High Resolution Melting (HMR) como nuevo metodo para la deteccionn simultanea y discriminacion de los herpesvirus bovinos tipo 1 (BoHV-1) y tipo 5 (BoHV-5).

MARIN, M; QUINTANA, S; LEUNDA, M; RECAVARREN, M; PAGNUCO, I; SPATH, E; PEREZ, S; ODEON, A

Tucuman

Jornada; XX Reunion Cientifico Tecnica de Laboratorios de Diangostico Veterinario; 2014

AALDV

Introduction Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are two closely related alpha-herpesviruses that infect cattle. Despite their high similarity, differentiation among BoHV-1 and BoHV-5 is not achieved by classical virological techniques. Thus, virus isolates and samples collected from affected animals must be analyzed using sensitive and reliable molecular methods. High resolution melting (HRM) analysis is a recently developed molecular technique. The development of HRM curve analysis, as an extension to Real time PCR, is based on the determination of changes in fluorescence as a result of melting double-stranded PCR products in response to temperature increase. The melting temperature (Tm) and the characteristic shape in the melting curve profile of the amplified products are highly dependent on the nucleotide sequence. There are no reports on the use of Real time PCR with HRM for the identification of bovine alpha-herpesviruses. The aim of this study was to develop a simple and reliable one-step Real time PCR assay with HRM analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5.   Materials and methods Reference strains LA (BoHV-1.1), Cooper (BoHV-1.1), N569 (BoHV-5a) and A663 (BoHV-5b), Argentinean field isolates (10 BoHV-1 and 1 BoHV-5 strains), 5 clinical samples in which BoHV DNA was detected by conventional PCR and 45 samples from the respiratory tract of experimentally infected animals were studied. DNA was extracted from viral isolates or tissue samples using a commercial kit (Qiagen Inc., Valencia, CA, USA). To identify the most effective set of primers for detecting and differentiating BoHV-1 and BoHV-5, three primers pairs were evaluated. The sequences of BoHV-1 and BoHV-5 obtained from GenBank database, corresponding to the amplified fragments, were aligned and compared by use of MAFFT multiple sequence alignment program. Assay conditions were optimized using varying primer concentrations, annealing temperature and time. Real Time PCR assays were carried out in a Rotor Gene Q thermocycler, in a final volume of 20 ìl using EvaGreen as intercalating fluorescent dye (KAPA HRM FAST). The cycling program consisted of an initial denaturation of 3 minutes at 95°C and 50 cycles of 15 seconds at 95°C, 15 seconds at 58°C and 20 seconds at 72°C. After amplification, a HRM curve analysis was performed and BoHV identification was performed using Rotor Gene Q software, version 1.7.94. The sensitivity and specificity of the assay was determined after amplification and genotyping of DNA from isolates and tissue samples. Moreover, the Real Time PCR-HRM was compared to virus isolation and conventional PCR (Campos et al., 2009) techniques using tissue samples from experimentally infected animals. Concordance between the three methodologies was assessed using Cohen?s Kappa coefficient. The difference among coefficients was calculated using the Chi-square test (P<0.05). All analyses were carried out with Epidat 3.0 software (Panamerican Health Organization [PHO]/World Health Organization [WHO]).   Results and Discussion According to the reaction efficiency and higher capacity for differentiation of BoHV-1 and BoHV-5, the primers described by Diallo et al. (2011) were selected. Within the amplified sequences, a few point nucleotides (14 in total) differed between both viruses. It was possible to obtain an efficient amplification strategy, a clear melting curve for differentiating BoHV-1 and BoHV-5 and appropriate melting peaks for both PCR amplicons. The melting points obtained for BoHV-1 and BoHV-5 were 89.7 °C and 91.1 °C, respectively. These results showed that BoHV types can be distinguished by the characteristic Tm shift in the melting curves. Moreover, from dual artificial mixes of BoHV-1 and BoHV-5 DNA it was possible to obtain distinguishable melting peaks for each BoHV type, confirming that the selected primer pair can differentiate mixed infections. BoHV was identified in all isolates and clinical samples from our collection when assessed by Real Time PCR-HRM assay, and BoHV types were properly differentiated. Regarding to virus detection in respiratory samples, BoHV-1 DNA was detected in 12/18 samples from BoHV-1-infected calves, including retropharyngeal and bronchial lymph nodes, the epithelium of nasal mucosa, trachea, bronchi and lungs. BoHV-5 DNA was detected in 7/18 samples from BoHV-5-infected calves, including retropharyngeal and mediastinal lymph nodes and epithelium of nasal mucosa, trachea and bronchi. Viral DNA was not detected in any of the 9 samples from uninfected control animals. Real time PCR-HRM assay allowed the detection even when DNA from 10 viral particles was assayed and there was no cross-reaction. The detection rate achieved with Real time PCR-HRM was higher when compared to virus isolation or conventional PCR. Out of 45 samples from the respiratory tract it was possible to detect BoHV DNA in 19 samples. However, by conventional PCR 14 samples were positive and infectious virus was only detected in 5 samples. The concordance coefficient between Real time PCR-HRM and conventional PCR was 0.76, between Real time PCR-HRM and virus isolation was 0.29 and between conventional PCR and virus isolation was 0.43. Significant differences were observed when comparing the three techniques against each other (P<0.05). Real Time PCR-HRM showed the highest sensitivity in relation to the other methodologies.There is a need for correctly identifying BoHV field isolates to achieve an accurate final etiological diagnosis and to better understand their epidemiology and pathogenesis. In this study, HRM was used as a novel procedure for analysis and differentiation of BoHV types in viral culture as well as in tissue samples. This method provides a rapid, sensitive and specific detection of bovine alpha-herpesviruses DNA. Furthermore, it demonstrated to be useful for the simultaneous detection of two herpesvirus types. Thus, this technique appears as an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.