suid herpesvirus 1 ( suhv-1 ) is the causative agent of aujeszky 's disease , a disease of great impact on the swine industry . due to the important economic losses caused by this disease , notification of any outbreak
infected young pigs suffer from neurological damage , whereas infected adults suffer from abortion and respiratory signals .
suhv-1 has only one serotype , and virus differentiation is made by molecular techniques , in particular by the restriction fragment length pattern ( rflp ) , allowing the differentiation of circulating viral strains .
the genotyping of samples by rflp was fundamental to understand the epidemiology of suhv-1 . despite the development of other molecular typing techniques , the main method of characterizing the suhv-1 is still digesting the full genome by the enzyme bamhi ( bamhi - rflp ) [ 48 ] .
this methodology was used by several authors to characterize suhv-1 isolates in europe , japan , argentina , and brazil [ 716 ] .
genotypes i and ii are distributed worldwide , and genotypes iii and iv , originally described in denmark and thailand , respectively , were no longer reported . currently , genotype i is prevalent in populations of wild boars in europe
. the disadvantages of bamhi - rflp are the need for virus isolation and a time - consuming electrophoresis .
the small amount of viral dna and the lack of facilities for virus isolation are also critical in the implementation of this technique .
the aim of this work was to develop a fast multiplex pcr followed by enzymatic restriction analysis using bamhi enzyme that allows the characterization of suhv-1 without the need of virus isolation , even in samples with small viral dna amounts .
primers were designed based on bamhi restriction sites found in the complete genome of suhv-1 available in genbank ( bk001744 ) , using the software webcutter ( http://rna.lundberg.gu.se/cutter2/ ) .
after detection of the best regions , the sequences were submitted to the program pimer3plus .
the amplicon sizes were chosen according to the restriction fragments that would be generated after enzymatic digestion and the possibility of their use in a multiplex pcr ( tables 1 and 2 ) .
eleven isolates of suhv-1 ( five of genotype i and six of genotype ii ) , previously characterized in other works [ 16 , 19 ] , the vaccine strain bartha , standard sample shope , and attenuated isolate nia-4 , were used for pcr development .
twenty clinical brain samples of pigs positive for suhv-1 by virus neutralization and virus isolation , collected from outbreaks in the state of santa catarina , brazil , in the years of 2002 and 2003 were used to standardize the technique in tissue sample .
ten negative samples of the same tissue were also used to determine whether the pcrs would be able to detect the virus without unspecific reactions .
total dna was extracted by commercial kit wizard genomic dna purification ( promega , usa ) and stored at 20c for later genotyping .
the reactions were developed by testing different parameters such as annealing temperature , concentration of the reagents ( primers , dna polymerase , mgcl2 ) , and duration of each stage .
the first tests were performed with separate primers for subsequent use in the multiplex .   
the multiplex pcrs were standardized using two sets of primers : mix a , which contained bamhi-1 - 738 , bamhi-3 - 256 , and bamhi-6/7 - 637 , and mix b , which contained primers bamhi-4 - 357 , bamhi-19/20 - 519 , and bamhi-16/17 - 799 , both of the mixes generate three bands prior to restriction enzyme .
a 20  l reaction was standardized with the following concentrations of reagents : 10  pmol of each primer , 4  l of 5x buffer gotaq green ( promega , usa ) , 2.0  mmol / l magnesium chloride , 6% dmso , 300  mmol / l dntp ( invitrogen , usa ) , and 3 u gotaq hotstart ( promega , usa ) .
the conditions used for both pcr were as follows : a denaturing stage at 95c for 5  min .
, 35 cycles at 95c for 50  s , 60c for 50  s , 72c for 1  min . , and a final stage extension at 72c for 7  min .. positive and negative controls were used in all the pcr mixes to check for contamination and unspecific amplifications .  
enzymatic restriction of the mpcr with bamhi was performed according to the manufacturer 's instructions ( new england biolabs , usa ) .
the gels were analyzed on 2.5% agarose gel stained with ethidium bromide at 0.5  mg  ml after one hour of running .
the primers chosen for pcr target nine restriction sites for bamhi detected in the in silico analysis of complete genome ( table 1 ) . the regions were chosen because they are the key points used to differentiate genomic types . among the chosen primer pairs , bamhi-1 - 738 and bamhi-4 - 357 are most important for the differentiation of all genotypes .
genotype ii has no restriction site in bamhi-1 - 738 , and genotype iii has no restriction site in bamhi-4 - 357 ( table 2 ) .
these same areas offer the opportunity to subtyping with the enzyme bsteii , which has a restriction site in bamhi-4 - 357 for genotype i. the other primers were selected because the regions can be used to differentiate some suhv-1 strains , like the standard strain shope , used in our laboratory for virus neutralization tests .
the bamhi - mpcr was first developed with a set of six primer pairs in one reaction , but good visualization of the results could only be possible after two hours of electrophoresis .
it was possible to differentiate genotypes i and ii using enzymatic restriction after dna amplification with mix a ( figure 1(a ) ) .
mix b was used to differentiate specific strains like standard strain shope ( figure 1(b ) ) . among the twenty clinical samples , seventeen amplified as expected .
three samples required the addition of a larger amount of dna for better visualization of bands in agarose gel .
all isolates previously characterized in other studies [ 15 , 16 ] showed the expected restriction patterns for genotype ii ( figure 1 ) .
the bamhi - rflp is one of the most used methods for molecular characterization of suhv-1 .
although sequencing methods are considered more accurate [ 8 , 18 ] , the restriction enzyme analysis is still important for genotyping of suhv-1 .
the biggest problems of rflp are the need of ultracentrifuges , the necessity of virus isolation and the electrophoresis that takes over eight hours under controlled temperature [ 14 , 16 ] .
the multiplex pcr followed by restriction with bamhi ( bamhi - mpcr ) developed in this study allows the analysis of restriction sites for genotyping of suhv-1 directly from clinical samples without the need of any equipment other than that of a basic molecular biology laboratory .  
the bamhi - mpcr was able to detect the virus in all the clinical samples , which demonstrated its importance for genotyping suhv-1 when there is a shortage of equipment or isolation is not possible .
we have previously developed a nested pcr for the same purpose in our laboratory , but it is a more laborious and time - consuming technique and very prone to contamination .  
pcr followed by restriction enzyme has been used characterize other viruses of the herpesviridae family , such as bovine herpesvirus 1 , 2 , and 5 , herpes simplex 1 [ 22 , 23 ] , varicella - zoster virus , and human cytomegalovirus .
these techniques are simple and easy to perform in molecular biology laboratories and are very useful in epidemiological studies .
genotyping of suhv-1 allows a better understanding of its epidemiology and helps to weigh the impacts of this infectious agent and , ultimately , to reduce or even avoid the losses it produces .
molecular techniques have been used with many objectives such as to verify the introduction of new genotypes in a country , to characterize field isolates derived from vaccines samples   and even to identify suhv-1 isolates from wild animals , such as the wild boar .
the bamhi - mpcr is a fast and useful method for genotyping that allows for comparison with results from the published data of the last twenty - five years since the beginning of the use of bamhi - rflp technique .
 suid herpesvirus 1 ( suhv-1 ) is the causative agent of aujeszky 's disease . 
 the infectious agent has only one serotype , but it was classified by restriction enzyme analysis of the whole genome into four genotypes , named i to iv . 
 the aim of this study was to standardize a rapid method for genotyping suhv-1 without virus isolation , using a multiplex - pcr followed by enzymatic restriction analysis . 
 the complete genome of the virus was analyzed in silico to determine the restriction sites for the enzyme bamhi . 
 primers were designed to flank sites with emphasis on certain points of differentiation of genotypes . 
 the standard pcrs were able to detect the suhv-1 and also to differentiate genotypes from brain tissue of infected pigs . 
 the bamhi - pcr is a rapid , practical , and sensitive way to genotype suhv-1 . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion