Diagnosis of ferlaviruses in snakes and characterization of isolates based on gene sequences

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). \nSamples 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.\nUnspecific 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.\nFerlaviruses 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. \nA 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.\nIn 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. \nOn 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. \n