gE Gene Research Articles

Isolation and Phylogenetic Analysis of Reemerging Pseudorabies Virus Within Pig Populations in Central China During 2012 to 2019.

To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European–American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.

Development of multiplex TaqMan qPCR for simultaneous detection and differentiation of eight common swine viral and bacterial pathogens.

It is laborious to diagnose the infections of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Suid herpesvirus 1 (SuHV-1) because of the similar clinical symptoms in piglets. Staphylococcus aureus (S. aureus), Streptococcus suis (S. suis), Salmonella choleraesuis (S. choleraesuis, serotype: 6,7:c:1,5), and Escherichia coli (E. coli) are common secondary bacterial pathogens in viral infections. Furthermore, the mixed infection of these viral and bacterial pathogens is more and more common in practical swine breeding. Therefore, a TaqMan multiplex qPCR method for simultaneous detection and differentiation of their pathogen was established in this study by designing specific primers and probes for the E2 gene of CSFV, the ORF7 gene of PRRSV, the ORF1 gene of PCV2 and the gE gene of SuHV-1, the nuc gene of S. aureus, the ef-tu gene of S. suis, the ivnA gene of S. choleraesuis, and the 23S rRNA gene of E. coli, and its specificity, sensitivity, and reproducibility were subsequently tested. The results showed that TaqMan multiplex qPCR method showed a high specificity with no cross reaction between different viruses, and a good repeatability with its coefficient of variation lower than 5%. Besides, the sensitivity of this method was also at least 10 times higher compared with conventional PCR. Overall, this study provided a reliable multiplex TaqMan qPCR method for the diagnosis and differentiation of the mentioned pathogens in pigs, laying a certain technical basis for disease prevention and control.

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.

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018-2019.

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.

Diagnosis and gI antibody dynamics of pseudorabies virus in an intensive pig farm in Hei Longjiang Province.

BackgroundPseudorabies (PR), caused by the pseudorabies virus (PRV), is an endemic disease in some regions of China. Although there are many reports on epidemiological investigations into pseudorabies, information on PRV gI antibody dynamics in one pig farm is sparse.ObjectivesTo diagnose PR and analyze the course of PR eradication in one pig farm.MethodsTen brains and 1,513 serum samples from different groups of pigs in a pig farm were collected to detect PRV gE gene and PRV gI antibody presence using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.ResultsThe July 2015 results indicated that almost all brain samples were PRV gE gene positive, but PRV gI antibody results in the serum samples of the same piglets were all negative. In the boar herd, from October 2015 to July 2018 three positive individuals were culled in October 2015, and the negative status of the remaining boars was maintained in the following tests. In the sow herd, the PRV gI antibody positive rate was always more than 70% from October 2015 to October 2017; however, it decreased to 27% in January 2018 but increased to 40% and 52% in April and July 2018, respectively. The PRV gI antibody positive rate in 100-day pigs markedly decreased in October 2016 and was maintained at less than 30% in the following tests. For 150-day pigs, the PRV gI antibody positive rate decreased notably to 10% in April 2017 and maintained a negative status from July 2017. The positive trend of PRV gI antibody with an increase in pig age remarkably decreased in three tests in 2018.ConclusionsThe results indicate that serological testing is not sensitive in the early stage of a PRV infection and that gilt introduction is a risk factor for a PRV-negative pig farm. The data on PRV gI antibody dynamics can provide reference information for pig farms wanting to eradicate PR.

Retrospective detection and phylogenetic analysis of pseudorabies virus in dogs in China.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years as a result of a recent outbreak of pseudorabies. The causative agent has a wide spectrum of hosts, including pigs, cattle, sheep, dogs, cats, bats, bears, and even some avian species. Although dog-related cases of pseudorabies have been reported regularly, many cases are overlooked, and few PRV strains are isolated because death occurs rapidly after PRV infection and veterinarians often do not test for PRV in dogs. Here, we performed a retrospective detection of PRV in dogs from July 2017 to December 2018. We found that PRV (including gE-deleted strains, classical strains, and variant strains) is prevalent in dogs regardless of season and region and that the epidemic PRV strains in dogs share high sequence similarity with gC and gE genes of swine epidemic strains and commercial vaccine strains. Collectively, our findings underscore the importance of PRV surveillance in dogs, which is beneficial for understanding the epidemiology of PRV in dogs and assists in efforts aimed at effectively controlling this disease.

Latent pseudorabies virus infection in medulla oblongata from quarantined pigs.

Pseudorabies virus (PRV) is a major pathogen in pig husbandry and is also a risk to human well-being. Pigs with latent PRV infection carry the virus lifelong, and it can be activated under conducive conditions. This poses a very important challenge to the control of the virus and may even prevent its elimination. To investigate latent infection with wild-type (wt) PRV, and also infection due to the use of live attenuated vaccines on farms, 80 pigs from two large-scale swine operations were traced. At 6months old, the quarantined pigs were slaughtered and brain samples were collected. A PCR assay targeting the gB and gE genes was developed to detect PRV DNA fragments in medulla oblongata. Five of the samples (6.3%) were gB and gE gene fragment double-positive, 60 of the samples (75%) were gB single-positive, and 15 samples (18.7%) showed double-negative. A portion of latency-associated transcripts (LATs), EP0 mRNA, were found to be present in the gB gene fragment positive samples. Furthermore, the five double-positive samples were transmitted blindly, and apparent cytopathic effects were found in three of the five samples in the fourth generation. By means of Western blotting, PCR and sequencing, two of the isolated viruses were found to be related to vaccine strain Bartha-K61. Another was closely related to domestic epidemic strains HN1201 and LA and relatively unrelated to other Asian isolates. These results suggest that the live vaccines are latently present in brains, in a manner similar to wt PRV, and this poses potential safety issues in the pig husbandry industry. Wt PRV and live vaccine viruses were found to co-exist in pigs, demonstrating that the live vaccines were unable to confer complete sterilizing immunity, which may explain outbreaks of pseudorabies on vaccinated farms.

Evaluation of glycoprotein E subunit and live attenuated varicella-zoster virus vaccines formulated with a single-strand RNA-based adjuvant.

IntroductionVaricella‐zoster virus (VZV), a human alphaherpesvirus 3, elicits both chickenpox and shingles and/or postherpetic neuralgia. A live attenuated vaccine (LAV) and glycoprotein E (gE) subunit vaccine were developed to prevent VZV‐induced diseases. We recently reported that single‐strand RNA (ssRNA) based on the intergenic region of the internal ribosome entry site of cricket paralysis virus (CrPV) is an effective adjuvant for protein‐based and virus‐like particle‐based vaccines. Here, Chinese hamster ovary expression system and an LAV from Oka/SK strains.MethodsWe appraised the adjuvant effect of the same CrPV ssRNA encoding the gE gene formulated in the two vaccines using VZV‐primed C57BL/6 mice and guinea pigs. Humoral immunity and cell‐mediated immunity were assessed by enzyme‐linked immunosorbent assay (ELISA) and ELISPOT in gE subunit vaccine and by ELISA and fluorescent antibody to membrane antigen in LAV.ResultsThe gE subunit vaccine‐induced gE‐specific antibodies and CD4+ T‐cell responses (indicated by interferon‐γ [IFN‐γ] and interleukin‐2 secretion) in the ssRNA‐based adjuvant containing the VZV gE gene. Therefore, an ssRNA adjuvant combined with gE antigen can trigger the innate immune response and induce an adaptive immune response to ultimately activate humoral and cell‐mediated responses. VZV LAV could also induce VZV‐specific antibodies and IFN‐γ stimulated by LAV, whereas the effect of ssRNA as a vaccine adjuvant could not be confirmed. However, the ssRNA adjuvant increased VZV‐specific neutralizing antibody response.ConclusionsTaken together, these results highlight that the gE subunit vaccine and LAV developed in this study can be functional VZV vaccines, and ssRNAs appear to function better as adjuvants in a subunit vaccine than in an LAV.

Genetic evolution analysis of novel recombinant pseudorabies virus strain in Sichuan, China

Pseudorabies is a disease that seriously endangers the pig industry in China. Recently, we successfully isolated a pseudorabies virus from the brain tissue of piglets at a farm in Sichuan, China, and named it the FJ62 strain. In order to understand the molecular biological characteristics of the strain, primers were designed for glycoproteins gB, gC, gD and gE, which were amplified by a polymerase chain reaction (PCR) and sequenced. After comparing the sequence with the GenBank 22 pseudorabies virus reference strains and establishing the genetic evolutionary tree, it was found that the gB gene of pseudorabies virus was highly homologous (up to 100%) with the MY-1 strain which is isolated from a wild boar in Japan (AP018925) but that homology with other strains in China was low. The gC gene was in the same branch as most of the representative strains in China, with 99.5% homology. The gD gene is in the same branch as the domestic strain LA in China (KU552118), and the homology was 99.9%. The gE gene was in the same branch as the domestic BJ/YT strain in China (KC981239), with 99.9% homology. The results showed that the FJ62 strain of the pseudorabies virus isolated here may be a variant strain of FJ62 isolated from a domestic pig after natural recombination of pseudorabies virus genotype I from wild boar and genotype II from pigs in China. There have been no similar reports in Sichuan. The discovery of the recombinant virus strain provides a reference basis for the prevention and control of pseudorabies and a design strategy for a vaccine in Sichuan, China, in the future.

Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant.

The prevalence of an emerging variant of the pseudorabies virus (PRV) has been causing serious losses to farmers in China. Moreover, the commercially available PRV vaccine often fails to provide thorough protection. Therefore, in this study, we generated a PRV-∆gC\\gE∆TK strain with defects in gC, gE, and TK of PRV. Compared to the parental PRV strain and the single gene deletion strains (PRV-∆gC, PRV-∆gE, and PRV-∆TK), PRV-∆gC\\gE∆TK grew slowly, and exhibited fewer and smaller plaques on swine testis (ST) cells. Furthermore, animal experiment results showed that mice that were immunized intramuscularly with PRV-∆gC\\gE∆TK, survived throughout the experiment with no observed clinical symptoms, and were completely protected against PRV challenge. Additionally, deletion of the gC, gE, and TK genes significantly alleviated viral damage in the brain. Furthermore, one-day-old weaned piglets immunized intramuscularly with PRV-∆gC\\gE∆TK elicited higher levels of gB antibodies against both the emerging PRV variant and the parental PRV, exhibited full protection against challenge with both variants, and showed neutralization capacity against PRV. These data suggest that PRV-∆gC\\gE∆TK is a promising vaccine candidate for the control of pseudorabies.

Functional interactions between herpes simplex virus pUL51, pUL7 and gE reveal cell-specific mechanisms for epithelial cell-to-cell spread

Herpes simplex virus spread between epithelial cells is mediated by virus tegument and envelope protein complexes including gE/gI and pUL51/pUL7. pUL51 interacts with both pUL7 and gE/gI in infected cells. We show that amino acids 30–90 of pUL51 mediate interaction with pUL7. We also show that deletion of amino acids 167–244 of pUL51, or ablation of pUL7 expression both result in failure of gE to concentrate at junctional surfaces of Vero cells. We also tested the hypothesis that gE and pUL51 function on the same pathway for cell-to-cell spread by analyzing the phenotype of a double gE/UL51 mutant. In HaCaT cells, pUL51 and gE function on the same spread pathway, whereas in Vero cells they function on different pathways. Deletion of the gE gene strongly enhanced virus release to the medium in Vero cells, suggesting that the gE-dependent spread pathway may compete with virion release to the medium.

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.

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.

Development of a qPCR for the detection of infectious laryngotracheitis virus (ILTV) based on the gE gene

ABSTRACT1. Infectious laryngotracheitis is a respiratory disease that affects the poultry industry worldwide. It is common in flocks with high-bird density, causing major economic losses.2. In this study, a SYBR® FAST polymerase chain reaction (PCR) double-strand DNA intercalating agent assay was performed for the detection of infectious laryngotracheitis virus (ILTV) in clinical samples in comparison with a conventional nested-PCR, both based on the glycoprotein E encoding gene. This assay amplified 56 bp and was capable of detecting 19 to 1 copies of virus.3. In total, 164 clinical samples were obtained from birds with respiratory problems from the period of 2009–2016. In the nested-PCR, there were 45.12% positive samples and 54.88% negative samples, while in the real-time PCR (qPCR), there were 81.1% positive samples and 18.9% negative samples.4. In conclusion, qPCR from the DNA double-strand intercalating agent SYBR® GREEN FAST was useful for the diagnosis of ILTV because it detected samples that were negative in nested-PCR. This assay has advantages, such as a shortened processing-time, and no need for post-amplification processing (electrophoresis) with additional reagents, such as MgCl2 and agarose. Hence, qPCR proved to be useful, rapid and low cost for use with clinical samples.

Polymorphisms affecting the gE and gI proteins partly contribute to the virulence of a newly-emergent highly virulent Chinese pseudorabies virus

An outbreak of a highly virulent pseudorabies virus strain, ZJ01, occurred in PRV-vaccinated pigs in China in 2011. In this study, ZJ01 caused fatal diseases, while the Chinese prototypic PRV strain LA caused mild respiratory disorders. Full-genome sequencing results indicate the two viruses can be classified into two sub-clusters that distinct from traditional European and US strains. To examine the potential role of the gE and gI proteins in ZJ01 virulence, we generated several recombinant viruses. In two chimeric viruses (rZJ01-LA/gEI and rLA-ZJ01/gEI), the gE and gI genes were swapped using corresponding genes from ZJ01 and LA. rZJ01-LA/gEI and the parental virus rZJ01 retained high virulence in piglets, although the survival time for rZJ01-LA/gEI infected piglets was obviously prolonged. In contrast, rLA-ZJ01/gEI exhibited higher virulence than its parental virus rLA. We conclude that changes in gE and gI proteins partly contribute to the enhanced virulence of ZJ01 strain.

Epidemiological investigation of pseudorabies in Shandong Province from 2013 to 2016.

In late 2011, a variant pseudorabies virus (vPRV) emerged in Bartha-K61-vaccinated pig herds, resulting in high morbidity and mortality of piglets in China. Since 2013, the autopsy lesions, histological examinations, virus isolation, phylogenetic analysis and selection pressure analysis of the gE gene of vPRV were recorded for 395 clinical cases, and 5,033 pig serum samples were detected by PRV gE-coated enzyme-linked immunosorbent assay. The major clinical symptoms were abortion in pregnant sows, fatal neurological signs in piglets and respiratory disease in growing pigs. Necrotic splenitis, hepatitis and lymphadenitis, haemorrhagic nephritis and non-suppurative encephalitis were observed by histopathological examination. Typical eosinophilic inclusion bodies were found in the nuclei of liver cells. Using PCR, 110 samples among 395 clinical cases tested positive for the gE gene. Fifteen vPRV strains were isolated and confirmed by sequencing and phylogenetic analysis of the gE gene. The strains shared 97.1%-99.9% nucleotide (nt) and 96.6%-99.5% amino acid (aa) homology with PRV reference strains. Selection pressure analysis showed that one site in the codons of glycoprotein E was under positive selection. Of the 5,033 serum samples, 2,909 were positive by ELISA for a positive rate of 57.8%. These results showed that vPRV was still prevalent in Shandong Province, indicating severe PRV infectious pressure. The preparation of new vaccines against PRV is extremely urgent.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 102 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas.

Detection of pseudorabies virus by duplex droplet digital PCR assay.

Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines. We compared this assay with a TaqMan real-time PCR (qPCR) using the same primers and probes. Both assays exhibited good linearity and repeatability; however, ddPCR maintained linearity at extremely low concentrations, whereas qPCR did not. Based on positive results for both gE and gB, the detection limit of ddPCR was found to be 4.75 copies/µL in contrast of 76 copies/µL for qPCR, showing that ddPCR provided a 16-fold improvement in sensitivity. In addition, no nonspecific amplification was shown in specificity testing, and the PRV wild-type was distinguished from a gE-deleted strain. The ddPCR was more sensitive when analyzing clinical serum samples. Thus, ddPCR may become an appropriate detection platform for PRV.

The herpevac trial for women: Sequence analysis of glycoproteins from viruses obtained from infected subjects.

The Herpevac Trial for Women revealed that three dose HSV-2 gD vaccine was 58% protective against culture-positive HSV-1 genital disease, but it was not protective against HSV-2 infection or disease. To determine whether vaccine-induced immune responses had selected for a particular gD sequence in strains infecting vaccine recipients compared with viruses infecting control subjects, genetic sequencing studies were carried out on viruses isolated from subjects infected with HSV-1 or HSV-2. We identified naturally occurring variants among the gD sequences obtained from 83 infected subjects. Unique or low frequency amino acid substitutions in the ectodomain of gD were found in 6 of 39 HSV-1-infected subjects and in 7 of 44 HSV-2-infected subjects. However, no consistent amino acid change was identified in isolates from gD-2 vaccine recipients compared with infected placebo recipients. gC and gE surround and partially shield gD from neutralizing antibody, and gB also participates closely in the viral entry process. Therefore, these genes were sequenced from a number of isolates to assess whether sequence variation may alter protein conformation and influence the virus strain’s capacity to be neutralized by vaccine-induced antibody. gC and gE genes sequenced from HSV-1-infected subjects showed more variability than their HSV-2 counterparts. The gB sequences of HSV-1 oral isolates resembled each other more than they did gB sequences rom genital isolates. Overall, however, comparison of glycoprotein sequences of viral isolates obtained from infected subjects did not reveal any singular selective pressure on the viral cell attachment protein or surrounding glycoproteins due to administration of gD-2 vaccine.

A high-temperature passaging attenuated Pseudorabies vaccine protects piglets completely against emerging PRV variant

Emerging variant of pseudorabies virus (PRV) have evaded the antiviral immunity of commercially available PRV vaccine and have led to PRV outbreaks in Chinese pig farms. Here, we attenuated a PRV variant strain by serial passages in vitro and evaluate the protective efficacy of the attenuated strain as a vaccine candidate. The virulent PRV variant strain JS-2012 was continuously passaged in Vero cells at 40°C and attenuated rapidly. After 90 passages in Vero cells, the passaged virus lost its ability to cause death in 2-week-old piglets. The 120th passage virus was avirulent in the sucking piglets. An attenuated strain, JS-2012-F120 derived from the 120th passage virus by three rounds of plaque cloning grew better than its parent strain JS-2012 in Vero cells and showed notably different cytopathic effects and plaque morphology from JS-2012. PCR combined with sequence analysis showed that JS-2012-F120 contained a 2307-bp deletion covering nucleotide 487 of gE gene to 531 of US2 gene. After inoculation with JS-2012-F120, young piglets were completely protected from challenge with the classical and emerging virulent PRVs. Moreover, the piglets did not develop specific gE antibodies. Thus, JS-2012-F120 appears to be a promising marker vaccine to control PRV variant circulating in Chinese pig farms, and the high-temperature passaging in vitro was an efficient method to attenuated alphaherpesvirus.