Browsing by Person "Marschang, Rachel E."

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Diagnosis of ferlaviruses in snakes and characterization of isolates based on gene sequences
(2013) Abbas, Maha Diekan; Marschang, Rachel E.
PMV are important pathogens for reptiles especially snakes and have been isolated from wild and private collections. During a period (2009-2011), a total of 495 clinical samples originating from 251 snakes of several families including Boidae, Pythonidae, and Colubridae were screened for the presence of PMV by RT-PCR described by Ahne et al. (1999) targeting a partial sequence of the L gene and virus isolation on the reptilian cell line viper heart cells (VH2). Samples with positive amplicons (566 bp, L gene) were subjected to RT-PCR targeting partial sequence of HN as described by Ahne et al. (1999) and Marschang et al. (2009) and U gene as described by Marschang et al. (2009). All RT-PCR positive amplicons were subjected to sequencing in order to exclude false positive results. Phylogenetic analyses using several programs (Phylip 3.36 and Mega 5.05) were carried out to explore the associations between the viruses detected and to broaden our understanding of their taxonomic relationships. Unspecific size products and specific size products with non specific amplicons were repeatedly obtained using the previously published protocol. Several trials were therefore carried out in an attempt to increase the specificity of the original RT-PCR protocol (Ahne et al., 1999) including optimization and sensitivity tests. Several concentrations of MgCl2 (1, 1.5, 2, 2.5) mM and different annealing temperatures (45, 48 and 51) Cº were used in order to eliminate the unspecific size products. Sensitivity tests using several ferlavirus isolates were conducted using new degenerate primers targeting the conserved L gene. Changes in annealing temperature and MgCl2 concentration did not decrease the number of unspecific reactions detected. Sensitivity tests showed that the RT-PCR protocol described by Ahne et al. (1999) has the highest sensitivity. However, this protocol has been shown to be highly unspecific. Sequencing of RT-PCR products is therefore necessary to ensure specific results. Ferlaviruses were detected in 5.97% of the snakes tested (15 of the 251 snakes screened). All ferlavirus positive snakes were from the families Colubridae and Pythonidae. The low infection rate might indicate a fluctuation in the infection rate. A total of six different partial L gene sequences were obtained from 19 RT-PCR products using RT-PCR (L gene) and verified by sequencing. Three of these products clustered within subgroup B isolates. The one detected in an Indian python was 97% similar to FDLV (AY141760.2) (Subgroup A). Two (Pangut GER09 and Hobuc HUN09) were not assigned to subgroup A or B. However, they clustered together forming the first two representatives of the novel subgroup C within the Ferlavirus genus extending its classification into three squamate subgroups; A, B and C. Concurrent viral infections (PMV, reo and AdV) were detected in a group of corn snakes in Germany which highlight the significance for testing for different pathogens and different organs and tissues. In order to assess the degree of genetic diversity within this group of viruses, complete CDS regions of the F and HN genes from nine ferlaviruses were sequenced and compared (on both nt and deduced aa sequence levels) with each other and with the corresponding sequences of other genera of the Paramyxovirinae. Phylogenetic analyses were conducted for each gene separately and for the concatenated sequences of F, HN and the extended portion (1544 nts) of L gene. On a genomic level, squamate ferlaviruses are closely related; however they are distributed into three different genogroups (A, B and C). Deduced animo acid sequences of both F and HN genes of all ferlavirus isolates revealed conserved domains corresponding to those described for other members of the Paramyxovirinae. However, the chelonid ferlavirus showed for both genes and for some motifs some differences to the squamate (snake and lizard) group.
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.