strain had the strongest pathogenicity to mice with an LD50 of 103.5, while the GXGG-2016 strain with the TK gene deletion was not pathogenic to mice. Taken together, our data provide direct evidence for the genomic recombination and natural TK gene deletion of PRVs, which may provide a reference for a better understanding of PRV evolution in China and contribute to the clinical control of PRV infection in pig farms.

Comparison of the protective efficacy between the candidate vaccine ZJ01R carrying gE/gI/TK deletion and three commercial vaccines against an emerging pseudorabies virus variant.

Pseudorabies virus (PRV) is a swine alpha-herpesvirus that mainly causes reproductive disorders in sows and neurological diseases in piglets. Vaccination is the most efficient method to prevent the disease. In China, since the emergence of PRV mutant strains in late 2011, the traditional commercial vaccines have not been providing complete protection. Our previous studies have demonstrated that PRV ZJ01 is a highly virulent strain, and its derivative, ZJ01R, which carries the gE/gI/TK gene deletion, could provide protection against the variant PRV challenge. However, the difference in immune efficacy between ZJ01R and other commercial vaccines remains unclear. In this study, the immune protection efficacy between ZJ01R and three commercial PRV vaccines (Bartha-K61, HB2000, and SA215) was evaluated in piglets. The safety of ZJ01R was shown to be equivalent to that of the three commercial vaccines. The titers of the neutralizing antibodies against the PRV classical strain LA in the four vaccine groups were similar, while the anti-PRV variant neutralizing antibody titers in the ZJ01R group were significantly higher than those in the Bartha-K61, HB2000, and SA215 strain groups. After the PRV challenge, ZJ01R, HB2000, and SA215 vaccinations could provide complete protection, whereas the Bartha-K61 vaccination could only provide 60 % protection. Importantly, the rectal viral excretion and PRV DNA loads in the lung tissues in the ZJ01R group were significantly lower than those in the Bartha-K61, HB2000, and SA215 groups. Altogether, these results indicated that ZJ01R could provide higher protection efficacy against the PRV virulent ZJ01 challenge than the three commercial PRV gene-deleted live vaccines derived from the classical vaccine strains, providing the potential to develop a new PRV vaccine to control the epidemic PRV variant strains in the future.

A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.

Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.

Pseudorabies Virus: From Pathogenesis to Prevention Strategies.

Pseudorabies (PR), also called Aujeszky's disease (AD), is a highly infectious viral disease which is caused by pseudorabies virus (PRV). It has been nearly 200 years since the first PR case occurred. Currently, the virus can infect human beings and various mammals, including pigs, sheep, dogs, rabbits, rodents, cattle and cats, and among them, pigs are the only natural host of PRV infection. PRV is characterized by reproductive failure in pregnant sows, nervous disorders in newborn piglets, and respiratory distress in growing pigs, resulting in serious economic losses to the pig industry worldwide. Due to the extensive application of the attenuated vaccine containing the Bartha-K61 strain, PR was well controlled. With the variation of PRV strain, PR re-emerged and rapidly spread in some countries, especially China. Although researchers have been committed to the design of diagnostic methods and the development of vaccines in recent years, PR is still an important infectious disease and is widely prevalent in the global pig industry. In this review, we introduce the structural composition and life cycle of PRV virions and then discuss the latest findings on PRV pathogenesis, following the molecular characteristic of PRV and the summary of existing diagnosis methods. Subsequently, we also focus on the latest clinical progress in the prevention and control of PRV infection via the development of vaccines, traditional herbal medicines and novel small RNAs. Lastly, we provide an outlook on PRV eradication.

Characterization of B cell receptor H-CDR3 repertoire of spleen in PRV-infected mice.

Pseudorabies virus (PRV), also known as suid Alphaherpesvirus 1 (SuHV-1), which is one of the most devastating infectious pathogen of swine industry worldwide. Vaccination is the safest and most effective PRV prevention and control strategy. B cell receptor (BCR) is membrane-bound immunoglobulin located on the surface of B cells capable of specifically binding foreign antigens, which is one of the most important molecules regulating the proliferation and function of B cells. Here, to assess the molecular diversity of BCR H-CDR3 repertoire after different PRV strains infection, we detected the IGHV, IGHD, IGHJ genes usage and CDR3 sequence changes of mice spleen with PRV vaccine strain (Bartha-K61), variant strain (XJ) and mock infection by high-throughput sequencing. We found that PRV-infected groups shared partial BCR sequences, which are most likely to be PRV-specific BCR candidates. However, there were still differences in the IGHV genes usage as well as the combined usage of IGHV and IGHJ genes between the Bartha-K61 strain and XJ strain infection groups. In addition, the CDR3 sequences exhibited large differences in the types and lengths in PRV infection groups. Our study contributes to a better understanding of the host adaptive immune response to PRV infection and provides a theoretical basis for further research on novel and efficient PRV vaccines in the future.

The Genetic Characterization of a Novel Natural Recombinant Pseudorabies Virus in China.

We sequenced the complete genome of the pseudorabies virus (PRV) FJ epidemic strain, and we studied the characteristics and the differences compared with the classical Chinese strain and that of other countries. Third-generation sequencing and second-generation sequencing technology were used to construct, sequence, and annotate an efficient, accurate PRV library. The complete FJ genome was 143,703 bp, the G+C content was 73.67%, and it encoded a total of 70 genes. The genetic evolution of the complete genome and some key gene sequences of the FJ strain and PRV reference strains were analyzed by the maximum likelihood (ML) method of MEGA 7.0 software. According to the ML tree based on the full-length genome sequences, PRV FJ strain was assigned to the branch of genotype II, and it showed a close evolutionary relationship with PRV epidemic variants isolated in China after 2011. The gB, gC, gD, gH, gL, gM, gN, TK, gI, and PK genes of the FJ strain were assigned to the same branch with other Chinese epidemic mutants; its gG gene was assigned to the same branch with the classic Chinese Fa and Ea strains; and its gE gene was assigned to a relatively independent branch. Potential recombination events were predicted by the RDP4 software, which showed that the predicted recombination sites were between 1694 and 1936 bp, 101,113 and 102,660 bp, and 107,964 and 111,481 bp in the non-coding region. This result broke the previously reported general rule that pseudorabies virus recombination events occur in the gene coding region. The major backbone strain of the recombination event was HLJ8 and the minor backbone strain was Ea. Our results allowed us to track and to grasp the recent molecular epidemiological changes of PRV. They also provide background materials for the development of new PRV vaccines, and they lay a foundation for further study of PRV.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.

Isolation and Characterization of Two Pseudorabies Virus and Evaluation of Their Effects on Host Natural Immune Responses and Pathogenicity.

Pseudorabies, caused by the pseudorabies virus (PRV), is an acute fatal disease, which can infect rodents, mammals, and other livestock and wild animals across species. Recently, the emergence of PRV virulent isolates indicates a high risk of a variant PRV epidemic and the need for continuous surveillance. In this study, PRV-GD and PRV-JM, two fatal PRV variants, were isolated and their pathogenicity as well as their effects on host natural immune responses were assessed. PRV-GD and PRV-JM were genetically closest to PRV variants currently circulating in Heilongjiang (HLJ8) and Jiangxi (JX/CH/2016), which belong to genotype 2.2. Consistently, antisera from sows immunized with PRV-Ea classical vaccination showed much lower neutralization ability to PRV-GD and PRV-JM. However, the antisera from the pigs infected with PRV-JM had an extremely higher neutralization ability to PRV-TJ (as a positive control), PRV-GD and PRV-JM. In vivo, PRV-GD and PRV-JM infections caused 100% death in mice and piglets and induced extensive tissue damage, cell death, and inflammatory cytokine release. Our analysis of the emergence of PRV variants indicate that pigs immunized with the classical PRV vaccine are incapable of providing sufficient protection against these PRV isolates, and there is a risk of continuous evolution and virulence enhancement. Efforts are still needed to conduct epidemiological monitoring for the PRV and to develop novel vaccines against this emerging and reemerging infectious disease.

A noval strategy of deletion in PK gene for construction of a vaccine candidate with exellent safety and complete protection efficiency against high virulent Chinese pseudorabies virus variant.

A variant of pseudorabies virus (PRV) with enhanced pathogenicity have emerged in many vaccinated swine herds in China since 2011. PRVΔTK&gE-AH02, a previously described TK/gE deletion PRV strain arising from the PRV variant AH02LA, has been shown to be safe for PRV antibody positive piglets, and could provide protection against emerging PRV variants. However, inoculation of PRVΔTK&gE-AH02 into PRV antibody negative neonatal piglets caused lethal infection. In the study, in order to attenuate the virulence of PRVΔTK&gE-AH02, an additional deletion of 1∼x223C13 bp of US3 (the serine/threonine kinase, PK) gene was performed to generate a TK/PK/gE deletion PRV variant (PRVΔTK&PK&gE-AH02). We found that the growth kinetics of PRVΔTK&PK&gE-AH02 was similar to that of PRVΔTK&gE-AH02. Mice inoculated with PRVΔTK&PK&gE-AH02 in different dose (104.0∼x223C107.0 TCID50) survived and showed no observable clinical symptoms. No virus was detected in the brains or lungs of the mice inoculated with PRVΔTK&PK&gE-AH02. Moreover, mice inoculated with PRVΔTK&PK&gE-AH02 and PRVΔTK&gE-AH02 showed similar survival against virulent PRV AH02LA strain. Importantly, safety test showed no clinical symptoms in PRV antibody negative neonatal piglets that were intranasally inoculated with PRVΔTK&PK&gE-AH02 at a dose of 106.5 TCID50, indicating that the virulence of PRVΔTK&PK&gE-AH02 was significantly mitigated. Piglets immunized with PRVΔTK&PK&gE-AH02 exhibited a high serum neutralization index. All piglets inoculated intramuscularly (I.M.) with 1 mL (105.0 TCID50) PRVΔTK&PK&gE-AH02 were completely protected against challenge intranasally (I.N.) with 2LD50 (106.5TCID50) PRV AH02LA strain. In summary, our results indicate that deletion of 1∼x223C13 bp of US3 (PK) can provide a useful way for further attenuation of PRV and the PRVΔTK&PK&gE-AH02 might be a promising vaccine candidate for controlling of the virulent PRV variants in China.

Effective cross-protection of a lyophilized live gE/gI/TK-deleted pseudorabies virus (PRV) vaccine against classical and variant PRV challenges.

Classical Bartha-K61 strains could not provide complete protection against the emerging highly virulent pseudorabies virus (PRV) variant strains, which has caused great economic losses to swine industry in China. In this study, a gE/gI/TK-deleted PRV vaccine strain based on a circulating PRV variant strain HeB12 was generated by serial passages in Vero cells and a lyophilized formulation was prepared as a live-attenuated PRV vaccine. Compared to commercial Bartha-K61 strains, vaccine efficacy in vivo experiments showed that the novel triple gene-deleted variant vaccine could provide complete cross-protection against the lethal challenges by the classical RA strain and variant TJ12 strain, indicating that it could be a better alternative product than the currently used Bartha-K61 strains for the control and eradication of epidemic pseudorabies in China.

A natural product, (S)-10-Hydroxycamptothecin inhibits pseudorabies virus proliferation through DNA damage dependent antiviral innate immunity.

Pseudorabies virus (PRV), a member of the subfamily alphaherpesvirinae, is one of the most important pathogenes that cause acute death in infected pigs and leads to substantial economic losses in the global swine industry. Recently, China's emerging PRV mutant strains resulted in the traditionally commercial vaccines not providing complete protection. Some studies reported that PRV could infect humans and cause endophthalmitis and encephalitis under certain circumstances. It is necessary to develop alternative manners to control the virus infection. Here, by screening a library of natural products, (S)-10-Hydroxycamptothecin (10-HCPT) was revealed to inhibit PRV replication with a selective index of 270.04. And 10-HCPT inhibited PRV replication by blocking the viral genome replication but not inhibiting the viral attachment, internalization, and release. RNA interference assay showed that 10-HCPT inhibited PRV replication by targeting DNA topoisomerase 1 (TOP1). Meanwhile, 10-HCPT treatment induced DNA damage response and stimulated antiviral innate immunity. Animal challenge experiments showed that 10-HCPT effectively alleviated clinical signs and hispathology, and increased INF-ß responses in lung and brain tissues of mice induced by PRV infection. The results demonstrate that 10-HCPT is a promising therapeutic agent to control PRV infection.

Detection of pseudorabies virus with a real-time recombinase-aided amplification assay.

Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV), which has caused great economic losses to the pig industry worldwide. Reliable and timely diagnose is crucial for the surveillance, control and eradication of PR. Here, a real-time fluorescent recombinase-aided amplification (real-time RAA) assay was established to detect PRV. Primers and probes were designed based on the conserved regions of the PRV gE gene. The assay was specific for the detection of wild-type PRV, showing no cross-reactivity with other important porcine viruses (including PRV gE-deleted vaccine strains). Analytical sensitivity of the assay was three 50% tissue culture infectious doses (TCID50 ) of PRV DNA per reaction with 95% reliability, which is comparable to that of a PRV-specific real-time PCR (qPCR) assay. In diagnosis of 206 clinical tissue samples, the diagnose accordance rate between the real-time RAA assay and qPCR assay was 97.57% (201/206). Interestingly, the amplified products of real-time RAA could be visualized under a portable blue light instrument, making it possible for the rapid detection of PRV in resource-limited settings and on-site screening. Therefore, our developed real-time RAA assay is a diagnostic method for the rapid detection of PRV in the field.

Quercetin as an antiviral agent inhibits the Pseudorabies virus in vitro and in vivo.

Pseudorabies virus (PRV) accounts for a critical swine disease incurring economic losses worldwide. Several PRV vaccines are commercially available but these vaccines are effective against only certain prevalent PRV strains in China. To prevent PRV-induced latent infection and decrease the pathogenicity, novel anti-PRV drugs are required to prevent PRV infection. Natural products show exceptional structural diversity representing an important source for developing novel therapeutic agents. Quercetin is a flavonoid with anti-oxidant, anti-cancer, anti-bacterial and anti-viral activities. This study involved quercetin for studying the anti-PRV function in vitro and in vivo. Quercetin was found to significantly decrease the PRV virulent strain HNX at a half-maximal inhibitory concentration (IC50) of 2.618 µM and selectivity index 229. This anti-PRV activity of quercetin was found to be dose-dependent. Furthermore, quercetin also inhibited a wide the infections by a spectrum of PRV strains like HNX, Ea, Bartha and Fa strain. These virucidal effects of quercetin suggest the interaction between these molecules and viral particles, and quercetin is responsible for inhibiting the adsorption of PRV infections. The silico assays suggesting that quercetin might interact with the gD-protein on the surface of the PRV important for viral infection. Additional, the quercetin plantar injection protected the mice from the lethal challenge, decreasing the PRV-infected mice's brain viral loads and mortality. These results provides a anti-PRV strategy and contribute to drug discovery and development.

Pathogenicity and immunogenicity of a gI/gE/TK/UL13-gene-deleted variant pseudorabies virus strain in swine.

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells.

Owing to viral evolution and recombination, emerging pseudorabies virus (PRV) strains have caused unprecedented outbreaks in swine farms even when the pigs were previously vaccinated, which might indicate that traditional vaccines were unable to provide effective protection. The development of safe and efficacious vaccines presents prospects to minimize the clinical signs and eventually eradicate the infection. In this study, we used an emerging PRV strain, HNX, as the parental strain to construct a recombinant PRV with TK/gE gene deletion and Fms-related tyrosine kinase 3 ligand (Flt3L) expression, named HNX-TK-/gE--Flt3L. HNX-TK-/gE--Flt3L enhanced the maturation of bone marrow derived dendritic cells (DCs) in vitro. Significantly more activated DCs were detected in HNX-TK-/gE--Flt3L-immunized mice compared with those immunized with HNX-TK-/gE-. Subsequently, a remarkable increase of neutralizing antibodies, gB-specific IgG antibodies, and interferon-gamma (IFN-γ) was observed in mice vaccinated with HNX-TK-/gE--Flt3L. In addition, a lower mortality and less histopathological damage were observed in HNX-TK-/gE--Flt3L vaccinated mice with upon PRV lethal challenge infection. Taken together, our results revealed the potential of Flt3L as an ideal adjuvant that can activate DCs and enhance protective immune responses and support the further evaluation of HNX-TK-/gE--Flt3L as a promising PRV vaccine candidate.