Serial high-resolution analysis of blood virome and host cytokines expression profile of a patient with fatal H7N9 infection by massively parallel RNA sequencing.
Hu Y,Ren X,Liu Y,Yang F,Liu H,Cao B,Jin Q
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases
Bloodstream infections and cytokine dysregulation are associated with high rates of morbidity and mortality among patients with influenza virus H7N9 infection. Here, massively parallel RNA sequencing was used to serially analyse plasma viruses and cytokine transcriptomes of a fatal H7N9-related case at successive times throughout the clinical course of infection. The results showed that acute viraemia with H7N9 virus occurred, and the virus was sensitive to antiviral therapy with oseltamivir. In addition, a lot of genome sequences from Acinetobacter baumannii were detected in blood on day 18 after illness onset when the bacteria were cultured. In parallel, longitudinal mRNA expression patterns of host cytokines associated with infection were analysed. This study illustrated the potential of RNA-sequencing to explore the plasma virome and expression profile of the infected host cytokines simultaneously in infectious disease.
Dynamic cerebrospinal fluid analyses of severe pseudorabies encephalitis.
Zheng Liheng,Liu Xiaojin,Yuan Deqin,Li Ruoxu,Lu Jianhua,Li Xiangdong,Tian Kegong,Dai Erhei
Transboundary and emerging diseases
We reported a severe human pseudorabies encephalitis case and described a dynamic clinical manifestation with cerebrospinal fluid analyses and cytological and serological evaluation, which may elucidate the mechanism of PRV infection and facilitate clinical diagnosis and treatment in human.
A Case of Human Viral Encephalitis Caused by Pseudorabies Virus Infection in China.
Yang HongNa,Han Hui,Wang Hao,Cui Yi,Liu Han,Ding ShiFang
Frontiers in neurology
We report a human case of viral encephalitis caused by pseudorabies virus (PRV) in China. A 43-year-old man with no previous medical history presented with high-grade fever, headache and tonic-clonic seizures as well as coma. Plain computer tomography (CT) brain imaging showed hypo-density in the bilateral basal ganglia, bilateral occipital lobe, bilateral limbic lobe, and left thalamic. Next-generation sequencing (NGS) confirmed the presence of PRV in cerebral spinal fluid (CSF). Regular polymerase chain reaction (PCR) was applied to confirm the presence of PRV in the CSF and blood. In addition, serological (immunological) tests were used to further validate the presence of PRV in the peripheral blood. This case suggested that it was possible for PRV to result in human central nervous system (CNS) infection, and it is necessary for people to increase awareness of self-protection when contacting animals.
[Clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies virus].
Zhao W L,Wu Y H,Li H F,Li S Y,Fan S Y,Wu H L,Li Y J,Lü Y L,Han J,Zhang W C,Zhao Y,Li G L,Qiao X D,Ren H T,Zhu Y C,Peng B,Cui L Y,Guan H Z
Zhonghua yi xue za zhi
To detect potential pathogens including pseudorabies virus in patients with encephalitis of unknown etiology in China and describe novel encephalitic entities. Patients with clinically suspected infectious encephalitis were enrolled in a multicenter study to identify the pathogens in PUMCH Encephalitis Program.Next-generation sequencing(NGS) of cerebrospinal fluid (CSF) was used in patients with encephalitis of unknown etiology enrolled from 2016 to 2017.The patients diagnosed as PRV encephalitis were studied to describe this novel entity. The four patients(3 male, 1 male, 38-54 years old) had occupational exposure to raw park when working in the production or marketing of pork and at least one got injured during pork-cutting.Two of them were confirmed with NGS of CSF, and anti-PRV antibodies were positive in 3 patients whose serum was available for serological analysis.They all presented with an acute onset of fever, convulsion, loss of consciousness and respiratory failure within 1 to 4 days and rapidly deteriorated even on extensive treatment.All the patients needed ICU admission and 3 needed mechanical ventilation.Two patients also had bilateral retinitis.Neuroimaging revealed symmetric gray matter lesions including limbic system, basal ganglia and midbrain without obvious hemorrhage.Lumbar puncture revealed elevated intracranial pressure and lymphocytic pleocytosis [(6-64)×10(6)/L] of CSF.The patients failed to response to the treatment of acyclovir combined with intravenous immunoglobulin and steroids.Modified Rankin Score was 3, 5, 5 and 6 (died) for the 4 patients respectively on last follow-up. PRV could be a cause of severe encephalitis.The patients with suspected pseudorabies encephalitis (PRE) need to be tested for PRV DNA timely.Severe encephalitis with bilateral involvement of limbic system, basal ganglion, thalamus and midbrain in patient with occupational exposure indicate this emerging infectious encephalitis.
Identification of molecular determinants for the nuclear import of pseudorabies virus UL31.
Li Meili,Jiang Si,Mo Chuncong,Zeng Zhancheng,Li Xiaowei,Chen Chunke,Yang Yanjia,Wang Jinlin,Huang Jinlu,Chen Daixiong,Peng Tao,Cai Mingsheng
Archives of biochemistry and biophysics
Herpes simplex virus 1 (HSV-1) UL31 is a multifunctional protein and important for HSV-1 infection. Pseudorabies virus (PRV) UL31 is a late protein homologous to HSV-1 UL31. Previous studies showed that PRV UL31 is predominantly localized to nucleus, however, the molecular determinants for its nuclear import were unclear to date. Here, by utilizing live cells fluorescent microscopy, UL31 fused with enhanced yellow fluorescent protein was transiently expressed in live cells and confirmed to exclusively target to the nucleus in the absence of other viral proteins. Furthermore, the nuclear import of UL31 was found to be dependent on the Ran-, importin α1-, α3-, α5-, α7-, β1-and transportin-1-mediated pathway. Therefore, these results would open up new avenues for depicting the biological functions of UL31 during PRV infection.
Bilateral Necrotizing Retinitis following Encephalitis Caused by the Pseudorabies Virus Confirmed by Next-Generation Sequencing.
Hu Feng,Wang Jiawei,Peng Xiao-Yan
Ocular immunology and inflammation
PURPOSE:The objective of this study was to report a case of bilateral necrotizing retinitis following viral encephalitis caused by the pseudorabies virus. CASE REPORT:A 49-year-old male had decreased bilateral visual acuity after the recovery of consciousness for one month. He had been in an unconsciousness status due to encephalitis for two months before the ocular symptoms developed. He was a pig slaughterer. Ocular ultrasound showed bilateral vitreous haze and retinal detachment. A vitrectomy and silicone oil tamponade were performed on the left eye. During surgery, massive periphery retinal necrosis appearing as a tattered fish net, and multiple retinal holes were observed. The pseudorabies virus was detected by next-generation sequencing in the vitreous specimen. CONCLUSION:The pseudorabies virus may cause bilateral necrotizing retinitis following viral encephalitis among those with close contact to pigs. Intraocular fluid provides a greater selection of samples and a longer time window for pathogenic detection.
Improved immune response to an attenuated pseudorabies virus vaccine by ginseng stem-leaf saponins (GSLS) in combination with thimerosal (TS).
Ni Jingxuan,Bi Shicheng,Xu Wei,Zhang Cenrong,Lu Yisong,Zhai Lijuan,Hu Songhua
Vaccination using attenuated vaccines remains an important method to control animal infectious diseases. The present study evaluated ginseng stem-leaf saponins (GSLS) and thimerosal (TS) for their adjuvant effect on an attenuated pseudorabies virus (aPrV) vaccine in mice. Compared to the group immunized with aPrV alone, the co-inoculation of GSLS and/or TS induced a higher antibody response. Particularly, when administered together with GSLS-TS, the aPrV vaccine provoked a higher serum gB-specific antibody, IgG1 and IgG2a levels, lymphocyte proliferative responses, as well as production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) from lymphocytes, and more importantly provided an enhanced cytotoxicity of NK cells and protection against virulent field pseudorabies virus challenge. Additionally, the increased expression of miR-132, miR-146a, miR-147 and miR-155 was found in murine macrophages cultured with GSLS and/or TS. These data suggest that GSLS-TS as adjuvant improve the efficacy of aPrV vaccine in mouse model and have potential for the development of attenuated viral vaccines.
Human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: A case report.
Wang Yuwei,Nian Hong,Li Ziwei,Wang Wenjuan,Wang Xin,Cui Yan
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
We report the case of a patient who presented with viral encephalitis and a pulmonary infection complicated with bilateral acute retinal necrosis after direct contact with diseased swine. Next-generation sequencing of the cerebrospinal fluid and vitreous humor detected pseudorabies virus (PRV) simultaneously. Intravenous acyclovir and dexamethasone treatment improved the symptoms of encephalitis, and vitrectomy surgery with silicone oil tamponade was used to treat the retinal detachment. This case implies that PRV can infect humans; thus, self-protection is imperative when there is contact with animals.
Antiviral activities of Radix isatidis polysaccharide against pseudorabies virus in swine testicle cells.
Tong Chao,Chen Zewen,Liu Fang,Qiao Yanyan,Chen Tong,Wang Xuebing
BMC complementary medicine and therapies
BACKGROUND:Radix isatidis has been used in China and other Asian countries for its antiviral and anti-inflammatory effects for thousands of years. However, the antiviral effect of Radix isatidis polysaccharide against pseudorabies virus (PRV) is still unknown. METHODS:The polysaccharide were isolated from extract of the roots of Radix isatidis. MTT assays were used to determine the preventive effect, inhibitory effect and antiviral effect of Radix isatidis polysaccharide on PRV in vitro. RESULTS:This study found that different concentrations of polysaccharides from this plant can inhibit PRV replication by 14.674-30.840%, prevent infection at rates of 6.668-14.923%, and kill this virus at rates of 32.214-67.422%. CONCLUSION:These results broaden the understanding of this traditional Chinese herb and provide a theoretical basis for further research. Moreover, Radix isatidis polysaccharide could be used for antiviral therapy.
Comparison of pseudorabies virus China reference strain with emerging variants reveals independent virus evolution within specific geographic regions.
Wang Xin,Wu Chang-Xian,Song Xian-Rong,Chen Huan-Chun,Liu Zheng-Fei
Pseudorabies virus (PRV) China reference strain Ea is genetically closely related to newly emerged variants; however, there is limited information about PRV Ea. Here, we compared PRV Ea with new variant strains by growth kinetics, genome sequencing, and protein expression analysis. Growth analysis showed that strain Ea forms smaller plaques than strain HNX. The full-length genome sequence of Ea revealed that it is clustered in the same subgroup as HNX. Ea and HNX strains exhibited similar extracellular virion protein polymorphisms, whereas strain Bartha expressed less VP26 and more GAPDH. In infected cells, strain Ea expressed high levels of IE180 protein, and Ea and HNX produced higher levels of UL21 protein than strain Bartha. These findings provide evidence that PRV China reference strain Ea is genetically closely related to the newly emerged variant strains, indicating that strain PRV China may have evolved independently leading to the emergence of a variant strain.
Porcine NK Cells Stimulate Proliferation of Pseudorabies Virus-Experienced CD8 and CD4CD8 T Cells.
De Pelsmaeker Steffi,Devriendt Bert,De Regge Nick,Favoreel Herman W
Frontiers in immunology
Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-γ, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8 and CD4CD8 PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies.
The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation.
Tanneti Nikhila S,Federspiel Joel D,Cristea Ileana M,Enquist Lynn W
Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.
Characteristics of human encephalitis caused by pseudorabies virus: A case series study.
Yang Xue,Guan Hongzhi,Li Chen,Li Ying,Wang Shengjun,Zhao Xiuhe,Zhao Yuying,Liu Yiming
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
BACKGROUND:Pseudorabies virus (PRV) has been thought to cause diseases only in animals. However, recent studies have shown that PRV can also cause illnesses in humans. METHODS:This was a case series study. The cases of five patients with clinical symptoms of acute encephalitis, which were confirmed to be caused by PRV infections, were reviewed. CASE PRESENTATION:The five patients all had jobs involving the handling of pigs. They had acute onset and rapid progression of clinical presentations, which were consistent with central nervous system infections. Four of them had respiratory failure, which required ventilator support. Brain magnetic resonance imaging showed abnormal signals in the bilateral temporal lobes and insular cortex in all five patients, bilateral frontal lobes in one patient, and caudate nucleus in one patient. Cerebrospinal fluid analysis results were consistent with a viral infection. Next-generation sequencing of the cerebrospinal fluid confirmed the presence of PRV. All patients received human immunoglobulin, glucocorticoids, antiviral agents, and symptomatic supportive treatments. All patients survived until discharge, but suffered from various sequelae. Pneumonia was the most common complication during the disease course. CONCLUSIONS:PRV encephalitis should be included in the differential diagnosis of patients with a clinical presentation of central nervous system infection, especially for those who have had recent contact with pigs.
Pseudorabies Virus Infection of Epithelial Cells Leads to Persistent but Aberrant Activation of the NF-κB Pathway, Inhibiting Hallmark NF-κB-Induced Proinflammatory Gene Expression.
Romero Nicolás,Van Waesberghe Cliff,Favoreel Herman W
Journal of virology
The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host. The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.
Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice.
Laval K,Vernejoul J B,Van Cleemput J,Koyuncu O O,Enquist L W
Journal of virology
Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents, and dogs. In these nonnatural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch," which is uncommon in swine. Thus far, the pathophysiological and immunological processes leading to the development of the neuropathic itch and the death of the animal are unclear. Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 h postinoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and dorsal root ganglia (DRGs) by hematoxylin and eosin staining. PRV load was detected in the foot, PNS, and central nervous system tissues by quantitative reverse transcription-PCR. Infected mice had elevated plasma levels of proinflammatory cytokines (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) and chemokines (Gro-1 and monocyte chemoattractant protein 1). Significant IL-6 and G-CSF levels were detected in several tissues at 82 hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain) did not develop pruritus at 82 hpi. PRV-Bartha also replicated in the PNS, and the infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation. Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus 1 and varicella-zoster virus (VZV). The natural host of PRV is the swine, but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch." The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not mice infected with attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.
Detection of Pseudorabies Virus Antibodies in Human Encephalitis Cases.
Li Xiang Dong,Fu Shi Hong,Chen Ling Yan,Li Fan,Deng Jun Hua,Lu Xuan Cheng,Wang Huan Yu,Tian Ke Gong
Biomedical and environmental sciences : BES
Pseudorabies virus (PRV), a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine, was recently reported to infect human and led to endophthalmitis and encephalitis. A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%, 14.25%, and 6.52% in 2012, 2013, and 2017, respectively. The virus neutralizing antibody titers of positive samples correlated well with ELISA results. The pseudorabies virus antibody positive rate of patients with encephalitis were higher than that of healthy people in 2017. The above results suggest that some undefined human encephalitis cases may be caused by PRV infection.
Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV.
Tong Wu,Zheng Hao,Li Guo-Xin,Gao Fei,Shan Tong-Ling,Zhou Yan-Jun,Yu Hai,Jiang Yi-Feng,Yu Ling-Xue,Li Li-Wei,Kong Ning,Tong Guang-Zhi,Li Ji-Chang
Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.
A gD&gC-substituted pseudorabies virus vaccine strain provides complete clinical protection and is helpful to prevent virus shedding against challenge by a Chinese pseudorabies variant.
Zhang Chuanjian,Liu Yamei,Chen Saisai,Qiao Yongfeng,Guo Mingpeng,Zheng Yating,Xu Mengwei,Wang Zhisheng,Hou Jibo,Wang Jichun
BMC veterinary research
BACKGROUND:Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gC) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gC was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions. RESULTS:The results showed that a BAC clone of Bartha-K61 and a B-gD&gC clone were successfully generated. The growth kinetics of PRV B-gD&gC strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gC strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gC and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gC strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group. CONCLUSIONS:Our results suggest that PRV B-gD&gC strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.
Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies.
Ji Chihai,Wei Yingfang,Wang Jingyu,Zeng Yuchen,Pan Haoming,Liang Guan,Ma Jun,Gong Lang,Zhang Wei,Zhang Guihong,Wang Heng
Pseudorabies, also known as disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.
Development of a blocking immunoperoxidase monolayer assay for differentiation between pseudorabies virus-infected and vaccinated animalss.
Wang Y B,Li Y H,Li Q M,Xie W T,Guo C L,Guo J Q,Deng R G,Zhang G P
Polish journal of veterinary sciences
Pseudorabies (PR) outbreaks have devastated many swine farms in several parts of China since late 2011. The outbreak-associated pseudorabies virus (PRV) variant strains exhibited some typical amino acid changes in glycoprotein E (gE), a diagnostic antigen used for discriminating between PRV-infected and vaccinated animals (DIVA). To counteract the potential impact of epitope variations on current serological diagnostics of PRV, we produced monoclonal antibodies (mAbs) against gE protein of one representative PRV variant strain and developed a blocking immunoperoxidase monolayer assay (b-IPMA) for DIVA. The b-IPMA was based on the inhibition of binding between PRV-infected cells and mAb by PRV-specific antibodies present in clinical swine sera and was validated by comparison with a commercial PRV gpI Antibody Test Kit (IDEXX Laboratories, USA). The diagnostic sensitivity, diagnostic specificity and agreement were determined to be 99.25%, 98.18% and 99.02% respectively upon testing 509 serum samples. b-IPMA detected only PRV-specific antibodies and showed no cross- -reactivity with antibodies elicited by gE-deleted vaccine or other common swine pathogens. Thus, b-IPMA has the potential to be used for high-throughput screening of PRV-infected animals in veterinary clinics.
Alpha/beta interferon receptor deficiency in mice significantly enhances susceptibility of the animals to pseudorabies virus infection.
Wei Jingyun,Ma Yanmei,Wang Long,Chi Xiaojuan,Yan Ruoxiang,Wang Song,Li Xinxin,Chen Xiaoyong,Shao Wenhan,Chen Ji-Long
Pseudorabies virus, one of the neurotropic viruses, can infect numerous mammals. In particular, pseudorabies virus infection of swine occurs worldwide, and is a major threat to swine industry. However, the mechanism underlying the interaction between pseudorabies virus and host innate immune system is not fully understood. Here, we investigated the involvement of interferon α/β (IFN-α/β) receptor (IFNAR) in the pathogenesis of pseudorabies virus in a mouse model. The results showed that IFNAR-deficient (IFNAR) mice were highly susceptible to the virus infection, as evidenced by markedly reduced survival rate of infected animals and increased viral replication. The expression of IFN-α/β and relevant interferon-stimulated genes in IFNAR mice was significantly lower than that in wild-type (WT) littermates after the viral infection. Moreover, in response to the virus challenge, IFNAR mice displayed elevated levels of inflammatory cytokines including interleukin 6 (IL-6) and IL-1β, and IFNAR cells showed increased phosphorylation of STAT3. Collectively, these data reveal that the IFNAR mice are more sensitive to pseudorabies virus infection than WT animals, and excessive IL-6/STAT3 response in IFNAR mice may contribute to the pathogenesis. Our findings suggest that type I IFNs/IFNAR-dependent homeostatic control of the innate immunity is required for host defense against pseudorabies virus infection.
Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.
Li Jianglong,Li Xiangmin,Hao Genxi,Zhang Huawei,Yang Huiling,Chen Huanchun,Qian Ping
Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.
Genetic evolution analysis of novel recombinant pseudorabies virus strain in Sichuan, China.
Huang Jianbo,Zhu Ling,Zhao Jun,Yin Xinhuan,Feng Yu,Wang Xvetao,Sun Xiangang,Zhou Yuancheng,Xu Zhiwen
Transboundary and emerging diseases
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.
Human Endophthalmitis Caused By Pseudorabies Virus Infection, China, 2017.
Ai Jing-Wen,Weng Shan-Shan,Cheng Qi,Cui Peng,Li Yong-Jun,Wu Hong-Long,Zhu Yi-Min,Xu Bin,Zhang Wen-Hong
Emerging infectious diseases
We report human endophthalmitis caused by pseudorabies virus infection after exposure to sewage on a hog farm in China. High-throughput sequencing and real-time PCR of vitreous humor showed pseudorabies virus sequences. This case showed that pseudorabies virus might infect humans after direct contact with contaminants.