Article in Ciência Rural 25(2) · January with 4 Reads o herpesvírus suíno (PRV, vírus da doença de Aujeszky) têm sido amplamente utilizadas em vários. doença de Aujeszky em sistema de baculovirus. Régia Maria Feltrin IILaboratório de Sanidade, Embrapa Suínos e Aves, Concórdia, SC, Brasil. IIICentro de. CLONING AND EXPRESSION OF AUJESZKY’S DISEASE VIRUS GLYCOPROTEIN E .. vírus da doença de Aujeszky de surtos em suínos no Estado de Santa.
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The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals.
The development, optimization and evaluation of a polymerase chain reaction PCR assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a aujesz,y conserved viral gD gene soena. Non-specific reactions were not observed when a related herpesvirus, other porcine DNA genome viruses and uninfected cells were used to assess PCR.
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The analytical sensitivity of the test was estimated to be 1. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. A rapid, sensitive and specific PCR-based diagnostic assay to detect pseudorabies virus in clinical samples is provided. The etiological agent of this disease is suid herpesvirus type 1, usually named pseudorabies virus PRVa pantropic alphaherpesvirus which causes fatal infections in baby pigs, respiratory disease and poor growth in fattening pigs and reproductive disorders in adults 28 The virus principally affects pigs, which are considered to be the natural host for PRV and the reservoir of the virus in nature, but also infects a broad range of wild and non-porcine mammals with the important exception of higher-order primates 8.
The viral agent following a primary replication can establish latent infection and develops a latency-reactivation infection which allows its perpetuation in pig populations 1012 Traditionally, PRV detection is based on direct virus isolation followed by confirmation using immunofluorescence, immunoperoxidase or neutralization tests with specific antiserum 2.
However, this method is time-consuming and false negative results may occur in submissions from latently infected animals The polymerase chain reaction PCR can be used to identify PRV genomes in secretions or organ samples and although some PCR assays for PRV detection with different sensitivities have been reported 37915 there is no standard procedure recommended so far 2.
This paper describes the development, optimization and performance assessment of a rapid and highly sensitive PCR test for detection of pseudorabies virus. Seven virus-negative tissues samples from clinically healthy animals were also included.
PRV specific primers were xujeszky using the Oligo 6. Primers sequences, genome positions and the size of PCR products are shown in Table 1. Primers designed for the specific amplification of the viral gD glycoprotein gene of the PRV genome. The Sma I restriction endonuclease site was used for additional specificity confirmation of the amplification products.
Thus, the optimal concentration of MgCl2 and primers were 1. The annealing temperature and number of cycles were determined experimentally. This was possible due to the high annealing temperature of the primers pair designed and contributes to the reaction efficiency.
Negative controls were run with each test.
Agarose gel electrophoresis was used to detect PCR products. PCR experiments were performed on serial ten-fold dilutions of a viral suspension of PRV isolate V with a titer of 10 6. The analytical sensitivity of the test was consistently observed to be 1. Specificity of the PRV amplicons was furthermore confirmed by Sma I restriction endonuclease analysis which generated the two expected fragments of and bp in length Fig.
Lanes 1 and 3 are amplification products, Lanes 2 and 4 are amplification products after digestion with Sma I. Moreover, the viral genomes of a related herpesvirus and other DNA genome porcine viruses as follow: Especially HVB1 is an important target for specificity assay because is a related herpesvirus which is known to infect swine BHV-1 4. Finally, nucleic acids from tissue homogenates samples derived from seven healthy pigs, and a non infected PK cell line were also tested showing no positive products data not shown.
Each one of the nine tissue field samples from pigs diagnosed as PRV infected based on clinical signs and laboratory methods yielded the corresponding PRV amplified product when analyzed. Seven tissue samples from clinically healthy animals were negative for PCR amplification data not shown. The virus primarily replicates in the respiratory tract, spreads along cranial nerves to the brains and via lymph and blood to internal organs, with the reproductive organs being affected.
Replication in the respiratory tract, central nervous system and reproductive organs is responsible for pathological changes causing different disorders In general, PRV infections must be considered in the differential diagnosis of respiratory, reproductive and nervous disorders.
Under typical conditions of intensive swine production, several clinically similar viral diseases can occur which require laboratory differential diagnosis.
A rapid and accurate diagnosis of PRV infection is important for the initiation of appropriate control strategies. Since the rapid detection of infected animals would reduce the potential transmission of the viruses to uninfected herds avoiding the spread of the diseases The polymerase chain reaction PCR is a rapid tool that can be used no only to detect acutely PRV infected pigs but it is the recommended test for detect PRV latent infection.
The trigeminal ganglion is the most consistent site for virus isolation, although latent virus is usually difficult to culture or even impossible 113 and PCR is the method recommended to detect viral genome present in this site.
The PCR for PRV genome detection is also an important method in screening pig specimens collected for xenotransplantation to increase the safety of organ transplantation 7 and to detect viral infection in a wide spectrum of species reported to be susceptible to PRV, through either natural or experimental infections 8. The nucleotide sequence amplified in this study corresponds to a bp fragment in the gD gene of the PRV genome This gene codes for an envelope glycoprotein named gD which plays and important role in binding cellular receptors and is critical for virus replication in different organs This region was highly conserved for all reported genomes as shown by aligning of these sequences.
Also, the BLAST search against nucleotide databases of different herpesvirus and random nucleotide sequences revealed this region is very specific for PRV genomes.
The possibility to perform annealing and elongation in one single step of the aujeszmy profile contributed to the specificity and the efficiency of the assay and allowed the use of a very fast PCR program. The assay specificity df demonstrated by the absence of amplifications in all heterologous viruses evaluated and in tissue samples derived from seven deona pigs.
The assay proved to be very sensitive due to as little as 1. In addition, positive amplifications were obtained in all the tissue samples, from PRV natural infected pigs, evaluated. The analysis directly from clinical samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. The PCR assay described here provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for pseudorabies infections.
National Center for Biotechnology InformationAujesxky. Journal List Braz J Microbiol v. Published online Sep 1. Author information Article notes Copyright and License information Disclaimer. All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License. Oligonucleotide primers and restriction endonuclease selection PRV specific primers were designed xuinos the Oligo dw. Table 1 Primers designed for the specific amplification of the viral gD glycoprotein gene of the PRV siinos.
Open in a separate window. Induction and inhibition of apoptosis by pseudorabies virus in the trigeminal ganglion during acute infection of swine. World Organization for Animal Health. Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR. The isolation and characterization of a strain of infectious bovine rhinotracheitis virus from stillbirth in swine. Can J Com Med. Diagnostic methods for detection of Classical swine fever virus—Status quo and new developments.
Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract. Iowa State University Press; Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs.
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Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and pigs with emphasis on respiratory tract. Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine. The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. Articles from Brazilian Journal of Microbiology are provided here courtesy of Elsevier. Support Center Support Center.