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Objective: To investigate the neural connections between Shenmen (HT7)-heart and the brain by observing the tracing viruses co-labeled brain nuclear groups after injection of the pseudorabies viruses (PRV), the reverse transsynaptic virus tracer carrying different fluorescent protein genes, into the myocardium and Shenmen (HT7) point, respectively.Methods: Pseudorabies virus 531 (PRV531) carrying the green fluorescent protein gene and pseudorabies virus 724 (PRV724) carrying the red fluorescent protein gene were injected into the left ventricular wall and Shenmen (HT7) point area of the left forelimb of six C57BL/6 mice, respectively. After 120 h, whole brain tissue was extracted under 4％ paraformaldehyde perfusion to prepare brain sections. Neuronal co-labeling with the tracing viruses was observed under fluorescence microscopy. Results: Co-labeled signals from the mouse ventricular wall and Shenmen (HT7) point region were found at all levels of the mouse central nervous areas, such as the cerebral cortex, hypothalamus, midbrain, pons, and medulla oblongata. The number of co-labeled neurons was higher in the primary motor area, the hypothalamic paraventricular nucleus, the subceruleus nucleus, and the paramedian reticular nucleus. Conclusion: There is a neural connection between Shenmen (HT7), the heart, and the brain, which may be most closely related to the autonomic nervous system.
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Objective:To explore the epidemiology, clinical features and prognosis of pseudorabies virus (PRV) infection in human.Methods:A case of human PRV encephalitis combined with acute retinal necrosis (ARN) in the First Affiliated Hospital of Zhengzhou University in May 2020 was reported. The epidemiology, clinical features, neuroimaging, cerebrospinal fluid (CSF), next-generation sequencing (NGS), treatment and prognosis of human PRV infection were summarized and analyzed with the previous published data.Results:The present case was a 38-year-old man who developed high fever, headache, cognitive decline, recurrent epileptic seizures after butchering a pig. Brain magnetic resonance imaging showed lesions in the insular lobes, temporal lobes, cingulate gyrus, frontal lobes, basal ganglia and hippocampus, with more significant signals on the left side. Afterwards, bilateral ARN occurred and resulted in his blindness. PRV DNA was detected from the aqueous humor. By literature review, a total of 20 cases (including this case) were analyzed. Most patients (95%, 19/20) had the history of direct contact with swine. The median incubation period was 7 days. The infection normally caused encephalitis (95%, 19/20), some cases with endophthalmitis (60%, 12/20). Based on the neuroimaging of the 19 patients, the lesions in neuroimaging were mainly in limbic system, especially in insular (17/19) and temporal lobes (17/19). The basal ganglia was often involved (9/19).The PRV-DNA was detected by NGS in CSF or intraocular fluid. Antiviral drugs and adjuvant treatment, including immunoglobulin and/or corticoid therapy, were effective to only few cases. Most patients (90%, 18/20) had the sequelae of severe impairment of daily living (modified Rankin Scale scores≥3).Conclusions:The cardinal clinical characteristics of human PRV infection are progressive panencephalitis and endophthalmitis, with an unfavorable outcome. The history of exposure to sick swine and typical neuroimaging suggest PRV infection. NGS of CSF and/or intraocular fluid is the dependable diagnostic method.
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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.
Subject(s)
Adult , Animals , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Viral , Blood , China , Encephalitis , Allergy and Immunology , Virology , Enzyme-Linked Immunosorbent Assay , Herpesvirus 1, Suid , Allergy and Immunology , Prevalence , Pseudorabies , Blood , Allergy and Immunology , Virology , Retrospective Studies , Seroepidemiologic StudiesABSTRACT
Objective: To investigate the effect of type I interferon receptor 1 (IFNAR1) on Pseudorabies virus (PRV) replication. Methods: A stable cell line in which porcine kidney epithelial cells (PK 15) knocked out the IFNAR1 gene was constructed using a lentiviral-mediated CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR) gene editing technique. The function of IFNAR1 was verified by cell viability assay, fluorescence observation, flow cytometry detection, titer determination, and Real-time PCR. Results: With the prolongation of PRV infection, knocking out the IFNAR1 gene can significantly promote transcription of PRV-TK mRNA, translation of PRV-gE protein, and virulence of progeny virus. Conclusion: IFNAR1 plays an important role in inhibiting the proliferation of PRV.
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Viral-encoded microRNAs (miRNAs) have vital roles in the regulation of virus replications and host immune responses. The results of previous studies have indicated that miRNA clusters are involved in the replication and virulence of the pseudorabies virus (PRV), which may potentially lead to immune escape or facilitation of PRV replication. This study's previous research revealed that prv-miR-LLT11a was differentially expressed during PRV infection. The present study's results have demonstrated that prv-miR-LLT11a could significantly inhibit PRV replication. It was further determined that SLA-1 was the target gene of prv-miR-LLT11a, and simultaneously, that overexpression of prv-miR-LLT11a could downregulate the mRNA and protein levels of SLA-1 in a dose-independent manner. Furthermore, the present study also observed that prv-miR-LLT11a can downregulate TAP1 expression. Our findings provide a better understanding of the molecular mechanism involved in the effects of prv-miR-LLT11a on SLA-1 and TAP1 as well as its involvement in immune system evasion of PRV.
Subject(s)
Herpesvirus 1, Suid , Immune System , MicroRNAs , Pseudorabies , RNA, Messenger , United Nations , Virulence , Virus ReplicationABSTRACT
Objective·To investigate the effectiveness of nerve transfer in repairing defecation function after spinal cord injury by the pseudorabies virus (PRV) retrograde tracing. Methods·The spinal cords were transected between L6 and S1 nerve root in 20 rats. The nerve transferring surgery was then conducted in 10 rats (Group B) and the remaining rats were control (Group A). After six months, all rats were injected with 6 μL PRV, sacrificed after 3 d and perfused with paraformaldehyde. Spinal cords were then harvested and frozen sections were prepared for observation. Results·There was no detectable infection of PRV proximal to the injury level in Group A, while infected neurons proximal to the injury level were widely observed in Group B.Conclusion·Nerve transfer has potent effect on defecation reconstruction after spinal cord injury in rats. PRV retrograde tracing can prove the existence of new neuron pathway.
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Objective·To investigate the effectiveness of nerve transfer in repairing defecation function after spinal cord injury by the pseudorabies virus (PRV) retrograde tracing. Methods·The spinal cords were transected between L6 and S1 nerve root in 20 rats. The nerve transferring surgery was then conducted in 10 rats (Group B) and the remaining rats were control (Group A). After six months, all rats were injected with 6 μL PRV, sacrificed after 3 d and perfused with paraformaldehyde. Spinal cords were then harvested and frozen sections were prepared for observation. Results·There was no detectable infection of PRV proximal to the injury level in Group A, while infected neurons proximal to the injury level were widely observed in Group B.Conclusion·Nerve transfer has potent effect on defecation reconstruction after spinal cord injury in rats. PRV retrograde tracing can prove the existence of new neuron pathway.
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To evaluate the gB antibody level of pseudorabies virus (PRV) in swine,a rapid test strip was developed.In the strip,the expressed protein of gB was labeled with colloidal gold,the staphylococcal protein A (SPA) and swine anti PRV antibody were blotted on the nitrocellulose membrane for the test and control lines,respectively.The specificity and sensitivity of the strip were detected with standard positive,negative and immunized sera of PRV,the results indicated that the strip was high specificity and sensitivity.Field swine serum samples were tested by the new strip and commercial IDEXX PRV gB ELISA kit,simultaneously.The agreement rate of the two methods was 91.04％.Furthermore,the dipstick assay based on the strip is rapid (5 min),sensitive and easy to perform.This suggests that the new strip is an acceptable alternative for field diagnosis.
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Outbreaks of pseudorabies (PR) have occurred in southern China since late 2011, resulting in significant economic impacts on the swine industry. To identify the cause of PR outbreaks, especially among vaccinated pigs, 11 pseudorabies virus (PRV) field strains were isolated from Guangdong province during 2013–2014. Their major viral genes (gE, TK, gI, PK, gD, 11K, and 28K) were analyzed in this study. Insertions or deletions were observed in gD, gE, gI and PK genes compared with other PRV isolates from all over the world. Furthermore, sequence alignment showed that insertions in gD and gE were unique molecular characteristics of the new prevalent PRV strains in China. Phylogenetic analysis showed that our isolates were clustered in an independent branch together with other strains isolated from China in recent years, and that they showed a closer genetic relationship with earlier isolates from Asia. Our results suggest that these isolates are novel PRV variants with unique molecular signatures.
Subject(s)
Asia , China , Disease Outbreaks , Genes, Viral , Herpesvirus 1, Suid , Pseudorabies , Sequence Alignment , SwineABSTRACT
The pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the exact function of EP0 is not clear. In this study, using polymerase chain reaction, a 1,104 base-pair sequence of the EP0 gene was amplified from the PRV Becker strain genome and identification of the EP0 gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV EP0 gene encoded a putative polypeptide with 367 amino acids. The encoded protein, designated as EP0 contained a conserved RING-finger superfamily domain and was found to be closely related with the herpes virus RING-finger superfamily and was highly conserved among the counterparts encoded by RING-finger genes. Multiple nucleic acid sequence and amino-acid sequence alignments suggested that PRV EP0 showed a relatively higher similarity with EP0-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV EP0 had a close evolutionary relationship with members of genus Varicellovirus, especially bovine herpesvirus 1 (BoHV-1) and BoHV-5. Antigen prediction indicated that several potential B-cell epitopes were located in EP0. Also, subcellular localization analysis demonstrated that EP0 was predominantly localized in the nucleus, suggesting that it might function as a nuclear-targeted protein.
Subject(s)
Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , Computational Biology , DNA, Viral/genetics , Herpesvirus 1, Suid/genetics , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Protein Structure, Secondary , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
Our investigation was conducted in order to verify a recent severe epidemic at several swine farms in northern China that indicated a newly emerging disease. Evidence confirmed that the epidemic was caused by a virulent Pseudorabies virus infection in swine herds.
Subject(s)
Animals , China/epidemiology , Enzyme-Linked Immunosorbent Assay/veterinary , Epidemics/veterinary , Herpesvirus 1, Suid/classification , Pseudorabies/epidemiology , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Swine , Swine Diseases/epidemiology , Vaccination/adverse effects , VirulenceABSTRACT
In the present study,we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses.Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses,indicated by codon adaptation index,effective number of codons (ENc) and GC3s value.The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus,with a strong bias towards the codons with C and G at the third codon position.Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions.ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content,as well as the gene length.In addition,comparison of codon preferences in the US1 gene of PRV with those of E.coli,yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast,49 between PRV and human,but 48 between PRV and E.coli.Although there were slightly fewer differences in codon usages between E.coli and PRV,the difference is unlikely to be statistically significant,and experimental studies are necessary to establish the most suitable expression system for PRV US1.In conclusion,these results may improve our understanding of the evolution,pathogenesis and functional studies of PRV,as well as contributing to the area of herpesvirus research or even studies with other viruses.
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To evaluate the value of the vaccinations with 3 strains of gene-deleted mutants from pseudorabies virus(PRV), PRV TK-, PRV gE-/gI- and PRV TK-/gE-/gI- after to exposure to the wild Fa strain, these mutant strains from the PVR reference isolate Fa were used to vaccinate 4 weeks old PRV-free pigs with a dosage of 105 PFU each ,and followed by nasally challenged by the parental Fa strain with a dosage of 107 PFU at 14 days post vaccination. The pathological changes, virus discharge and distribution were evaluated after vaccination and challenge. It was found that the histopathological observations in the 10 collected samples including cerebrum, cerebellum, heart, liver, lungs, spleen, kidneys, tonsils, lymph nodes and trigeminal ganglion from these 3 mutant strains showed that the rates of occurrence of pathological changes in various organs were 4/10, 3/10 and 4/10 respectively, whereas that of the positive controls were 9/10. The damage in lungs was more serious in pigs vaccinated with PRV TK-mutant and positive control in comparison with other groups of pigs inoculated, and the damages in cerebrum, cerebellum and trigeminal ganglion in positive controls were more serious than those of pigs vaccinated with the 3 gene-deleted mutants. However, the tonsils, the main organ for latent infection were damaged mildly in the pigs inoculated with these 3 gene-deleted mutants in comparison with that of the positive controls. As demonstrated by Southern blot analysis, all the vaccinated pigs could discharge viruses by secretion through nasal cavity, but the soldier pigs were not infected successively by the gene-deleted mutants and the gene-deleted mutants were also unable to establish infection in cerebrum and cerebellum. Nevertheless, they could not effectively block discharge of PRV Fa after exposure to Fa virus, but could block effectively the virulent Fa virus invading into cerebrum and cerebellum. From these observation, it is evident that the deleted mutants of the TK, gE/gI , TK/gE/gI genes can block the invasion of virulent Fa virus into cerebrum and cerebellum and lessen the damages on multiple organs or tissues ,indicating that the deleted mutant of TK/gE/gI gene may be the most promising candidate of vaccine strain for development of the commercial vaccine.
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PURPOSE: c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV). MATERIALS AND METHODS: Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons. RESULTS: Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/ section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4+/-10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/ section, and 2.7+/-1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder- specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder. CONCLUSION: We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.
Subject(s)
Animals , Female , Rats , Herpesvirus 1, Suid/physiology , Immunohistochemistry , Interneurons/cytology , Neurons/cytology , Proto-Oncogene Proteins c-fos/metabolism , Rats, Sprague-Dawley , Spinal Cord Injuries/physiopathology , Urinary Bladder/cytologyABSTRACT
lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.
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@# ObjectiveTo determine the effect of acute and chronic spinal cord injury (SCI) resulted from thoracic cord transection on the urinary bladder spinal neural pathway.Methods76 adult Sprague-Dawley rats were randomly divided into 4 groups, non-SCI, SCIa, SCIb and SCIc respectively. The non-SCI rats underwent no surgical procedure except Pseudorabies virus (PRV) tracer injection into the bladder tissue, while the rats of other groups were spinalized and given PRV injection at different time after SCI. Transcardiac perfusion fixation was done in appropriate survival periods after PRV injection. Then sections of dorsal root ganglion (DRG) and spinal cord were processed for visualization of virus by the Streptavidin-Peroxidase (SP) immunohistochemical procedure. All sections were measured with the Olympus Cue-2 image analysis system.ResultsThe bladder weights in SCIb and SCIc groups markedly increased (P<0.001). The time-ordered flow charts of PRV tracing were similar in the non-SCI rats and in the SCI rats. The cross-sectional area of the labeled DRG cell profiles increased significantly after SCI (P<0.001). The number of labeled cells in dorsal horn in L6 and S1 segments 3 days after PRV injection markedly increased in chronic SCI rats, and so did the number of labeled motor neurons 4 days post-injection. ConclusionThe acute and chronic SCI have little effect on the process of virus transneuronal transport below the level of lesion. Subsequent to chronic SCI, marked reorganization of the micturition reflex pathways occurs.
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Purpose: No ideal method for subdividing and assessing changes in neurons of the spinal cord during specific conditions has been established. We attempted to develop a method for subdividing spinal neurons using immunohistochemical and fluorescent staining, which is an important key towards understanding the mechanism of reflex voiding. Materials and Methods: Thirty Sprague-Dawley rats, weighting 200-300g, were divided into five groups. A cystometrogram was performed during saline or acetic acid instillation. We identified the neuronal pathway associated with the detrusor by injecting a pseudorabies virus (PRV) into the detrusor muscle and inspecting the changes in relation to different time sequences. An immunohistochemical staining method was used to stain the fos-protein encoded by the c-fos gene. Immunofluorescent staining was performed to evaluate changes in the neurons in relation to the voiding reflex, and the neurons then subdivided. Results: We confirmed pseudorabies virus (PRV) infection of the cells in the sacral parasympathetic nucleus through immunohistochemical staining two days after injection. On detection of an increase in c-fos positive cells after dividing the c-fos positive area of the L6 and S1 spinal cord into 4 sections, significant increases were observed in the sacral parasympathetic nucleus (SPN) and dorsal commissure (DCM). Double staining was performed to detect the neurons associated with the voiding reflex in the SPN and DCM areas showing overexpression of c-fos. Conclusions: The establishment of a method for detecting morphological changes, and subdividing neurons by immunohistochemical and fluorescent staining, may provide an important key towards understanding the mechanism of various neuromodulations of clinically applied treatments. (Korean J Urol 2005;46:487-494)
Subject(s)
Acetic Acid , Genes, fos , Herpesvirus 1, Suid , Neurons , Proto-Oncogene Proteins c-fos , Rats, Sprague-Dawley , Reflex , Spinal CordABSTRACT
The nerves innervating the sublingual gland of the rat was investigated using PRV (pseudorabies virus) as a neural tracer. The neural tracer was injected into left sublingual gland of the rat. In the central nervous system, PRV immunoreactive neurons were labeled bilaterally and tended to be more densely labeled in the left side. PRV immunoreactive neuronal cell bodies and fibers were observed in insular cortex, paraventricular nucleus, deep mesencephalic nucleus, spinal trigeminal tract, lateral paragigantocellular nucleus, parvicellular reticular nucleus, raphe obscurus, gigantocellular reticular nucleus and gigantocellular reticular nucleus, alpha. The more densely labeled PRV immunoreactive neurons were found in the deep mesencephalic nucleus, spinal trigeminal tract and lateral paragigantocellular nucleus. These results may provide a neuroanatomical data on the nerves innervating the sublingual gland in the rat brain.
Subject(s)
Animals , Rats , Brain , Central Nervous System , Herpesvirus 1, Suid , Neurons , Paraventricular Hypothalamic Nucleus , Pseudorabies , Raphe Nuclei , Sublingual Gland , Trigeminal Nucleus, SpinalABSTRACT
The hippocampus is known as involved in learning and memory functions and the entorhinal cortex plays a crucial role as a gateway connecting the several areas and hippocampal formation. Entorhinal cortex lesions have been employed in numerous studies as the Alzheimer's disease model. The purpose of this study were to identify the CNS hip-pocampal and cholinergic pathway and to investigate the morphological changes of the hippocampal cholinergic inner-vations by using the Pseudorabies virus injection into the hippocampus after entorhinal cortex lesions. The pseudorabies virus and double labelled neurons (ChAT and PRV) were distributed at several different nuclei including agranular insular cortex, bed nucleus of stria terminalis, central amygdala, globus pallidus, lateral segment, lateral hypothalamic area, laterodorsal tegmental nucleus, medial septal nucleus, mesencephalic reticular nucleus, periaqueductal gray matter and substantia innominata The morphological changes were observed in the hippocampal cholinergic innervation after entorhinal cortex lesions. These data suggested that the hippocampal cholinergic innervation showed morphological changes throughout the whole brain areas after entorhinal cortex lesion.
Subject(s)
Animals , Rats , Alzheimer Disease , Amygdala , Brain , Entorhinal Cortex , Globus Pallidus , Herpesvirus 1, Suid , Hippocampus , Hypothalamic Area, Lateral , Learning , Memory , Neurons , Periaqueductal Gray , Septal Nuclei , Substantia InnominataABSTRACT
The nerves innervating the auricle of the rat were investigated using PRV (pseudorabies virus) as a neural tracer. The neural tracer was injected into rostral part of the right auricle of the rat. The PRV immunoreactive neurons were labeled bilaterally and more densely labeled in the brain than spinal cord. In the brain, PRV immunoreactive neuronal cell bodies and fibers were observed in thalamus, periaqueductal gray matter, reticular formation, spinal trigeminal nucleus, spinal tract of trigeminal nerve and facial nucleus. The more densely labeled PRV immunoreactive neurons were found in thalamus, reticular formation, spinal tract of trigeminal nerve and facial nucleus. In the spinal cord, PRV immunoreactive neurons were extended from T8 to L2 segments. The more densely labeled PRV immunoreactive neurons were found from T11 to L2 segments. Above results, the nerves innervating the auricle of the rat were widely distributed in brain and spinal cord and may have many connections with other nerves. These results may provide a neuroanatomical data on the nerves innervating the auricle of the rat in the central nervous system.