Detection of Pseudorabies Virus Antibodies in Human Encephalitis Cases / 生物医学与环境科学（英文）

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.

Functional analysis of prv-miR-LLT11a encoded by pseudorabies virus

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.

Neurologic Mechanisms Underlying Voiding Dysfunction due to Prostatitis in a Rat Model of Nonbacterial Prostatic Inflammation / 대한배뇨장애요실금학회지

PURPOSE: The neurological molecular mechanisms underlying the voiding dysfunction associated with nonbacterial chronic prostatitis/chronic pelvic pain syndrome remain poorly understood. In this study, we assessed whether prostate inflammation activated bladder afferent neurons, leading to bladder dysfunction, and sought to elucidate the underlying mechanisms. METHODS: Thirty male Sprague-Dawley rats were divided into 3 groups: sham-saline, formalin-injected, and capsaicin-pretreated and formalin-injected. Chemical prostatitis was induced by 0.1 mL of 10% buffered formalin injected into the ventral prostate. Capsaicin was injected subcutaneously to desensitize capsaicin-sensitive nerves. In each group, conscious cystometry was performed, and c-fos expression within the spinal cord was determined immunocytochemically. Double immunofluorescent staining with c-fos and choline acetyltransferase (ChAT) was performed. On the third day after pseudorabies virus (PRV) infection, c-fos and PRV double-staining was performed. RESULTS: Intraprostatic formalin significantly increased the maximal voiding pressure and decreased the intercontraction interval, compared with controls. Pretreatment with capsaicin significantly reversed these effects. More c-fos-positive cells were observed in the sacral parasympathetic nucleus (SPN) and dorsal gray commissure (DCM) in the prostatitis group than in the sham group. c-fos-positive cells decreased in the capsaicin-pretreated group. Preganglionic neurons labeled by c-fos and ChAT were observed in the SPN in rats with prostatitis. Interneurons labeled by c-fos and PRV were identified in the DCM after PRV infection. CONCLUSIONS: Our results suggest that prostate inflammation activates afferent nerve fibers projecting to the lumbosacral spinal cord, producing reflex activation of spinal neurons innervating the bladder and bladder hyperreflexia. This is mediated by capsaicin-sensitive prostate afferent neurons.

Detección serológica de Brucella suis, virus de influenza y virus de la enfermedad de Aujeszky en criaderos porcinos familiares de menos de 100 madres en Argentina / Serological detection of Brucella suis, influenza virus and Aujeszky's disease virus in backyard and small swine holders in Argentina

Los criaderos porcinos de menos de 100 madres representan más del 99% de los de todo el país; sin embargo, existen escasos reportes sobre su situación sanitaria y productiva. Se recabó información productiva y se tomaron muestras para detectar anticuerpos contra Brucella suis (Bs), virus de la enfermedad de Aujeszky (VA) y virus de influenza (VI) en 68 establecimientos de menos de 100 madres ubicados en la región norte, centro y sur del país. El 80% de los establecimientos fueron positivos al VI H1 pandémico 2009, el 11% al H3 clúster 2, mientras que el 11,7% presentó anticuerpos contra el VA y el 6% contra Bs. Ninguno de los productores conocía los factores de riesgo para la transmisión de enfermedades del cerdo al humano. El 47% compra sus reproductores a pares o en ferias. En lo que respecta a normas de bioseguridad, solo el 16% de los establecimientos tenía cerco perimetral y el 37% de las granjas contaba con asesoramiento veterinario. Los resultados de este estudio demuestran que la caracterización productiva y el relevamiento sanitario son de suma importancia para mejorar la productividad y reducir el riesgo de transmisión de enfermedades. El conocimiento de la situación sanitaria y de los factores de riesgo es necesario para conseguir un mejor control y la erradicación de enfermedades en sistemas de baja tecnificación. Se deberían llevar a cabo estudios más representativos a nivel país para detectar los agentes circulantes y, sobre la base de esta información, implementar medidas de prevención y control.

Farmers raising less than 100 sows represent more than 99% of swine producers in Argentina, although little is known about their sanitary status and productive characteristics in the country. Sanitary and productive information was obtained. Furthermore, samples for serological studies were taken to detect antibodies against Brucella suis (Bs), Aujeszky's disease virus (AV) and influenza virus (IV) in 68 backyard and small producers with less than 100 sows located in the north, central and south regions of Argentina. Antibodies against H1 pandemic were detected in 80% of the farms while 11%, 11.7% and 6.0% of the producers were positive to influenza H3 cluster 2, AV and Bs, respectively. None of the producers was aware of the risk factors concerning the transmission of diseases from pigs to humans. A percentage of 47% of them buy pigs for breeding from other farmers and markets. With regard to biosecurity measures, only 16% of the farms had perimeter fences. The results of this study demonstrate that productive characterization and disease surveys are important to improve productivity and to reduce the risk of disease transmission among animals and humans. The study of sanitary status and risk factors is necessary for better control and eradication of diseases in backyard or small producers. More representative studies at country level should be carried out to detect the pathogensthat circulate and, with this knowledge, to implement prevention and control measures.

Immune Response of Recombinant Pseudorabies Virus rPRV-VP2 Expressing VP2 Gene of Porcine Parvovirus in Mice / 病毒学报

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.

An oral Aujeszky's disease vaccine (YS-400) induces neutralizing antibody in pigs

PURPOSE: Aujeszky's disease (AD) is an economically important disease affecting both wild and domestic pigs of the species Sus scrofa. A previous study yielded serological evidence of AD in Korean wild boars, which could spread AD to other animals. A new Aujeszky's disease virus (ADV) bait vaccine is required to prevent AD outbreaks in swine. In the present study, we investigated the safety and immunogenicity of a gE-deleted marker vaccine, strain YS-400, in young domestic pigs. MATERIALS AND METHODS: The YS-400 strain was propagated in Vero cells, and the trial ADV bait vaccine (a vaccine blister in a matrix including an attractant) was prepared. Pigs were orally immunized with the vaccine (2 mL, 10(7.5) TCID(50)/mL) delivered using a syringe or in the bait vaccine. The animals were observed for 9 weeks after vaccination, and immunogenicity was assessed using a virus neutralization (VN) test and enzyme linked immunosorbent assay. RESULTS: The YS-400 strain was non-pathogenic to pigs when given orally and induced high VN titers (1:32-1:128) 6 weeks post-administration. Of the pigs given the ADV bait vaccine twice or three times, 40% were seropositive by 2 weeks, and 100% were seropositive by 7 weeks after the first dose. Pigs that consumed the AD bait vaccine three times developed VN titers that were slightly higher than those of pigs given the vaccine twice. CONCLUSION: Domestic pigs given the trial ADV bait vaccine exhibited no adverse effects and developed high VN titers against ADV, indicating that the YS-400 strain is safe and can prevent ADV infection in domestic pigs.

Seroprevalence and associated risk factors of pseudorabies in Shandong province of China

A cross-sectional serological study was conducted in Shandong province of China to determine the seroprevalence and risk factors associated with seropositivity due to pseudorabies virus (PRV) infection in small- and medium-sized farrow-to-finish herds following outbreaks of variant PRV strains. A total of 6,035 blood samples from 224 randomly selected herds were screened. The results showed that 25.0% of the herds and 56.7% of the serum samples were seropositive for field strains of PRV. Herds consisting of 50–100 breeding sows had higher herd seroprevalence and serum sample seroprevalence than larger herds. Both the highest herd seroprevalence and highest serum sample seroprevalence were observed in western Shandong, followed northern Shandong. Based on univariate analysis, the following risk factors were utilized in subsequent multivariable logistic regression analysis: region, herd size, weight of purchased gilts, and all-in/all-out practice. Upon multivariate analysis, region, herd size, weight of purchased gilts and all-in/all-out practice were significantly associated with PRV herd seropositivity. These findings indicate that we are facing a serious situation in the prevention and control of pseudorabies. The results could help predict the next outbreak and set out control measures.

Molecular characterization and phylogenetic analysis of pseudorabies virus variants isolated from Guangdong province of southern China during 2013–2014

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.

Innate Immune Evasion Mechanisms of Pseudorabies Virus / 病毒学报

Pseudorabies is an economically important disease in a variety ot animals caused by pseudorabies virus. Since 2011, pseudorabies outbreaks occurred in many regions of China. Related researches on this virus become a hot topic in virology and veterinary. One of the difficulties for pseudorabies prevention and control is innate immune evasion. Explorations on this issue are conducive to the development of vaccine and drugs. Therefore, this review summarized the recent research progress on the mechanisms of pseudorabies virus innate immune evasion. Theoretical direction was provided on effetive prevention and control of pseudorabies owing to this review.

Molecular cloning and characterization of Pseudorabies virus EP0 gene.

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.

Seroepidemiological Survey of Aujeszky's Disease Virus in Wild Boar (Sus scrofa) and Raccoon Dogs (Nyctereutes procyonoides koreensis) in Korea

Aujeszky's disease caused by Aujeszky's disease virus (ADV) is one of the most important diseases in the pig industry. In this study, we conducted a seroepidemiological survey of ADV in wild boars and raccoon dogs in South Korea. In total, 217 wild boar sera collected between March and August 2013, and 96 raccoon dogs between 2011 and 2012 were screened for the presence of antibodies against ADV. The sero-positive rates in wild boars and raccoon dogs tested for ADV were found to be 3.55% (8/225) and 0% (0/96), respectively. The presence of virus neutralization antibody titer against ADV means that small number of wild boars was infected with ADV and AD may be circulated continuously in Korean wild boar populations, and that wild boars may act as a potential reservoir of ADV. Therefore, to achieve the declaration of AD free, effective preventive measures to block transmission of AD should be taken to the wild boars.

Studies on neuronal tracing with pseudorabies virus / 病毒学报

With its abilities of trans-synaptic tracing and self-replication and wide host range, pseudorabies virus (PRV) has been applied in the field of neuroanatomy since the 1970s. Four decades of PRV application have made many advances in researches on neuronal tracing with PRV. Mechanism studies focused on investigating infection of primary neurons and tracing direction in secondary neurons, while application studies focused on development of new pathological strains and innovation of tracing techniques. To date, the mechanism and application of viral tracing are not completely figured out yet. Integration of molecular biology technology will improve the efficiency in related researches.

Identification of nuclear localization signals of pseudorabies virus gene UL49 / 病毒学报

Tegument protein VP22 is encoded by Pseudorabies Virus (PRV) UL49. To identify the nuclear localization signals of UL49, it is necessary to determine the transport mechanism and biological functions of the VP22 protein. In this study, we identified two nuclear localization signals from UL49, NLS1 (5RKTRVA ADETASGARRR21) and NLS2 (241PGRKGKV247). The functional nuclear localization signal (NLS) of UL49 was identified by constructing truncated or site-specific UL49 mutants. The deletion of both NLS1 and NLS2 abrogated UL49 nuclear accumulation, whereas the deletion of NLS1 or NLS2 did not. Therefore, both NLS1 and NLS2 are critical for the nuclear localization of UL49. And our resuts showed that NLS2 is more important in this regard.

Investigation of etiology of massive infection with porcine pseudorabies virus in Henan and neighboring Provinces / 病毒学报

In early 2011, the serious outbreak of porcine pseudorabies virus (PRV) infection suddenly recurred in Henan and neighboring Provinces. To investigate the etiology of massive infection with PRV, 16 800 serum samples, 905 porcine epidemic diarrhea virus (PEDV) back-feeding tissues, and 56 PR gene deleted live vaccines were colleted from January 2011 to May 2013 to detect PRV field infection using a PRV gE antibody test kit. The gE and TK genes of 11 new epidemic PRV strains were sequenced by PCR, and their molecular characteristics were analyzed. Moreover, virus titer determination, protective test against PRV, and vaccine potency testing were performed. The results showed that the detection rate of PRV field infection-positive pig farms was 68.06%, and the overall positive rate of PRV field infection in serum was 38.47%; the positive rates in breeding sows, breeding boars, reserve pigs, and commercial pigs were 40.12%, 30.88%, 54.67%, and 26.52%, respectively. The new epidemic strains were in the same evolutionary branch and belonged to the virulent strain group. Compared with the classical PRV strain, the virulence of new epidemic strains changed a little. The length of gE gene was 1 787 bp, and the length of TK gene was 963 bp. The nucleotide homologies of gE and TK genes to Chinese reference strains were 98.2%-99.8% and 98.90%-99.6%, respectively, and the amino acid homologies were 97.1%-99.8% and 97.5%-99.4%, respectively. Commercial vaccine had a 100% protective effect against the new epidemic strains. The positive rate of PRV field infection was 0% in vaccine and 40.44% in back-feeding tissues. The results confirmed that PRV field infection rates were rising sharply among pigs in Henan and neighboring Provinces after 2011. The main virulence genes of new epidemic PRV strains did not change significantly over the years. PR gene deleted live vaccines had no PRV field infection and could completely resist the attack of new strains. The virus carriage of breeding boars and reserve pigs and the serious PRV field infection in PEDV back-feeding tissues were the main causative factors for massive infection with PRV and epidemic outbreak in Henan and neighboring Provinces from 2011 to 2013.

Emergence of virulent pseudorabies virus infection in Northern China

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.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.

Atividade antiviral de extratos de plantas medicinais disponíveis comercialmente frente aos herpesvírus suíno e bovino / Antiviral activity of commercially available medicinal plants on suid and bovine herpesviruses

O presente trabalho teve como objetivo pesquisar a atividade antiviral in vitro de plantas medicinais disponíveis comercialmente sobre herpesvírus suíno (SuHV-1) e bovino (BoHV-1). As espécies adquiridas foram Mikania glomerata, Cymbopogon citratus, Equisetum arvense, Peumus boldus, Solanum paniculatum, Malva sylvestris, Piper umbellatun e Solidago microglossa. A citotoxicidade dos extratos foi avaliada na linhagem celular MDBK pelas alterações morfológicas das células e obtenção da concentração máxima não citotóxica (CMNC) de cada planta. A atividade antiviral foi realizada com os extratos em suas respectivas CMNC e avaliada com base na redução do título viral e expressos em porcentagem de inibição. Os extratos aquosos de Peumus boldus e Solanum paniculatum apresentaram atividade antiviral sobre o SuHV-1 com 98% de inibição viral enquanto o de Peumus boldus inibiu apenas o BoHV-1 em 99%.

This paper aims to find commercially available medicinal plants showing antiviral activity in vitro on suid and bovine herpesviruses. The following species were tested: Mikania glomerata, Cymbopogon citratus, Equisetum arvense, Peumus boldus, Solanum paniculatum, Malva sylvestris, Piper umbellatun and Solidago microglossa. The cytotoxicity was evaluated by morphological changes in cells determining the maximum not cytotoxic concentration (MNCC). The antiviral activity was evaluated by viral title reduction. The extracts from Peumus boldus and Solanum paniculatum showed antiviral activity against SuHV-1 with 98% of inhibition. The extract of Peumus boldus also showed activity against BoHV-1 with 99% of inhibition.

Identification of CNS neural circuitry involved in the innervation of C7 spinal nerve: a viral transsynaptic tracing study / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>Contralateral C7 spinal nerve transfer is a useful operation for the treatment of brachial plexus root avulsion. The recovery of the independent function at the ipsilateral side, however, depends on neural circuitry reorganization in the central nervous system (CNS). This study tried to locate the CNS neuronal elements involved in the innervation of C7 spinal nerve.</p><p><b>METHOD</b>Pseudorabies virus (PRV, TK/gG(-), 2 microlitre), which expressed green fluorescent protein (GFP), was injected into the left C7 spinal nerve in 20 adult Sprague Dawley rats. After rats survived for 6 h, 12 h, 24 h and 36 h, the C1-C7 segments of the spinal cord and brain were processed using a polyclonal immunohistochemical antibody against PRV.</p><p><b>RESULTS</b>PRV-labeled neurons were found mainly in gray matter of the C1-C7 segments of the spinal cord and at the following structures of the brain: lateral vestibular nucleus, lateral paragigantocellular nucleus, A5 cells, red nucleus, primary and secondary motor cortexes, primary and secondary somatosensory cortexes. Although located bilaterally, the PRV-labeled neurons existed predominantly in the ipsilateral side of the spinal cord and the contralateral side of the brain at 6-12 h after injection (p.i.). The number of PRV-labeled neurons in the CNS was increasing with rat's survival time and the distribution of these neurons turned bilateral with no obvious dominance to either side at 24 h and 36 h (p.i.).</p><p><b>CONCLUSION</b>By use of transsynaptic tracing technique with PRV, the anatomically connected set of neurons, which modulates the activity of C7 spinal nerve, is located successfully in the CNS.</p>

Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.