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.

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.

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.

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.

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.

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.

Soroprevalência de pseudorraiva, peste suína clássica e brucelose em suínos do estado do Piauí / Seroprevalence of pseudorabies, classical swine fever and brucellosis in swine in the state of Piauí

Este trabalho teve como objetivo determinar a soroprevalência de pseudorraiva, peste suína clássica (PSC) e brucelose suína em suínos do estado do Piauí, Brasil. Foram coletadas amostras sanguíneas de 384 suínos de criações intensivas e extensivas do estado. Anticorpos anti-Brucella spp. foram detectados pelo teste do antígeno acidificado tamponado e confirmados pelo teste 2-mercaptoetanol, enquanto a detecção de anticorpos contra os vírus da PSC e pseudorraiva foi realizada por ensaio imunoenzimático (ELISA), utilizando-se kits comerciais específicos. Anticorpos anti-Brucella spp. foram detectados em 1,04% (2/192) dos suínos de criações intensivas. Dos rebanhos avaliados, 0,78% (3/384) dos animais exibiram anticorpos contra o vírus da PSC, sendo 1,04% (2/192) de criações intensivas e 0,52% (1/192) de criações extensivas. Anticorpos contra o vírus da pseudorraiva foram detectados apenas em suínos de criação extensiva, com prevalência de 5,2% (10/192). Esses são os primeiros dados sobre a soroprevalência da brucelose suína, pseudorraiva e PSC em rebanhos do Piauí. A detecção de 10 amostras positivas para pseudorraiva causa preocupação sobre a possibilidade da circulação viral na população suídea desse estado e revela uma necessidade premente de realização de estudos mais extensos para melhor compreender a importância dessas enfermidades de notificação obrigatória em estados da região Nordeste brasileira.

This study aimed to determine the prevalence of Pseudorabies, Classical Swine Fever (CSF) and Swine Brucellosis in swine in the state of Piauí, Brazil. Blood samples were collected from 384 pigs from intensive and small outdoor systems in the state. Anti-Brucella spp. antibodies were detected by Buffered Acidified Antigen Test and positive results confirmed by 2-Mercaptoethanol Test. Detection of antibodies against CSF and Pseudorabies virus were performed by Enzyme-Linked Immunosorbent Assay (ELISA) using specific commercial kits. Only two samples (1.04% - 2/192) from the intensive system were seropositive to Brucella spp. In the evaluated herds, 0.78% (3/384) of animals had antibodies against CSF virus, two from outdoor pigs (1.04% - 2/192) and one from intensive systems (0.52% - 1/192). Antibodies against the Pseudorabies virus were detected only in outdoor pigs, with seroprevalence of 5.2% (10/192). This is the first report on seroprevalence of Pseudorabies, CSF and Brucellosis in hog farms in Piauí, Brazil. The detection of 10 positive cases raises a concern regarding Pseudorabies virus circulation in the swine population in the state and reveals a need for further studies to better understand the real situation and status of obligatory notified diseases in the swine herds in the Northestern states of Brazil.

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.

Neurological disorder in cattle associated with bovine herpesvirus 4 / Desordem neurológica em bovinos associada ao herpevírus bovino 4

A nested PCR assay was used to diagnose bovine encephalitis through herpesviruses including bovine herpesvirus 5 (BHV-5), bovine herpesvirus 1 (BHV-1), Aujeszky's disease virus (SHV-1), and ovine herpesvirus 2 (OHV-2) in 14 fragments of central nervous system (CNS) from cattle that died with neurological signs. In addition, as some samples of bovine herpesvirus type 4 (BHV-4) have been isolated from neural tissue, it was also tested by nested PCR. The cases of encephalitis occurred in isolation at different times of the year and did not present any seasonality. The duration of the clinical course ranged between 1 to 15 days, and in 64.3 percent of the cases it manifested between 1 to 2 days. The most frequently observed neurological signs were ataxia, recumbency, unsteadiness and inability to stand, opisthotonus, paddling movements, nystagmus and ptyalism. In the nested assay, there was no evidence of: BHV-1, SHV-1 or OHV-2 in the DNA obtained from the CNS in any of the samples. But the presence of BHV-4 was found in all fragments of the CNS in cattle which died presenting neurological signs. Moreover, BHV-5 was found in association with BHV-4 in two of these samples.

Nested PCR foi utilizada para o diagnóstico de encefalite bovina por herpesvírus incluindo o herpesvírus bovino 5 (BHV-5), o herpesvírus bovino 1 (BHV-1), o vírus da doença de Aujeszky (SHV-1) e o herpesvírus ovino 2 (OHV-2) em 14 fragmentos do sistema nervoso central (SNC) de bovinos que morreram com sinais neurológicos. Embora o BHV-4 não seja reconhecido como vírus neurotrófico, foi detectado nos casos de encefalite que ocorreram isoladamente em diferentes épocas do ano e não apresentaram nenhuma sazonalidade. A duração do curso clínico variou entre 1 e 15 dias, e em 64,3 por cento dos casos manifestou-se entre 1 e 2 dias. Os sinais neurológicos mais freqüentemente observados foram ataxia, apatia, instabilidade, opistótono, movimentos de pedalagem, nistagmo e sialorréia. Nos ensaios de PCR nested realizados a partir do DNA obtido do SNC, não foi encontrado evidência de: BHV-1, SHV-1 ou OHV-2 em nenhuma das amostras. Mas, a presença de BHV-4 foi encontrada em todos os fragmentos do SNC de bovinos que morreram com sinais neurológicos. Além disso, o BHV-5 foi encontrado em associação com o BHV-4 em duas dessas amostras.

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.

Construction of an infectious clone of pseudorabies virus strain ZJ genome maintained as a bacterial artificial chromosome / 病毒学报

pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.

Erradicação da doença de Aujeszky em Santa Catarina: importância da condição sanitária das leitoas de reposição / Aujeszky's diasease eradication in Santa Catarina State: relevance of sanitary status of replacement gilts

A doença de Aujeszky (DA) é uma infecção causada por um herpesvírus, o vírus da DA (VDA), primariamente em suínos. Esta doença está presente no Estado de Santa Catarina (SC) desde 1984. Devido ao impacto da DA no mercado exportador de carne suína, no comércio de reprodutores e nas perdas de produtividade, um programa de erradicação, financiado por uma parceria entre a indústria e a associação de produtores tem tido sucesso em eliminar gradualmente a DA de rebanhos suínos de SC. O último caso de DA no estado foi identificado em julho de 2004. Durante o processo de despovoamento/repovoamento, foi detectado um rebanho suíno positivo para o VDA localizado no oeste de SC. Estudos de rastreabilidade da origem daqueles animais indicaram que a fonte era uma granja que distribuía reprodutores ilegalmente sem certificação sanitária. Este suinocultor mantinha um sistema de integração que incluía 40 diferentes produtores para quem eram comercializados reprodutores e/ou suínos para terminação. Testes de soroprevalência detectaram anticorpos anti-VDA em 12 daqueles rebanhos. Devido a sua localização dentro do raio de 2,5km do foco inicial, uma outra granja, onde havia uma central de inseminação artificial que distribuía sêmen suíno para outras 5 granjas do mesmo proprietário, foi testada e também resultou positiva. Os objetivos deste artigo são descrever as condições sanitárias frente ao VDA naquelas granjas que receberam suínos ou sêmen suíno daqueles suinocultores, as medidas para controlar e eliminar o VDA dos rebanhos positivos e a situação atual decorrente deste trabalho. Além disso, o artigo busca alertar que medidas de vigilância ativa e normas sanitárias para comércio e distribuição de material genético devem ser seguidas, caso contrário, a DA pode recrudescer e tornar-se fora de controle como ocorria antes do início deste programa.

Aujeszky's disease (AD) is an herpesvirus infection, caused by the pseudorabies virus (PRV), primarily in swine and present in Santa Catarina State (SC) since 1984. Due to the impacts of AD in the pork export, breeder's trade and productivity losses, an eradication program, financed by industry and swine producers association, has successfully eradicated the AD of swine herds in SC State. The last case of AD in the State was identified in July of 2004. During the depopulation/repopulation process, a PRV positive swine herd located in the west region of SC State was detected. Traceability studies of the origin of those animals indicated that the source was a swine farm which illegally distributed breeders without sanitary certification. This swine producer maintained an integration system which included 40 different producers, to whom were commercialized breeders and/or finishers. Serum-prevalence tests detected PRV antibodies in 12 of those herds. Due to the location into the 2.5km of radius from the initial outbreak, another swine farm, which had an artificial insemination center that distributed swine semen to another 5 herds of the same owner was tested positive as well. The objectives of this paper are to describe the sanitary status related to AD on the farms which have received pigs or swine semen from these swine producers, the measures to control and eliminate ADV from positive herds and the outcome of this work. Beyond that, to alert that measures of active surveillance and sanitary rules for commerce and distribution of genetic materials must be properly fulfilled, otherwise, AD can reactivate and become out of control as occurred before the eradication program.

Diagonsis establishment of fluorescen quantitative PCR assay for pseudorabies wild-type virus and vaccine virus / 生物工程学报

We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.

Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-gamma and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.