Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 1, 2002, pp. 109-111

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(1) 2002, pp. 109-111
SHORT COMMUNICATION  
A Heminested Polymerase Chain 
  Reaction for the Detection of Brazilian Rabies Isolates from Vampire Bats and 
  Herbivores   
RM Soares++, F Bernardi*, SM Sakamoto**/++, 
  MB Heinemann**, A Cortez**, LM Alves**/++, AD Meyer+++, 
  FH Ito**, LJ Richtzenhain**/+ 
Instituto de Ciências Biomédicas, 
  Universidade de São Paulo, São Paulo, SP, Brasil *Faculdade de 
  Veterinária, Universidade Santo Amaro, São Paulo, SP, Brasil **Faculdade 
  de Medicina Veterinária e Zootecnia, Universidade de São Paulo, 
  Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, 05508-900 
  São Paulo, SP, Brasil 

  +Corresponding author. Fax: +55-11-3818-7928. E-mail: leonardo@usp.br 
  

  ++Fapesp fellowship 

  +++ Capes fellowship 
Received 6 March 2001 

  Accepted 26 September 2001 
Code Number: oc02020 
A heminested-PCR (hn-PCR) using primers to 
  the nucleoprotein-coding gene in a nested set was evaluated in the detection 
  of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein 
  sequences belonging to genotype 1 were aligned. Based on such alignment, primers 
  were directed to highly conserved regions. All 42 clinical samples positive 
  by both fluorescent antibody and mouse inoculation tests were also positive 
  by the hn-PCR. Brain tissue that had been left to decompose, obtained from an 
  experimentally inoculated mouse was tested by hn-PCR and yielded positive results. 
  In conclusion, primers designed here were capable of amplifying Brazilian RV 
  isolates obtained from a rural epidemiological cycle.  
Key words: rabies virus - polymerase chain reaction 
  - detection of viral RNA - rabies diagnosis - Brazil 
Rabies is a widespread zoonosis, which has been 
  of great concern due to its ability to determine a fatal acute encephalomyelitis 
  when the host is bitten by an ill animal (Wilkinson 1988). The causing agent 
  is a virus that belongs to the Lyssavirus genus in the Rhabdoviridae 
  family. Based on phylogenetic analyses of the nucleoprotein-coding gene (N gene), 
  the Lyssavirus genus has been divided into seven genotypes. Genotype 
  1 includes rabies virus (RV) strains (Bourhy et al. 1993). RV genome is composed 
  of a single-stranded, negative-sense, non-segmented RNA that codes for five 
  separate proteins: nucleoprotein, glycoprotein, phosphoprotein, membrane protein 
  and polymerase (Tordo 1991).  
The World Health Organization (WHO) recommends 
  that the fluorescent-antibody test (FAT) and mouse inoculation test (MIT) carried 
  out simultaneously should be used for the detection of RV (Meslin et al. 1996). 
  FAT is a rapid and low cost technique, which may show positive results, as it 
  is able to detect viral antigens whether they are viable or not (Meslin et al. 
  1996). However, FAT's efficacy may be jeopardized when decomposed tissue is 
  used. In such cases, PCR may be used as a surrogate due to its more appropriate 
  performance (Sacramento et al. 1991, Kalmovarin et al. 1993, Whitby et al. 1997). 
   
PCR based on N gene has been widely used for 
  diagnostic purposes since it is one of the most conserved fractions in RV (Smith 
  et al.1992, Kalmovarin et al. 1993, Heaton et al. 1997, Crepin et al. 1998, 
  Heaton et al. 1999, Black et al. 2000).  
The aim of this study was to design primers based 
  on RV genotype 1 N gene and to evaluate the performance of a hn-PCR for direct 
  RV detection in clinical samples. Primers were based on the sequences of genotype 
  1 strains, including one Brazilian isolate (Kissi et al. 1995). Virus sequences 
  were aligned by using the Clustal X computer program (Thompson et al. 1997) 
  and the oligonucleotides were designed to recognize regions with high degree 
  of similarity within the N genes. The segments that were selected for primer 
  design are located in the middle of the N gene. Kissi et al. (1995) have demonstrated 
  that such gene fraction shows a striking level of similarity among RV isolates. 
  The physical and chemical properties of the primer were predicted by using the 
  Oligo 4.0 computer program (Rychlik & Rhoads 1989).  
Primers with a maximum of three mismatches with 
  any aforementioned sequence and no mismatch in the 3' terminal nucleotide were 
  chosen. As the efficiency of PCR is inversely related to the length of the sequence, 
  primers were chosen based on the shortest flanking distance. Primer sets P510/P784 
  and P510/P942 (P510:ATA GAG CAG ATT TTC GAG ACA GC; P784:CCT CAA AGT TCT TGT 
  GGA AGA; P942:CCC ATA TAA CAT CCA ACA AAG TG) define 295 and 455 base pairs, 
  respectively.  
Samples were 20% (w/v) homogenate of brain material 
  in PBS which had been stored at -20ºC and previously tested by FAT and 
  MIT (FAT/MIT), as prescribed by the WHO (Meslin et al. 1996). These samples 
  came from distinct geographical areas within the Brazilian territory and from 
  multiple species of herbivores and vampire bats (Table). 
  Total RNA was directly extracted from the samples by the TRIzol LS method, according 
  to manufacturer's instructions (Gibco BRL). Reverse transcription was performed 
  with 7 µl of the extracted product added to a final volume of 20 µl 
  containing 1 mM of each dNTP, 20 pmols of primer P510, 1 x RT buffer (Gibco 
  BRL), 1 mM dTT, 200 units of M-MLV reverse transcriptase (Gibco, BRL) and 0.01% 
  DEPEC treated ultra pure water. The mixture was incubated for 1 h at 42ºC. 
   
A primary amplification was performed in 5 µl 
  of the reverse transcripted-cDNA template in a final volume of 50 µl, 
  containing 0.2 mM of each dNTP, 25 pmols of primer P510, 25 pmols of primer 
  P942, 1.5 mM of MgCl2, 1 x PCR buffer (Gibco, BRL), 1.25 units of 
  Taq DNA polymerase (Gibco, BRL) and ultra pure water. The amplification was 
  performed on a MJ Research PTC-200 Thermal Cycler. The heminested amplification 
  was performed in 5 µl of primary amplification template and primers P510 
  and P784. The following cycling conditions for the primary amplification were 
  adopted: initial heating at 94°C/3min, 35 cycles at 94°C/45 sec, 55°C/60 
  sec, 72°C/90 sec and a final extending step at 72°/10 min. The thermal 
  cycles for the nested assay were the same, except for an amplification phase 
  of 25 cycles. PCR products were run in 2% agarose gel electrophoresis in standard 
  TBE and stained with ethidium bromide 0.5 µg/ml (Sambrook et al. 1989). 
  Gels were observed under UV light and photographed.  
Amplicons yielded by both primer pairs are shown 
  in the Figure. The Table 
  shows hn-PCR results in samples from several Brazilian localities positive for 
  rabies by FAT/MIT. The ten samples which yielded negative results by FAT/MIT 
  also yielded negative results by PCR (results not shown). Total concordance 
  between hn-PCR and FAT/MIT was observed. Considering that other kind of rabies 
  virus may be found in rural environment, 5 canine and 1 feline rabid samples 
  were tested by the hn-PCR, yielding positive results in all cases.  
Detection threshold for the hn-PCR method was 
  evaluated using normal brain homogenates of a mouse spiked with serial ten-fold 
  dilutions of Pasteur virus strain. Briefly, brain homogenates from experimentally 
  RV infected mouse containing 105.4 LD 50/ml were ten-fold serially 
  diluted with brain homogenates from a non-infected mouse. Titration of the brain 
  homogenates from the experimentally infected mouse was performed following the 
  method described elsewhere (Reed & Muench 1938). Brain homogenates containing 
  from 10 4.4 LD50/ml to 100.4 LD50/ml were tested by PCR. 
   
The performance of hn-PCR on RV-infected brain 
  tissues at various stages of decomposition was also evaluated. Brain homogenates 
  from a mouse experimentally inoculated with PV strain were left at room temperature 
  for 24, 48, 72 and 96 h, and then tested by hn-PCR. The hn-PCR was able to detect 
  104.4, 103.4, 102.4 and 101.4 LD 
  50/ml (25000, 2500, 250 and 25 LD 50/ml). In the Figure, 
  the results of hn-PCR on 103.4, 102.4, 101.4 
  and 100.4 LD50/ml are shown. The hn-PCR was also capable of amplifying 
  RV genetic sequences from all the decomposed brain tissue samples which have 
  been left 24, 48, 72 and 96 h at room temperature.  
False positive results are the main problem associated 
  with nested-PCR due to contamination with amplified DNA from the primary reaction 
  (Forghani & Erdman 1995). In order to avoid false positive results, some 
  extensive precautions should be taken: (i) each step of sample handling (RNA 
  extraction, first and second amplification steps) should be performed in different 
  laboratories; (ii) use of disposable gloves; (iii) analyses of no more than 
  ten samples per reaction; (iv) inclusion of negative control for each five samples. 
  The hn-PCR described in this paper was performed in three steps, one for cDNA 
  synthesis and two for both amplification assays.  
As the detection of PCR products on agarose gel 
  may reveal spurious bands sometimes (results not shown), good candidates for 
  further improvements in the sensitivity, specificity and rapidity of the hn-PCR 
  are the combination of reverse transcription and first amplification in a single 
  step, and the standardization of methods other than agarose gel separation for 
  the detection of PCR products.  
At present, simultaneous use of FAT and MIT is 
  irreplaceable for RV detection. However, their performance may occasionally 
  be impaired in decomposed tissues and/or samples containing no viable virus. 
   
The goal of this study was to assess the fine 
  performance of the primers designed for Brazilian RV isolates obtained in rural 
  epidemiological cycle. Hn-PCR results presented here unequivocally provide an 
  additional tool for rabies diagnosis in some special cases, such as: (i) samples 
  which have been inadequately stored and then sent for laboratorial analysis; 
  (ii) when FAT alone yield negative results, FAT is impracticable to perform, 
  and results are urgent (for the entire hn-PCR procedure is accomplished in 24 
  h); and (iii) when dealing with small amount of sample as those obtained from 
  bats and laboratory animals in experimental studies.
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  Instituto Oswaldo Cruz - Fiocruz

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