Bats host major mammalian paramyxoviruses

Drexler, Jan Felix; Corman, Victor Max; Müller, Marcel Alexander; Maganga, Gael Darren; Vallo, Peter; Binger, Tabea; Gloza-Rausch, Florian; Rasche, Andrea; Yordanov, Stoian; Seebens, Antje; Oppong, Samuel; Sarkodie, Yaw Adu; Pongombo, Célestin; Lukashev, Alexander N.; Schmidt-Chanasit, Jonas; Stöcker, Andreas; Carneiro, Aroldo José Borges; Erbar, Stephanie; Maisner, Andrea; Fronhoffs, Florian; Buettner, Reinhard; Kalko, Elisabeth K.V.; Kruppa, Thomas; Franke, Carlos Roberto; Kallies, René; Yandoko, Emmanuel R.N.; Herrler, Georg; Reusken, Chantal; Hassanin, Alexandre; Krüger, Detlev H.; Matthee, Sonja; Ulrich, Rainer G.; Leroy, Eric M.; Drosten, Christian

Phylogenetic analyses. Nucleic acid alignments based on amino acid code were done in Mega4 (www.megasoftware.net). Gap-free coding nucleotide sequence alignments were generated containing the novel viruses as well as reference strains, using the complete deletion option in which any site containing gaps was deleted from the data set (see Supplementary Table S3 for a list of all reference strains with isolation years, host and GenBank accession number). The data set used for inference of Paramyxovviridae phylogenies comprised 559 nucleotide (nt). An additional analysis was done for only those rubulaviruses for which only a shorter 217 nt fragment could be amplified (see Fig. 2a , note that Supplementary Fig. S1 includes only rubulaviruses with complete 559 nt coverage). Bayesian phylogenies were calculated with MrBayes V3.2 56 using amino acid sequences (WAG + G model) and nucleotide sequences (GTR + I + G model). Both analyses yielded identical topologies ( Fig. 2 and Supplementary Fig. S1 ). Convergence of chains was confirmed by the PSRF statistic in MrBayes 57 , as well as by visual inspection of individual traces using TRACER from the BEAST package 58 . Outgroups were Rabies virus (GenBank, NC_001542) for phylogenies, including the complete family Paramyxoviridae, Newcastle Disease virus for phylogenies of the genus Rubulavirus (GenBank, NC_002617) and Human Parainfluenzavirus 1 (GenBank, NC_003461) for the genera Henipa-and Morbillivirus. In parallel to the Bayesian analyses, maximum likelihood algorithms (WAG + G substitution model, 5 gamma categories and 1,000 bootstrap replicates) were applied using PhyML V3.0 59 . Trees were visualised with FigTree V1.3.1 from the BEAST package 58 . Supplementary Tables S6 and S7 . Supplementary Fig. S6 summarises the numbers of GenBank entries of PV by ordinal host groups. Threshold amino acid distance values for classifying phylogenetic branches were estimated by comparing the maximum amino acid distances within and between established PV species in the corresponding sequence fragments. Measles virus, mumps virus and RSV were selected to determine the maximum within-species distances per fragment, based on their good coverage in sequence databases. Maximum amino acid diversity within all publicly available mumps virus sequences was 1.6% in the translated 559 nt fragment, 5.4% for measles virus and 6.1% for RSV. For comparison, the amino acid divergence between the species HeV and NiV ranged from 7.0 to 7.5% in this fragment. Only taxa exceeding 7.0% amino acid distance were therefore counted as separate viruses.