Bartha-K61 Vaccine Research Articles

Emergence and etiological characteristics of novel genotype Ⅱ pseudorabies virus variant with high pathogenicity in Tianjin, China

Pseudorabies (PR) is a highly infectious disease caused by pseudorabies virus (PRV). This study aimed to detect and identify recent outbreaks of genotype Ⅱ PRV, and further analysis it's etiological characteristics and pathogenicity. The brain tissues with suspected PRV infection were isolated and the main virulence-related genes of the isolated PRV strain were amplified and sequenced for phylogenetic analysis. In addition, the pathogenicity of the isolated PRV strain to 6-week-old mice and 9-days-old suckling piglets were evaluated. The results showed that a PRV strain was successfully isolated and named PRV TJbd2023 strain, which could proliferate in PK-15 cells and TCID50 of the 6th generation virus reached 107.57/0.1 ml. Phylogenetic trees and amino acids analysis were constructed based on full-length gE sequences, which showed that PRV TJbd2023 strain was clustered into genetype Ⅱ PRV variant with a characteristic 21-nucleotide insertion (encoding 63AASTPAA69) in gC gene, and some amino acid point mutations were also found in other virulence-related genes, including gB protein R223H and E836K, gD protein R320S, and gE protein T242A. Animal experiments showed that TJbd2023 could cause acute neurological symptoms with 103.41/mL LD50 on KM mice, and intranasal inoculation of suckling piglets with 2 ml of TJbd2023 strain(106.57/0.1 ml) led to a mortality rate of 66.70 %. Emerging genotype Ⅱ PRV variant such as isolated in our research named TJbd2023 with high pathogenicity might be responsible for recent outbreaks of PRV and immunization failure of Bartha-K61 vaccine in Tianjin, China.

Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Characterization of Pseudorabies Virus Associated with Severe Respiratory and Neuronal Signs in Old Pigs

Pseudorabies virus (PRV) represents a leading threat to the global pig industry. Generally, pigs exhibit a pronounced age resistance against PRV, and the virus generally does not cause severe clinical signs and even death in old pigs. However, we characterized two PRV strains (HeN21 and HuB20) associated with severe respiratory and neuronal signs in old pigs. Among these two strains, HeN21 was isolated from the tonsil of a 24-week-old pig that died from severe neuronal and respiratory signs in a PRV-outbreak farm where a commercial PRV attenuated vaccine developed based on a PRV variant was used; while, HuB20 was isolated from the lung and lymph node of a 20-week-old with symptoms in another farm where Bartha-K61 vaccine was used. In vitro evaluations in different cell models demonstrated that HeN21 and HuB20 led to similar cytotoxic effects to those caused by PRV variants on PK-15, Vero, and SK-N-SH cells after 30 hours of inoculation. However, HeN21 possessed a higher titer than the other PRV variants from the first to the fifth passage on PK-15 cells and induced plaques with larger size. In vivo assessments in mouse and fattening pig models showed that inoculations of HeN21 and HuB20 caused higher morbidity and mortality and severe pathological damages in tested animals. In particular, challenge of HeN21 led to severe respiratory and neuronal signs in 90-day-old pigs. These two strains displayed higher virus loads on the main organs of challenged mice and pigs. Phylogenetic analysis revealed that HeN21 and HuB20 belonged to genotype II. In addition, recombinant events were identified in the genomes of HeN21 and HuB20, and several events were located within genes associated with PRV virulence. Our data herein may suggest the emergence of novel PRV strains in China.

The Immune Efficacy of Inactivated Pseudorabies Vaccine Prepared from FJ-2012ΔgE/gI Strain.

An emerging pseudorabies virus (PRV) variant has been reported on Bartha-K61-vaccinated farms since 2011, causing great economic losses to China’s swine-feeding industry. In this study, two vaccines, FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG, were administered to piglets for immune efficacy investigation. Humoral immunity response, clinical signs, survival rate, tissue viral load, and pathology were assessed in piglets. The results showed that both vaccines were effective against the PRV FJ-2012 challenge, the piglets all survived while developing a high level of gB-specific antibody and neutralizing antibody, the virus load in tissue was alleviated, and no clinical PR signs or pathological lesions were displayed. In the unimmunized challenged group, typical clinical signs of pseudorabies were observed, and the piglets all died at 7 days post-challenge. Compared with commercial vaccines, the Bartha-K61 vaccine group could not provide full protection, which might be due to a lower vaccine dose; the inactivated vaccine vPRV* group piglets survived, displaying mild clinical signs. The asterisk denotes inactivation. These results indicate that FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG were effective and could be promising vaccines to control or eradicate the new PRV epidemic in China.

Protective efficacy of intranasal inactivated pseudorabies vaccine is improved by combination adjuvant in mice.

Pseudorabies virus (PRV) not only causes great economic loss to the pig industry but also seriously threatens the biosafety of other mammals, including humans. Since 2011, PRV mutant strains have emerged widely in China, and the classical Bartha-K61 vaccine cannot confer complete protection for pigs. PRV mainly infects pigs via the respiratory tract. Intranasal immunization with PRV has received more attention because intranasal vaccination elicits systemic and mucosal immune responses. To induce systemic and mucosal immune responses against PRV, we developed a combination adjuvant as a delivery system for intranasal vaccine, which was formulated with MONTANIDE™ Gel 01 and CVCVA5. In comparison to naked antigen of inactivated PRV, single Gel 01 adjuvanted inactivated antigen and single CVCVA5 adjuvanted inactivated antigen, intranasal inactivated PRV vaccine formulated with the combination adjuvant induced greater mucosal IgA immunity and serum antibody responses (IgG, IgG1, and IgG2a). Furthermore, the production of the Th1-type cytokine IFN-γ and the Th2-type cytokine IL-4 indicated that the cellular and humoral responses to the intranasal vaccine were improved by the combination adjuvant. In addition, the intranasal vaccine formulated with the combination adjuvant induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L–CD44+) memory subsets of both CD4 and CD8 T cells in nasal-associated lymphoid tissue. Intranasal challenge with virulent PRV in mice showed that the protective efficacy of the intranasal PRV vaccine was improved by the combination adjuvant compared with the other single-adjuvanted vaccines. In summary, these data demonstrated that Gel 01 combined with the CVCVA5 adjuvant induced a synergistic effect to improve mucosal immunity and protective efficacy of the intranasally inactivated PRV vaccine in mice. It represents a promising vaccination approach against PRV infection.

Serological Investigation and Genetic Characteristics of Pseudorabies Virus between 2019 and 2021 in Henan Province of China

In late 2011, severe pseudorabies (PR) outbreaks occurred among swine herds vaccinated with the Bartha-K61 vaccine in many provinces of China, causing enormous economic losses for the pork industry. To understand the epidemic profile and genetic characteristics of the pseudorabies virus (PRV), a total of 35,796 serum samples were collected from 1090 pig farms of different breeding scales between 2019 and 2021 in the Henan province where swine had been immunized with the Bartha-K61 vaccine, and PRV glycoprotein E (gE)-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA). The results reveal that the overall positive rate for PRV gE antibodies was 20.33% (7276/35,796), which decreased from 25.00% (2596/10,385) in 2019 to 16.69% (2222/13,315) in 2021, demonstrating that PR still existed widely in pig herds in the Henan province but displayed a decreasing trend. Further analysis suggested that the PRV-seropositive rate may be associated with farm size, farm category, quarter, region and the cross-regional transportation of livestock. Moreover, the gE gene complete sequences of 18 PRV isolates were obtained, and they shared a high identity (97.1–100.0%) with reference strains at the nucleotide level. Interestingly, the phylogenetic analysis based on the gE complete sequences found that there were both classical strains and variant strains in pig herds. The deduced amino acid sequence analysis of the gE gene showed that there were unique amino acids in the classical strains, the variant strains and genotype Ⅱ strains. This study provides epidemiological data that could be useful in the prevention of pseudorabies in Henan, China, and this finding contributed to our understanding of the epidemiology and evolution of PRV.

Same Dosages of rPRV/XJ5-gI−/gE−/TK− Prototype Vaccine or Bartha-K61 Vaccine Similarly Protects Growing Pigs Against Lethal Challenge of Emerging vPRV/XJ-5 Strain

Variant pseudorabies viruses (vPRV) have constantly emerged in China since late 2011. In the present study, a 1 × 106.0 TCID50 per-animal dosage of a commercially available Bartha-K61 vaccine and an rPRV/XJ5-gI−/gE−/TK− prototype vaccine freshly extracted from the vPRV/XJ-5 at the same dose were administered to evaluate the immune effectiveness thereof on growing pigs to prevent lethal strikes caused by vPRV/XJ-5. The results suggest that the Bartha-K61 vaccine at a dose of 1 × 106.0 TCID50 per animal and the same dosage of the rPRV/XJ5-gI−/gE−/TK− prototype vaccine protected growing pigs against the lethal challenge of vPRV/XJ-5 strain with 100% survive rate. Furthermore, the outcome of the clinical score, virus shedding, weight gain, and viral loads in different pig tissues in these two groups demonstrates that either the Bartha-K61 vaccine or the rPRV/XJ5-gI−/gE−/TK− prototype vaccine at the same dose exhibited parallel efficacy in pigs against the lethal challenge with the XJ-5 strain of vPRV.

Bartha-K61 vaccine protects nursery pigs against challenge with novel european and asian strains of suid herpesvirus 1

The present study investigates the pathogenicity of two recently isolated strains of Suid herpesvirus 1 (SuHV1), the Greek strain Hercules and the Chinese strain HeN1, in unvaccinated pigs and in pigs vaccinated with a Bartha-K61 strain. In an experiment performed in negative pressure kiosks (isolators), 45-day old seronegative pigs previously oronasally /intramuscularly vaccinated with the Bartha-K61 vaccine strain, along with unvaccinated controls, were challenged either with the Hercules strain or the HeN1 strain of SuHV1. All animals were observed daily for clinical signs and body temperature and nasal swabs, faeces, blood and bodyweight were collected up to a maximum period of 20 days post-challenge (dpc). The results showed that, in the unvaccinated pigs, HeN1 strain was more virulent than the Hercules strain, with increased mortality, shorter time to death and higher group clinical score (p < 0.05). However, after vaccination with the Bartha-K61 vaccine, there was a drastic reduction in morbidity, mortality, bodyweight loss and virus excretion to almost a similar extent in both strains (p < 0.05). No significant differences were seen among the pigs of the two vaccinated groups compared to unvaccinated unchallenged controls, except a slight elevation in body temperature and in clinical score in the HeN1 vaccinees at 2 and 3 dpc, while bodyweight gain was similar to that of the negative control pigs. Our study showed that despite differences in virulence, the standard vaccination scheme with the Bartha-K61 strain could equally protect nursery pigs against both the European and Chinese strains.

Assessment of an Aujeszky's Disease Control Strategy in a Highly Prevalent Pig Farm Based on Systematic Vaccination With an Inactivated gE-Negative Marker Vaccine.

Aujeszky's disease (AD) is endemic in Argentina. In 2016, an inactivated gE- negative marker Bartha K61 vaccine (AUSKIPRA® BK) was launched for use, making Argentina the only country to carry out a control strategy plan with it. In the present article, we describe the results of a control program in a farrow-to-finishing farm with high initial AD prevalence (33% in sows), based on the systematic vaccination, detection, and elimination of seropositive pigs, the replacement of sows with vaccinated gilts, and the instauration of artificial insemination. The program was suitable for diminishing the incidence and the prevalence at levels consistent with virus eradication. This situation has been sustained over time. This is the first report of AUSKIPRA® BK efficacy under field conditions.

Construction of a quadruple gene-deleted vaccine confers complete protective immunity against emerging PRV variant challenge in piglets

BackgroundPseudorabies virus (PRV) causes Aujeszky’s disease or pseudorabies (PR) in pigs worldwide, which leads to heavy economic losses to the swine industry. Pigs are the natural host, meanwhile, animals such as dogs, cats, foxes, rabbits, cattle and sheep are susceptible to infection. In 2011, the emerging PRV variant led to the outbreak of PR in Bartha-K61 vaccinated pigs. The PR outbreaks demonstrated that the Bartha-K61 vaccine did not provide full protection against the emerging PRV variant. It is widely believed that PRV live attenuated vaccine could control PRV infection.MethodsIn this study, we developed a novel PRV live attenuated vaccine by deleting its gI, gE, US9, and US2 genes through CRISPR/Cas9, which was named PRV GDFS-delgI/gE/US9/US2.ResultsSafety experiments confirmed that PRV GDFS-delgI/gE/US9/US2 was safe for 5- to 7-day-old suckling piglets. Piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine did not produce PRV gE-specific antibodies but could generate PRV gB-specific antibodies and high neutralizing titers against the PRV GDFS strain (variant PRV strain) or PRV Ea strain (older PRV strain). After challenge with the emerging PRV GDFS variant, none of the piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine showed any clinical signs, and their rectal temperatures were normal. Moreover, the autopsy and histopathological analyses revealed that the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine group did not show apparent gross or pathological lesions. Furthermore, the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine groups did not present weight loss. According to the criteria of the OIE terrestrial manual, the results of the experiment confirmed that the PRV GDFS-delgI/gE/US9/US2 vaccine could provide full protection against the emerging PRV variant strain in piglets.ConclusionsThe PRV GDFS-delgI/gE/US9/US2 strain is a potential new live attenuated vaccine against emerging PRV variant strain infections in China.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha K61: A Brief Review on the Knowledge Gathered During 60 Years of Research.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily of the herpesviruses and is the causative agent of Aujeszky’s disease in pigs, causing respiratory, neurological, and reproductive symptoms. Given the heavy economic losses associated with Aujeszky’s disease epidemics, great efforts were made to develop efficacious vaccines. One of the best modified live vaccines to this day is the attenuated Bartha K61 strain. The use of this vaccine in extensive vaccination programs worldwide has assisted considerably in the eradication of PRV from the domesticated pig population in numerous countries. The Bartha K61 strain was described in 1961 by Adorján Bartha in Budapest and was obtained by serial passaging in different cell cultures. Ever since, it has been intensively studied by several research groups, for example, to explore its efficacy as a vaccine strain, to molecularly and mechanistically explain its attenuation, and to use it as a retrograde neuronal tracer and as a vector vaccine. Given that the Bartha K61 vaccine strain celebrates its 60th birthday in 2021 with no sign of retirement, this review provides a short summary of the knowledge on its origin, characteristics, and use as a molecular tool and as a vaccine.

Glycoproteins C and D of PRV Strain HB1201 Contribute Individually to the Escape From Bartha-K61 Vaccine-Induced Immunity.

The newly emerged pseudorabies virus (PRV) novel variants can escape from the immunity induced by the classical vaccine Bartha-K61. Here we investigated the underlying mechanisms by constructing chimeric mutants between epidemic strain HB1201 and the Bartha-K61 vaccine. Our analyses focused on three viral envelope glycoproteins, namely gB, gC, and gD, as they exhibit remarkable genetic variations and are also involved in induction of protective immunity. The corresponding genes were swapped reciprocally either individually or in combination by using CRISPR/Cas9 technology and homologous recombination. The rescued chimeric viruses exhibited differential sensitivity to neutralizing antibodies in vitro, and gC was found to be the major contributor to inefficient neutralization against HB1201 by anti-Bartha-K61 serum. When tested in the 4-week-piglet model, substitution with HB1201 gC enabled Bartha-K61 to induce a protective immunity against HB1201 at a high challenge dose of 107 TCID50. Interestingly, despite a relatively lower cross-neutralization ability, the gD exchange also enabled Bartha-K61 to protect piglets from lethal challenge. In both cases, clinical signs and microscopic lesions were eased, and so was the viral tissue load with the exception of brain. A better protection could be achieved when both gC and gD were swapped in terms of reducing viral load in brain and virus-induced microscopic lesions. Thus, our studies not only revealed individual roles of gC and gD variations in the immune escape and also suggested a synergistic effect of both proteins on induction of protective immunity. These findings have important implications in novel vaccine development for PRV control in China.

Efficacy of the Bartha-K61 vaccine and a gE−/gI−/TK− prototype vaccine against variant porcine pseudorabies virus (vPRV) in piglets with sublethal challenge of vPRV

Pseudorabies has caused huge economic losses in China's pig industry and recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Therefore, the prevention and control of this recurrence of pseudorabies in China has been given priority. In a previous study, we showed that a suitable dose [1 × 106.3 50% tissue culture infectious dose (TCID50) per animal] of commercial Bartha-K61 vaccine protects growing pigs against lethal challenge by the emerging vPRV strain XJ5. In this study, different doses of the Bartha-K61 vaccine and our newly developed rPRV-gI−/gE−/TK− prototype vaccine derived from the vPRV strain XJ5 were used to evaluate immune protection against sublethal challenge by the vPRV strain XJ5. Pigs vaccinated with high doses of the Bartha-K61 vaccine or rPRV-gI−/gE−/TK− prototype vaccine showed no differences in their humoral immune responses, clinical symptoms, body weight gains, viral shedding, or gross and histological lesions after sublethal challenge by the vPRV strain XJ5. Therefore, we concluded that the Bartha-K61 vaccine at a dose of 1 × 105 TCID50 per animal protects pigs against sublethal challenge by the vPRV strain XJ5 and performs equally well as the same dose of the rPRV-gI−/gE−/TK− vaccine, whereas lower doses of the Bartha-K61 vaccine alone do not protect pigs from this challenge. These findings provide useful information for vaccination interventions and the ultimate eradication of pseudorabies caused by vPRV strains emerging in China.

Characterization of a moderately pathogenic pseudorabies virus variant isolated in China, 2014

In this study, we reported a moderately pathogenic pseudorabies virus (PRV) variant isolated from one Bartha-K61-vaccinated pig farm in Weifang, Shandong Province, China, 2014. The sick piglets in the farm were characterized by anorexia, weight loss and neurologic symptoms but did not die. Sequence alignment of the gE gene indicated that it belonged to a new mutated PRV strain and about 15% amino acid sites had mutations, deficiencies and insertions compared to the other PRV strains. The gD gene had two amino acid insertions and ten amino acid mutations in comparison with the Bartha-K61 vaccine strain. The TK and gM genes were the same as one highly pathogenic PRV TJ strain. Evidence from virus isolation, laboratory challenge, serological detection and histopathologic examination confirmed that the etiological agent of the disease is PRV SD1404, which is a moderately pathogenic strain and causes piglets to be sick but not to die. PRV SD1404 strain is different from other reports and should be paid more attention to avoid economic losses.

Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus

BackgroundRecently, pseudorabies (PR) outbreaks have been reported in a large number of swine herds vaccinated with the Bartha-K61 vaccine in China, the current pseudorabies virus (PRV) belonging to Genotype II is differential genetically from Bartha-K61 vaccine belonging to Genotype I. Furthermore, it has been proved that the Bartha-K61 vaccine cannot provide sufficient protection against the current PRVs in China. Therefore, the accurate and rapid identification of PRVs is essential. The objective of this study is to develop a duplex fluorescence melting curve analysis (FMCA) capable of rapid, simple, high-throughput differentiation of Chinese, European/American and Bartha-K61 vaccine strains of PRV.ResultsPrimers 6F/6R and probes P1/P2, combined with three recombinant plasmids p-B (Bartha-K61), p-N (Genotype I), and p-H (Genotype II), were used to establish the Bicolor FMCA. FAM Tm values (probe P1) and HEX (probe P2) channels of p-B were used as reference values. Tm differences (ΔTm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had ΔTm values of ±1 °C in both FAM and HEX channels, Genotype I samples had ΔTm values of ±1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel, and Genotype II samples had ΔTm values of 6.52 ± 1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel. The minimum detection limit of the duplex FMCA was approximately 1 × 100 copies per reaction for p-B, p-N, and p-H. The duplex FMCA technique was used to detect and different 198 suspected clinical samples, of which 18 (9%) were positive for Genotype II strains and eight (4%) were positive for Bartha-K61 vaccine strains, and the results were compared with sequencing and phylogenetic analyses, which confirmed that the Bicolor FMCA worked correctly for all samples.ConclusionsIn this study, we developed a duplex FMCA of dual-labeled, self-quenched probes that was performed for rapid detection and differentiation of Genotype I, II and Bartha-K61 vaccine strains of PRV. The duplex FMCA was rapid, simple, and high-throughput, and will likely prove useful for molecular epidemiological investigations and pathogen surveillance of PRV.

Epidemiological and genetic characteristics of swine pseudorabies virus in mainland China between 2012 and 2017

The outbreak of pseudorabies (PR) in many Bartha-K61 vaccinated farms in China in late 2011 has seriously damaged the pig industry of one of the largest producers of pork products in the world. To understand the epidemiological characteristics of the pseudorabies virus (PRV) strains currently prevalent in China, a total of 16,256 samples collected from pig farms suspected of PRV infection in 27 Provinces of China between 2012 and 2017 were evaluated for detection of PRV. Since the extensive use of gE-deleted PRV vaccine in China, the PRV-gE was applied for determining wild-type virus infection by PCR. Of the 16,256 samples detected, approximately 1,345 samples were positive for the detection of PRV-gE, yielding an average positive rate of 8.27%. The positive rates of PRV detection from 2012 to 2017 were 11.92% (153/1284), 12.19% (225/1846), 6.70% (169/2523), 11.10% (269/2424), 5.57% (147/2640), and 6.90% (382/5539), respectively. To understand the genetic characteristics of the PRV strains currently circulating, 25 PRV strains isolated from those PRV-gE positive samples were selected for further investigation. Phylogenetic analysis based on gB, gC, and gE showed that PRV strains prevalent in China had a remarkably distinct evolutionary relationship with PRVs from other countries, which might explain the observation that Bartha-K61 vaccine was unable to provide full protection against emergent strains. Sequence alignments identified many amino acid changes within the gB, gC, and gE proteins of the PRVs circulating in China after the outbreak compared to those from other countries or those prevalent in China before the outbreak; those changes also might affect the protective efficacy of previously used vaccines in China, as well as being associated in part with the increased virulence of the current PRV epidemic strains in China.

Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes

BackgroundSince the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses. An ideal vaccine candidate is expected to have a balanced safety and immunogenicity.ResultsFrom the infectious clone of PRV AH02LA strain, a TK deletion mutant was generated through two-step Red mutagenesis. After homologous recombination with a transfer vector, a TK&gE dual deficient mutant PRV (PRVΔTK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRVΔTK&gE-AH02 reached a titer of 107.5 TCID50 /mL on ST cells.The PRVΔTK&gE-AH02 at a dose of 106.0 TCID50 /animal was not virulent in mice or 1-day-old piglets with maternal PRV antibodies. No clinical signs or virus shedding were detected in 28~ 35-day-old piglets without maternal PRV antibodies after nasal or intramuscular administration with a dose of 106.0 TCID50, although it caused one death of four 1-day-old piglets without maternal PRV antibodies. In the efficiency test of PRVΔTK&gE-AH02, all four 28~ 35-day-old piglets without PRV antibody in the challenge control showed typical clinical symptoms and virus shedding, and two died at 4~ 5 days post challenge. All piglets in 105.0, 104.0 and 103.0 TCID50/dose PRVΔTK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRVΔTK&gE-AH02 stopped virus shedding in these piglets. In contrast, all four piglets in PRV Bartha K61 vaccine group developed high body temperature (≥40.5 °C) and viral shedding, despite they had mild or even no clinical symptoms.ConclusionsThe constructed TK&gE dual deletion mutant PRVΔTK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRVΔTK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRVΔTK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Genetic characterization and mutation analysis of Qihe547 Aujeszky\u2019s disease virus in China

BackgroundAujeszky’s disease virus (ADV) can cause neurologic disease in young pigs, respiratory disease in older pigs and abortion or birth of mummified fetuses or stillborn neonates. The re-emergence of Aujeszky’s disease (AD) in pig farms vaccinated with live vaccine (Bartha-K61) caused substantial economic losses to Chinese pig industry since late 2011. A field ADV, named Qihe547, was isolated from pigs that exhibited suspected AD clinical symptoms. To better understand the genetic characteristics and mutations of Qihe547 ADV, the whole genome was sequenced and analyzed.ResultsThe genomic length of Qihe547 ADV was 143,404 bp, with 73.59% G + C contents. Phylogenetic analysis based on the whole genome of ADV strains revealed that Chinese ADV strains were located to one group with three subgroups. Qihe547 ADV was closely related to these novel ADV strains isolated in China since 2012. Qihe547 presented numerous hypervariable regions compared with oversea ADV strains. In 34 genes of Qihe547 ADV, amino acid (AA) insertion or deletion were observed. In addition, numerous AA mutations were found in the main protective antigen genes (gB, gC and gD genes). The differences of potential antigenic peptides in the main protective antigens between Qihe547 ADV and ADV Bartha were discovered in the dominant antigenic regions of gB (AA59-AA126, AA507-AA734),the extracellular region of gC and gD.ConclusionHigh diversity was observed between Qihe547 and foreign ADV isolates. The AA variations and the differences of potential antigenic peptides in the important functional regions of the main protective antigen (gB, gC and gD) of ADV Qihe547 may contribute to immune evasion of the virus and may be partial reason that the virus escapes from the vaccination of Bartha-K61 vaccine. In a word, the effect of the variations obviously requires further research.

Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus

Since late 2011, pseudorabies (PR) has resurfaced in many large pig farms, causing great economic loss for the swine industry in China. The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause, and much attention has been focused on how to prevent and control the reoccurrence of this disease in China. In this study, two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61vaccine against both variant PRV (XJ5) and classical PRV (Ra) strain challenge. Humoral immunity response, clinical signs, survival rate, body weight, virus shedding and pathology were assessed in commercial pigs. The results showed that Bartha-k61vaccine, administered either once or twice, was effective against the PRV variant (XJ5) challenge, while no significant differences were observed between single and prime-boost vaccinated pigs. However, pigs vaccinated twice had better body weight gains than those vaccinated once, following challenge with the classical PRV strain (Ra) (p<0.01). Therefore, the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV strains.

Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system

Currently, pseudorabies virus (PRV) variant strains are outbreaking in China; these variants belong to genotype II PRV. The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains. Therefore, rapid attenuation of current epidemic strains is needed for effective PRV control. In this study, we report a rapid method for editing the PRV genome using the CRISPR-Cas9 system. We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain, because mice were more susceptible to PRV infection, we then evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated. Furthermore, the attenuated strain also induced immune protection in response to a parental PRV challenge. Overall, we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology, which will be critical for PRV control, especially when new variant PRV strains emerge.