Browsing by Subject "RNA viruses"

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    Establishment of reverse transcriptase polymerase chain reaction methods for the detection of newly described RNA viruses in reptiles : picornaviruses in tortoises, reptarenaviruses in snakes, and sunshinevirus in snakes
    (2018) Aqrawi,Tara; Marschang, Rachel E.
    The purpose of this study was to establish conventional reverse-transcriptase PCRs for the detection of recently described RNA viruses in reptiles. The viruses studied included picornaviruses of tortoises (torchivirus or virus “X”), reptarenaviruses and sunviruses in snakes. Picornaviruses are detected frequently in tortoises of various species in Europe. Until recently, tortoise picornaviruses (previously designated as virus “X”) could only be detected by isolation in Terrapene heart (TH1) cells in which they cause cell lysis, and nothing was known about the relationships of various isolates to one another. Clinical signs that have been described in picornavirus infected tortoises include softening of the shell in juvenile tortoises, rhinitis, conjunctivitis, kidney failure, and sudden death, but these viruses have also been detected in clinically healthy animals. This group of picornaviruses is able to infect a wide range of species in the family Testudinidae and has been detected in tortoises in many different European countries. In this study, a conventional RT-PCR was developed and established for the detection and identification of picornaviruses in clinical samples. To test the reliability of this RT-PCR as a diagnostic method and the several primer sets which were designed for this purpose (Table 4.7), 37 picornaviruses isolated from swabs and tissue samples collected in Germany and Italy between 1997 and 2012 were screened. The primer pair FKei2 (CTACCATCAGGATGCAGTT) - RKei2 (AAGCCAATCCTGCAACACT) gave the highest number of positive results from the chosen isolates (70%). 308 nucleotide long sequences of the amplified products of 26 picornavirus isolates were obtained which represent a small part of the viral polyprotein gene. Alignment of the obtained sequences from the amplified products revealed a close genetic relationship among the detected tortoise picornavirus isolates confirmed by the high identity values between 79.2 – 100% (Table 11.3 in appendix). Phylogenetic analysis clearly shows two main clusters, which together form a single monophyletic cluster. The obtained viral sequences of the polyprotein gene were compared with previously described picornaviruses and the highest similarities were observed with the corresponding gene sequences of picornavirus family members including: Canine picornavirus, Human enterovirus 109, Human rhinovirus C, and Human coxsackievirus A13. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. BLASTn analysis of the sequences confirmed that each of the PCRs with the different primer sets was specific. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise picornaviruses and to help prevent viral spread among animal collections. A conventional RT-PCR was also established for the detection of arenaviruses in snakes and used to screen clinical samples from live and dead animals for these viruses. The reptarenaviruses are considered to be the causative agent of inclusion body disease (IBD) which is a chronic progressive disease affecting captive boas and pythons worldwide. Samples from animals screened for virus detection were also screened for the presence of IBD typical inclusions in blood smears or histological preparations of 63 organs. The primer combinations MDS-400 (5’-TTCATTTCTTCATGRACTTTRTCAATC-3’) and MDS-402 (5’-GGSATAACAAAYTCACTTCAAATATC-3’) targeting part of the glycoprotein gene were used for the detection of reptarenavirus. 49 of 170 snakes tested (28.8%) were arenavirus positive. While 17 of 20 IBD positive snakes (85%) were arenavirus positive by RT-PCR, 17 of 43 IBD negative animals (39.5%) were arenavirus positive. Arenavirus was found in both boas (Boa constrictor) and pythons (Python regius, Malayopython reticulatus, Python molurus, and Morelia viridis). Alignment of the obtained sequences of a small portion of the glycoprotein gene from the detected arenaviruses showed high identity values between 71- 100% with previously described reptarenaviruses. Phylogenetic analysis indicated that a majority of the detected reptarenavirus strains clearly clustered with GGV, Boa AV NL B3 and UHV. None of the detected viruses clustered with CASV. Furthermore, this work includes a study concerning the first detection of sunshinevirus in snakes in Europe using RT-PCR as a diagnostic method. The first description of sunshinevirus was in 2008 after an outbreak of neuro-respiratory disease in an Australian collection of 70 pythons. The RT-PCR used in this study resulted in the detection of sunviruses in three out of 12 snakes tested (25%). The obtained sequences were compared with the corresponding portion of the sunshinevirus genome and the identity values were between 95- 98 %. The viruses were found in oral/ cloacal swabs, lung lavage and skin sample of ball pythons, all other tested snakes were negative (two boas, one anaconda and one Indian python). Clinical signs reported in the animals infected with sunshinevirus vary significantly, but mostly respiratory problems were reported.