Evaluation of immunogenicity of gene-deleted and subunit vaccines constructed against the emerging pseudorabies virus variants.

BACKGROUND: Pseudorabies (PR) (also called Aujeszky's disease, AD) is a serious infectious disease affecting pigs and other animals worldwide. The emergence of variant strains of pseudorabies virus (PRV) since 2011 has led to PR outbreaks in China and a vaccine that antigenically more closely matches these PRV variants could represent an added value to control these infections. METHODS: The objective of this study was to develop new live attenuated and subunit vaccines against PRV variant strains. Genomic alterations of vaccine strains were based on the highly virulent SD-2017 mutant strain and gene-deleted strains SD-2017ΔgE/gI and SD-2017ΔgE/gI/TK, which constructed using homologous recombination technology. PRV gB-DCpep (Dendritic cells targeting peptide) and PorB (the outer membrane pore proteins of N. meningitidis) proteins containing gp67 protein secretion signal peptide were expressed using the baculovirus system for the preparation of subunit vaccines. We used experimental animal rabbits to test immunogenicity to evaluate the effect of the newly constructed PR vaccines. RESULTS: Compared with the PRV-gB subunit vaccine and SD-2017ΔgE/gI inactivated vaccines, rabbits (n = 10) that were intramuscularly vaccinated with SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine showed significantly higher anti-PRV-specific antibodies as well as neutralizing antibodies and IFN-γ levels in serum. In addition, the SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine protected (90-100%) rabbits against homologous infection by the PRV variant strain. No obvious pathological damage was observed in these vaccinated rabbits. CONCLUSIONS: The SD-2017ΔgE/gI/TK live attenuated vaccine provided 100% protection against PRV variant challenge. Interestingly, the subunit vaccines with gB protein linked to DCpep and PorB protein as adjuvant may also be a promising and effective PRV variant vaccine candidate.

Comparative proteomic analysis of PK15 swine kidney cells infected with a pseudorabies pathogenic variant and the Bartha-K/61 vaccine strain.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease and is communicable across species. In particular, the emergence of PRV variants in 2011 have resulted in serious economic losses to the Chinese pig industry. In this study, we used tandem mass tag (TMT) quantitative protein analysis to identify differentially expressed proteins between the PRV variant strain SD-2017 and the vaccine strain Bartha-K/61 in the swine kidney cell line PK15. Overall, we identified 4690 proteins for SD-2017 infection compared with the mock-infected control cells. We found 162 differentially expressed cellular proteins including 41 up- and 121 down-regulated proteins. SD-2017-infected PK15 cells differential proteins were primarily related to gap junctions, the phagosome, antigen processing and presentation, cell adhesion molecules and peroxisome pathways. Compared to Bartha-K/61-infected PK15 cells, SD-2017-infected cells displayed differentially expressed proteins involved in tryptophan metabolism, mitophagy and Notch signaling. Western blot analysis of MARK2, TSR1 and TMED1 three representative proteins validated the reliability of the TMT data. This study is an initial at-tempt to compare the proteomes of PK15 cells infected by a PRV variant and a vaccine strain using TMT technology to provide new insights into the mechanisms of PRV pathogenesis and immune evasion.

Analysis of laboratory and serological test results in patients with acute brucellosis during follow-up.

BACKGROUND: The laboratory test results and serum-specific antibodies of patients with acute brucellosis initial infection were followed up and analyzed. METHODS: 70 patients in Hohhot City, Inner Mongolia Autonomous Region, with acute brucellosis were followed up for 360 days. Serum samples were collected at 0, 15, 30, 60, 90, 180, and 360 days after diagnosis and analyzed by Rose Bengal plate test (RBPT), colloidal gold test paper (GICA), and test tube agglutination test (SAT). The serum-specific antibodies IgG and IgM were detected. RESULTS: RBPT results: False negative (-) gradually increased with the extension of the course of disease, with the largest change in 30-60 days after diagnosis, and the constituent ratio increased by 12.9%. GICA results: The false negative increased with the course of disease, and the constituent ratio of false negative was 20.0% after 180 days of diagnosis. SAT results: 1:100 positive showed a ladder like decrease with the increase in the course of disease, and the largest decrease was 90-180 days, with a decrease of 34.3% in the constituent ratio. 360 days after diagnosis, the constituent ratio of positive was only 14.3%. During the follow-up period, the IgG average value fluctuated and the average IgM value decreased. CONCLUSION: The false-negative results of RBPT, GICA, and SAT increased with the course of disease, and the false-negative rates were higher than 20% after half a year. IgM level is beneficial to the early diagnosis of brucellosis, while IgG level is helpful to the judgment of brucellosis stage.

Genetic analysis of porcine circovirus type 2 from dead minks.

Circovirus infection is a growing problem in the field of veterinary and public health. It is associated with enteric diseases in both mammalian and avian hosts. In this study, we detected and isolated porcine circovirus strains in the tissue samples of minks that died from diarrhoea in Shandong Province, China. We sequenced the whole genome of two porcine strains of Circovirus, designated as MiSD-1 and MiSD-2， which had a 97.34% similarity on nucleotide sequence and were closely related to porcine circovirus type 2 (PCV2), but distantly related to mink circoviral species. Phylogenetically MiSD-1 and MiSD-2 are a part of the PCV2b genotype cluster, which is a highly prevalent genotype worldwide. The closer relationship of MiSD-1 and MiSD-2 to PCV2 from pigs than to other mink circoviral species may be evidence of cross-species transmission and considerable zoonotic potential.

Pathological and immunological characteristics of piglets infected experimentally with a HP-PRRSV TJ strain.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to swine industry all over the world. The aim of this study was to investigate the mechanism of pathogenesis and immune responses caused by a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). RESULTS: All piglets experimentally infected with a HP-PRRSV TJ strain virus developed typical clinical signs of PRRS. The percentages of CD3+, CD4+, and CD8+ lymphocytes significantly decreased in the infected group as compared to the uninfected control animals (p < 0.01). Total WBC dropped in the infected animals during the experiment. The level of ELISA antibody against PRRSV increased in 7-10 days after infection and then started to decline. Pathological observations demonstrated various degree lesions, bleeding and necrosis in the lungs of the infected piglets. CONCLUSIONS: These results clearly indicated that HP-PRRSV TJ strain infection would activate host humoral immune response at the early period post infection and cause severe pathological damages on lungs and inhibit cellular immune response after infection.

Effect of Nonstructural Protein 2 Hypervariable Regions in the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Marc-145 Cells.

BACKGROUND: Highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) causes prolonged high fever, red discoloration of the body, blue ears and a high mortality. Previously, we found that the PRRSV vaccine strain TJM contained a deletion of 120 amino acids (aa 628-747) in nonstructural protein 2 (Nsp2). We aimed to explore the replication features of PRRSV after adding the transiently expressed product of these 120 aa in vitro. METHODS: We constructed seven eukaryotic expression plasmids containing different parts of the 120-aa sequence, transfected them into Marc-145 cells and then inoculated the cells with 103 TCID50 TJM per well. We detected virus replication at mRNA and protein level by real-time RT-PCR and Western blotting, respectively, and determined the virus titer. RESULTS: The transiently expressed 120 aa and one of its truncated polypeptides inhibited PRRSV TJM propagation on Marc-145 cells. The complete 120-aa sequence induced a remarkable decrease in PRRSV replication, causing a reduction in structural protein levels between 36 and 48 h after infection. Additionally, aa 628-727 partly reduced the replication of PRRSV on Marc-145 cells. CONCLUSIONS: The 120 aa from Nsp2, especially aa 628-727, play a negative role in PRRSV TJM proliferation.

Novel amdoparvovirus infecting farmed raccoon dogs and arctic foxes.

A new amdoparvovirus, named raccoon dog and fox amdoparvovirus (RFAV), was identified in farmed sick raccoon dogs and arctic foxes. Phylogenetic analyses showed that RFAV belongs to a new species within the genus Amdoparvovirus of the family Parvoviridae. An RFAV strain was isolated in Crandell feline kidney cell culture.

Complete genome sequence and pathogenesis of bovine viral diarrhea virus JL-1 isolate from cattle in China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a pathogen found worldwide in calves. It can cause significant economic losses in agriculture. Many BVDV strains have been isolated in China. However, the pathogenesis and complete gene characteristics of BVDV isolate have yet not been reported in China. Here, a BVDV isolate was isolated and its pathogenesis and complete genome were studied. RESULTS: A new isolate of bovine viral diarrhea virus, named JL-1, was isolated from the spleen of a sick cow with diarrhea using MDBK cell culture. The complete genome of JL-1 is 12,276 nucleotides and contains a 5'-UTR of 382 nucleotides, a 3'-UTR of 188 nucleotides, and a large ORF encoding a polyprotein consisting of 3,901 amino acids. Genomic comparison and phylogenetic analyses of complete genomic sequence clearly showed that JL-1 fell into the BVDV-1b subtype. The result of pathogenesis of JL-1 strain showed that all infected calves developed clinical signs of elevated rectal temperatures, decreased leucopenia, and viral discharge. Viral antigen was detected in infected animal tissues using immunohistochemistry. Animals in the mock were normal. These results demonstrated that BVDV JL-1 was a virulent strain. CONCLUSIONS: This is the first study to report the pathogenesis and complete gene characterization of the BVDV strain in China. This report may set a good foundation for further study of BVDV in China.

Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

Evolution Characterization and Pathogenicity of an NADC34-like PRRSV Isolated from Inner Mongolia, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that causes severe abortions in sows and high piglet mortality, resulting in huge economic losses to the pig industry worldwide. The emerging and novel PRRSV isolates are clinically and biologically important, as there are likely recombination and pathogenic differences among PRRSV genomes. Furthermore, the NADC34-like strain has become a major epidemic strain in some parts of China, but the characterization and pathogenicity of the latest strain in Inner Mongolia have not been reported in detail. In this study, an NADC34-like strain (CHNMGKL1-2304) from Tongliao City, Inner Mongolia was successfully isolated and characterized, and confirmed the pathogenicity in pigs. The phylogenetic tree showed that this strain belonged to sublineage 1.5 and had high homology with the strain JS2021NADC34. There is no recombination between CHNMGKL1-2304 and any other domestic strains. Animal experiments show that the CHNMGKL1-2304 strain is moderately virulent to piglets, which show persistent fever, weight loss and high morbidity but no mortality. The presence of PRRSV nucleic acids was detected in both blood, tissues, nasal and fecal swabs. In addition, obvious pathological changes and positive signals were observed in lung, lymph node, liver and spleen tissues when subjected to hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). This report can provide a basis for epidemiological investigations and subsequent studies of PRRSV.

Complete genome sequence of a mink calicivirus in China.

We report the complete genome sequence of a novel calicivirus isolated from a diseased mink in China. The complete viral genome is approximately 8.4 kb in length and consists of three open reading frames. The availability of the complete genome sequence is helpful for further investigation into the molecular characteristics and epidemiology of calicivirus in mink.

Complete genome sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus NM1 strain from northern China.

NM1 is a highly pathogenic North American-type porcine reproductive and respiratory syndrome virus (PRRSV). The complete genome sequence shows that NM1 shares high sequence identity (99.2 to 99.4%) to other HP-PRRSV isolates, containing two discontinuous deletions, a 1-amino-acid deletion at position 481 and a 29-amino-acid deletion at positions 533 to 651, in nonstructural protein 2.

Glycosylated protein 4-deficient PRRSV in complementing cell line shows low virus titer.

Porcine Reproductive and Respiratory Syndrome (PRRS) threats the swine industry seriously. The spread of live vaccine virus leads to the emergence of recombinant virus, which brings biosafety problems. The replication-deficient virus as a vaccine candidate would avoid this problem. In the present study, the recombinant lentiviral plasmid pLV-EF1α-EGFP-2A-ORF4 was co-transfected with lentivirus in HEK293FT cells. The transfection mixture was harvested and transduced into Marc-145 to screen a cell line stably expressing the PRRSV ORF4 with puromycin. The cell line Marc-145-GP4 was confirmed with PCR, RT-PCR, IFA, and Western blotting using a monoclonal antibody against Glycoprotein 4 (GP4) of PRRSV. To obtain a replication-deficient PRRSV, Western blotting the recombinant plasmid pNM09-ΔORF4 was constructed by Overlap PCR and DNA recombinant technology with the pNM09 as a backbone plasmid. The pNM09-ΔORF4 was transfected into Marc-145-GP4 with electroporation after transcription in vitro. The replication-deficient virus was rescued on Marc-145-GP4 cells with trans-complementation of ORF4 gene and verified by RT-PCR and IFA. The results indicated that a cell line Marc-145-GP4 stably expressed PRRSV ORF4 was obtained. The recombinant GP4 was successfully expressed and obtained a monoclonal antibody Anti-A-GP4-70, which can specifically react with the virus. Finally, the replication-deficient virus rNM09-ΔORF4 can be rescued with low titer and could only reproduce on the Marc-145-GP4 cells. Unfortunately, the rNM09-ΔORF4 showed too low virus replication titer to determine it. This study lays the foundation for the rapid detection of PRRS and the functional study of GP4 and provides experience for replication-deficient PRRSV.

A follow-up study of 100 patients with acute brucellosis for its prognosis and prevention.

Objective: To prevent chronic brucellosis, this study analysed the changes in patient antibody titers, and the trajectories of biochemical indicators at different stages of brucellosis, identified relevant biomarkers, and explored risk factors affecting the prognosis of brucellosis patients. Methods: A prospective cohort study was conducted to follow 100 patients with acute brucellosis. Laboratory serological test results [taken with a serum (tube) agglutination test (SAT)] and biochemical parameters (liver function, renal function, and hematological system) were measured repeatedly at four-time points: 0 weeks-baseline survey, 6 weeks after the first treatment, 12 weeks after the second treatment, and 3 months after the third treatment. The changes in the antibody titres and biochemical parameters at each time point were analysed for trend changes. Results: One hundred patients with acute brucellosis were enrolled in this follow-up study, with 100% retention in follow-up. By the third follow-up, 21 patients had turned subacute and 11 had turned chronic. One-way repeated measures analysis of variance results showed statistically significant differences (p < 0.01) across the time points for the following five indicators: alanine aminotransferase, aspartate aminotransferase, total bilirubin, serum creatinine (SCr) and platelet count. The clinical symptoms of patients in the acute stage were mainly joint pain, fatigue, and fever, while those in the chronic stage complained primarily of joint pain and fatigue. The results of multivariate logistic analysis showed that joint pain [odds ratio (OR) = 3.652, 95% confidence interval (CI) =1.379-9.672], monoarticular pain (OR = 6.356, 95% CI = 4.660-8.669), elevated SCr (OR = 15.804, 95% CI = 1.644-151.966) and elevated haemoglobin (Hb) (OR = 1.219, 95% CI = 1.065-1.736) were risk factors for poor prognosis (not cured or chronic) in patients with brucellosis. Conclusion: The trajectory of changes in patient SAT posirates and antibody titers can be used to distinguish patients with chronic brucellosis. The brucellosis is preventable and treatable, and the standard treatment can be effective in reducing the clinical symptoms of affected patients. If patients are not treated in a timely manner, joint pain, monoarticular pain, and elevated SCr are risk factors for patients who are not cured. Therefore, the treatment cycle for these patients should be extended.

Rabies virus expressing dendritic cell-activating molecules enhances the innate and adaptive immune response to vaccination.

Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 10(7) fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.

Phylogenetic analysis of the bovine parainfluenza virus type 3 from cattle herds revealing the existence of a genotype A strain in China.

In 2009, a bovine parainfluenza virus (BPIV3), named as NM09, was isolated using MDBK cell culture from the nasal swabs of normal cattle in China. The NM09 isolate was characterized by RT-PCR and nucleotide sequence analysis. Its complete genome was 15,456 nucleotides in length. Similar to other sequenced PIV strains, the NM09 virus consisted of six non-overlapping genes, which were predicted to encode nine proteins with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and trinucleotide intergenic sequences. Nucleotide phylogenetic analysis of matrix and hemagglutinin-neuraminidase gene demonstrated that this NM09 isolate belonged to BPIV3 genotype A instead of the previously reported BPIV3 genotype C in China. It is implicated that the different genotypes A and C might coexist infection for a long time in China.

Subunit vaccine based on glycoprotein B protects pattern animal guinea pigs from tissue damage caused by infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.

Recombinant Bovine Herpesvirus Type I Expressing the Bovine Viral Diarrhea Virus E2 Protein Could Effectively Prevent Infection by Two Viruses.

Bovine respiratory disease complex (BRDC) is a comprehensive disease in cattle caused by various viral and bacterial infections. Among them, bovine herpesvirus type I (BoHV-1) and bovine viral diarrhea virus (BVDV) play important roles and have caused huge financial losses for the cattle industry worldwide. At present, vaccines against BRDC include trivalent attenuated BoHV-1, BVDV-1, and BVDV-2 live vaccines, BoHV-1 live attenuated vaccines, and BoHV-1/BVDV bivalent live attenuated vaccines, which have limitations in terms of their safety and efficacy. To solve these problems, we optimized the codon of the BVDV-1 E2 gene, added the signal peptide sequence of the BoHV-1 gD gene, expressed double BVDV-1 E2 glycoproteins in tandem at the BoHV-1 gE gene site, and constructed a BoHV-1 genetics-engineered vectored vaccine with gE gene deletion, named BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE. This study compared the protective effects in BoHV-1, BoHV-1 ΔgE, BoHV-1 gE/E2-Linker-E2+, and BVDV-1 inactivated antigen immunized guinea pigs and calves. The results showed that BoHV-1 gE/E2-Linker-E2+ could successfully induce guinea pigs and calves to produce specific neutralizing antibodies against BVDV-1. In addition, after BoHV-1 and BVDV-1 challenges, BoHV-1 gE/E2-Linker-E2+ can produce a specific neutralizing antibody response against BoHV-1 and BVDV-1 infections. Calves immunized with this type of virus can be distinguished as either vaccinated animals (gE-) or naturally infected animals (gE+). In summary, our data suggest that BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE have great potential to prevent BVDV-1 or BoHV-1 infection.

Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing.

Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5' and 3' ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3' end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP+ or EGFP- will lay the foundation for research on BoHV-1 and vaccine development in the future.

Recent advances in the study of NADC34-like porcine reproductive and respiratory syndrome virus in China.

Since porcine reproductive and respiratory syndrome virus (PRRSV) was first described in China in 1996, several genetically distinct strains of PRRSV have emerged with varying pathogenicity and severity, thereby making the prevention and control of PRRS more difficult in China and worldwide. Between 2017 and 2021, the detection rate of NADC34-like strain in China increased. To date, NADC34-like strains have spread to 10 Chinese provinces and have thus developed different degrees of pathogenicity and mortality. In this review, we summarize the history of NADC34-like strains in China and clarify the prevalence, genomic characteristics, restriction fragment length polymorphisms, recombination, pathogenicity, and vaccine status of this strain in China. In so doing, this study aims to provide a basis for the further development of prevention and control measures targeting the NADC34-like strain.