DETECTION OF PESTIVIRUS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN AMPLIFICATION OF THE 5' cDNA UNTRANSLATED REGION
DETECCIÓN DE PESTIVIRUS POR REVERSO TRANSCRIPCIÓN ACOPLADA A REACCIÓN EN CADENA DE LA POLIMERASA DE LA REGIÓN 5' cDNA NO TRADUCIDA
Heidy Díaz de Arce y L.J. Pérez
Laboratorio de Virología Animal del Centro Nacional de Sanidad Agropecuaria (CENSA), Apartado 10, ]]> San José de las Lajas, La Habana, Cuba
ABSTRACT
Pestivirus genus of the family Flaviviridae includes different viral species that cause significant losses to the livestock industry worldwide affecting cattle, sheep, goats and pigs. A rapid, sensitive, and specific detection of these agents is therefore essential for diagnosis and control of the viral infections. An RT-PCR assay was developed and standardized for the specific panpestivirus detection in serum based on a generic pair of primers that amplifies a 119 pb DNA fragment from a highly conserved region in the 5´-UTR RNA genome. The RT-PCR assay showed to be a specific, rapid, sensitive and highly repeatable purpose.
Key words: diagnosis; pestivirus; PCR
RESUMEN
El género Pestivirus de la familia Flaviviridae incluye diferentes especies virales que causan pérdidas importantes a la industria ganadera en todo el mundo afectando bovino, ovino/caprinos y cerdos. La detección rápida, sensible y específica de estos agentes es por lo tanto esencial para el diagnóstico y control de estas infecciones virales. Se desarrolló y estandarizó un ensayo de RT-PCR para la detección específica de pestivirus en suero basado en una pareja de cebadores que amplifica un fragmento de 119 pb de una región altamente conservada en el extremo 5´UTR del genoma ARN del virus. Este ensayo mostró ser propósito específico, sensible y altamente repetible.
Palabras clave: diagnóstico; pestivirus; PCR
INTRODUCTION
Viruses that comprise the Pestivirus genus of de family Flaviviridae cause significant losses to the livestock industry worldwide (1,2). Based on sequence analysis, currently 4 distinct genotypes, that have the taxonomic status of viral species, are identified within the genus Pestivirus (3); Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV). Pestiviruses are able to cross the species barrier, CSFV can be transmitted to cattle, BVDV can naturally cross-infect cattle, sheep, goats and pigs and BDV is an ovine pathogen that occasionally infects pigs. Pigs in particular are infected by all the pestivirus species (4).
The pestivirus genome is ~12.5 kb and comprises a single large open reading frame flanked by untranslated regions (UTRs) at the 5' and 3' ends. Translation results in a ~4000 amino acid polyprotein which is cleaved into four structural and eight non-structural proteins. Structural proteins derive from the N-terminal end of the polyprotein (4).
BVDV infection is widespread in cattle worldwide and it causes important economic losses 1. Moreover, BVDV is a contaminant of commercial foetal calf serum preparations and susceptible mammalian cell lines are often found inadvertently infected (4). BVDV has recently been recognized as a cause of iatrogenic disease in cattle due to contaminated vaccine (5) and detected in live vaccines for human use (6). In addition, Classical swine fever (CSF) is a highly contagious, widespread disease of swine that produces important losses for pig industry all over the world (4). All pestivirus species can infect pigs (4), so an accurate and rapid pestivirus detection is of great importance for the development of control measures in swine farming.
Infections caused by pestiviruses are detected by immunohistochemical techniques and are confirmed by virus isolation. RT-PCR assay has been proved to be a rapid and sensitive method to detect viral nucleic acids in clinical material and this technique has been used by several researchers for the detection of pestiviruses using oligonucleotide primers located in conserved regions of the viral genome (7,8,9).
However, the recent availability of several four distinct genotypes of pestivirus nucleotide sequences allows to take advantage of this information for the design of primer pairs for more sensitive and specific RT-PCR assays.
The sequence of the 5' UTR is highly conserved among pestiviruses and it is often used for molecular diagnosis and genotyping (4). The aim of this study was to develop an RT-PCR test that allowed the detection of a genomic fragment common for all pestivirus strains.
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MATERIALS AND METHODSViruses and cell cultures
Different pestivirus strains were selected for the development and evaluation of the RT-PCR assay. BVDV reference strain NADL and BDV reference strain Morendum were kindly provided by the Community Reference Laboratory (CRL) for CSF, Hannover, Germany. BVDV reference strains Oregon and Singer and the CSFV strains Alfort, Ames and PAV-250 were obtained from CISA/INIA, Valdeolmos, Spain. A collection of pestivirus field strains isolated in Cuba was also analyzed (Table 1). For evaluation of the assay analytical specificity, the gastroenteritis virus (TGEV) reference strain Purdue 115, kindly supplied by Professor Dong Wang You (Pathobiology laboratory, Gelph University, Canada), the viral strain from Intervet TGE vaccine and encephalomyocarditis virus EMCV field isolates form our collection were also used (Table 1). BVDV strains were cultured in Madin-Darby bovine kidney (MDBK) cells (ATCC CCL 22) and CSFV strains in porcine kidney cell line (PK-15) (ATCC CCL 33).
Clinical samples
A panel of 40 pig serum, from two International Inter-laboratory Comparison Tests 2006 and 2007, conducted by the CRL, Hannover, Germany, aimed to evaluate the quality and diagnostic performance of RT-PCR assays in different laboratories for the detection of pestivirus infections in pigs was used.
RNA extraction and cDNA synthesis
Total RNA was extracted from 250 uL amounts of samples (cell cultures, serum), with a commercial reagent (TRI Reagent LS, SIGMA, San Louis, Missouri, USA), as recommended by the supplier. RNA was resuspended in 10 uL of nuclease free water (Promega, Madison, WI, USA).
First strand complementary DNA (cDNA) was synthesized using Moloney-Murine leukemia virus reverse transcriptase (M-MLV RT) (Promega, Madison, WI, USA) in 20 uL final reaction volume. Briefly, 5 uL of RNA were incubated with 1 uL of random primers (50 ng/uL) (Promega, Madison, WI, USA) and 4 uL of nuclease free water (Promega, Madison, WI, USA) in 10 uL final reaction volume at 70ºC for 10 min. and then cooled on ice to accomplish nucleic acid denaturing. After incubation on ice 3.5 uL of nuclease free water, 4 uL of 5X reaction buffer [250 mM Tris_HCl (pH 8.3 at 25_C), 375 mM KCl, 15 mM MgCl2, 50mM DTT], 1 uL 10mM of each deoxynucleoside triphosphate, 0.5 uL of 40 U/uL RNAsin ribonuclease inhibitor and 1 uL of 200U/uL M-MLV RT was added and the reaction mixture was further incubated at 37ºC for 60 min.
PCR primers design
Panpestivirus specific primers were designed in the conserved 5' UTR of viruses from different pestiviral genotypes and subgenotypes, available in GenBank. Nucleotide sequences belonging to genotypes BVDV1 (BVDV1a and BVDV1b), BVDV2, CSFV and BDV as well as hepatitis C virus were aligned using Clustal W 1.8 software and manually examined for oligonucleotides corresponding to pestivirus. Primers were designed from highly conserved nucleotide region using the Oligo 6.31 program, (Molecular Biology Insights, Inc., USA). A BLAST search at National Center for Biotechnology Information (NCBI) site (http://www.ncbi.nlm.nih.gov) was performed using blastn algorithm for calculating sequence similarity with primers selected as query sequences against nucleotide databases of different pestivirus genotypes. ]]>
Primer sequences, genome positions and the size of PCR products are shown in Table 2.Polymerase chain reaction
A number of experiments was performed to optimize the RT-PCR protocol, including concentration of reagents and PCR cycling parameters the assay was finally optimized as follows: for PCR amplication, the reaction mixture was prepared in a volume of 50 uL comprised of 2 ul of cDNA, 1x GoTaq Green Master Mix (Promega) [200uM of each dNTP, 1.5mM MgCl2 (pH 8.5)] and 1 uM of each primer. The PCR reaction was done under the following conditions in a thermal cycler (Eppendorf Mastercycler): 1 cycle of 2 min at 95ºC; 35 cycles of denaturation at 94ºC for 30 s, annealing at 50ºC for 30 s, and elongation at 72ºC for 30 s; and 1 cycle of 5 min at 72ºC. The amplicons (10 ul) were visualized by electrophoresis on 2.0% agarose gel in TBE buffer (90mM Tris_borate, 2 mM EDTA).
As a minimum, one negative serum control for each batch of RNA extracted samples tested together and one water control for each sample are included in the PCR.
RESULTS
Optimization of the RT-PCR assay and primer selection.
The optimization of the RT-PCR reaction components and cycling parameters was performed, respectively. The primers, as well as magnesium chloride, were titrated in chequerboard assays to determine the most suitable concentrations. Different annealing/elongation temperatures and times were evaluated. The optimized concentrations of reaction components and cycling parameters were established as previously described.
With the panpestivirus specific primers, designed in a highly conserved region from the 5' UTR RNA genome from different pestiviral genotypes, PCR products of the expected sizes (119 bp) were amplified from all of the Examined pestivirus strains and isolates (Table 1). In heterologous RNA viruses affecting swine (Table 1) and in negative controls no DNA was amplified. Example of PCR product from CSFV reference strain Alfort and from negative controls separated in agarose gels are shown in Fig. 1.
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Sensitivity assay
The analytical sensitivity of the RT-PCR assay was evaluated by testing sequential 10- fold dilutions of Alfort strain with a titre of 107,86 TCID50/mL in negative serum. Purified viral RNAs were obtained using TriReagent (Sigma), as described in Section 2. The sensitivity value was obtained bearing in mind the volume of sample (0.25 ml) and the fraction of nucleic acid extract used in each reaction (5%) (represented by TCID50 per reaction).The detection limit of the PCR for negative serum spiked with serially diluted virus suspensions was equivalent to 0,89 TCID50 per amplification reaction (Fig. 2).
Specificity assay ]]>
The analytical specificity of the RT- PCR assay was evaluated by the analysis of viral RNA from a collection of 26 Pestivirus strains and isolates (Table 1). All the tested pestiviruses provided the specific amplicon of 119 bp. There was no cross-reaction to TGEV and EMCV tested. Example of PCR product from pestivirus strains and from negative controls separated in agarose gels is shown in Fig. 3.
RNAs extracted from serial dilutions, in serum, of CSFV strain Alfort strain viral suspension with a titre of 107,86 TCID50/ml were employed under reaction conditions described in Section 2. M: molecular weight marker 100 pb (Promega).
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Repeatibility
The repeatability of PCR assay was evaluated from multiple runs with replicated samples in each run. One negative serum sample from an uninfected pig and two positive serum samples from naturally infected pigs were used. Each sample was tested in triplicate in 10 consecutive PCR assays. Agreement between replicates within and between runs of the assay was obtained. ]]>
Panpestivirus detection in serum samplesTo determine the quality of the assay and the competence of the RT-PCR test performance in clinical sample, 40 standardized pig sera samples from two European inter-laboratory comparison tests 2006 and 2007 for Classical Swine Fever conducted by the Community Reference Laboratory, Hannover, Germany were evaluated.
The serum samples were obtained from experimentally infected animals at different post-inoculation days with different Pestivirus strains. Besides, BVDV positive fetal calve serum and negative pig serum were included. The samples contain a full representation of the analyte's concentration to test sensitivity and they were sent in duplicates in order to test reproducibility.
The results for the PCR part of the international ring test were fully in line with the Reference Laboratory expectations.
DISCUSSION
The 5´-UTR of the pestiviruses includes motifs necessary for viral gene expression and RNA replication, which are usually composed of a combination of primary, secondary and terciary structures (25). The nucleotide sequence of the 5´-UTR is very similar and well conserved among the members of the Pestivirus genus, thus providing a useful region for accurate suitable and virological diagnosis.
In the present study, analysis of the 5´-UTR from different pestivirus genotype and subgenotype sequences and from hepatitis C virus, a related genome belonging to the Flaviviridae family, was performed in order to find a pair of primers with a broad pestivirus specificity and that no cross reacts with related virus. To ensure specificity, genus-primers were designed to detect as many viral sequences as necessary to encompass genetic diversity.
For selective amplification of related cDNA sequences, the choices and working conditions for the PCR primer pairs are important. Beyond the general requirements of similar melting temperatures, low tendencies of hybridization to self or to other parts of the targeted or host genome, the number and positions of mismatches between the primers and the target sequences define the discriminating properties of the primers.
Our findings of positive amplifications with different pestivirus reference strains and isolates together with the detection of all pestivirus strains in clinical samples from two proficiency tests indicate wide specificity of the generic pestivirus primers and a correct optimization of the assay. ]]>
The current PCR for the panpestivirus detection should be considered as a "general", or "universal" PCR system because it is designed to detect a wide range of related viruses, members of a viral genus. Swine can develop infection with any of the pestivirus species, frequently showing only mild or innapparent symptoms (4). This panpestivirus PCR allows to screen the herds, in order to exhibit or exclude the presence of any pestivirus, in a single PCR assay. Therefore, the samples, selected as pestivirus positives by the "general" PCR, can then be further tested with the virus specific PCR assays, in order to identify the pestivirus, species which is causing the infection. The combined use of "general" and specific PCR assays provides a rapid and effective diagnosis (10).Otherwise the RT-PCR assay optimized showed a low detection limit of less than 1 TICD50/ml. This is an expected result because RT-PCR assay has been found to be the most sensitive method for detection of CSFV (11,12) and other pestivirus infections (13,14). In comparison to virus isolation in cell culture, the viral nucleic acid can be detected earlier after infection and for a longer period in cases where the pigs recover (15).
An advantage of RT-PCR is that due to the assay high sensitivity, pooled samples can be tested reducing diagnostic costs and time (16). This tool makes possible to detect very small virus doses that are below the detection limit of the conventional diagnostic techniques such as virus isolation and this allows preclinical diagnosis and supports early detection systems for CSF (11).
Another advantage from RT-PCR over conventional diagnosis is that it has been demonstrated that it becomes positive significantly earlier than the conventional virus isolation techniques (17,18).
CSF often has an incubation period of some weeks, on a herd basis, requiring several cycles of amplification before it becomes clinically apparent. ''Pre-clinical'' detection would therefore be of enormous benefit to disease control (19).
RT-PCR researches represent a novel diagnostic approach with a clearly enhanced sensitivity compared to the conventional antigen detection. Irrespectively of the strain specific virulence (20), RT-PCR assays are recommended for the detection of pigs infected with CSFV low virulent field virus strains. The presence of CSFV genomes in blood samples of field virus infected pigs up to 70 days after infection (21,22).
The validation of the assay in clinical sample enables serum samples to be examined and if the assay is sufficient automated, it could be used as screening test in alive animals to confirm pestivirus infections and might be usable as a means of certifying that pigs at an abattoir were non-viraemic at slaughter (11).
PCR generates large numbers of amplicons, therefore it carries a high risk of contamination. The RT-PCR assay demands a high level of technical organization, setting up laboratory space correctly and good laboratory habits. Besides, positive and negative controls in the isolation and amplification steps are crucial to obtain reliable results (23).
The excellent test performance in two proficiency tests conducted by the Community Reference Laboratory for CSFV diagnostic provides evidences of accurate and reliable results of the present RT-PCR assay.
To summarize an RT-PCR assay was developed and standardized for panpestivirus detection in swine serum. The RT-PCR assay was purpose-specific, rapid (result within 24-48 hours), sensitive, highly repeatable and provide a very useful tool for the diagnostic of virologists. ]]>
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(Recibido 7-12-2007; Aceptado 20-7-2008) ]]>