MIRTRIYKPTYTTTTPPTCHTPIKMEEDPREKMHPQSMWRLVRLRAQRLLSYSESTDLSTREFLEDVSKS I...R...M...D.A...I.G.
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1 A. FDLV iiiiiiiiiiiiiii1 AnaPV(1110RN043)iiii1 AnaPV(1110RN047)iiii1 AnaPV(1201RN001)I ii1 AnaPV(1201RN003) iii1 AnaPV(1203RN009) iii1 Biti-CA98 iiiiiiiiii1 Dasy-GER00 iiiiiiiii1 Xeno-USA99 iiiiiiiii1 Gono-GER85 iiiiiiiii1 PanGut-GER09 IIIIIII1 HoBuc-HUN09 IIIIIIIi1 Orth-GER05 iiiiiiiii1 Igu-GER00 iiiiiiiiii1 MIRTRIYKPTYTTTTPPTCHTPIKMEEDPREKMHPQSMWRLVRLRAQRLLSYSESTDLSTREFLEDVSKS ---MYT..SVHI.A.SS...S...V...R.Y...ND...I.R V...R.Y...ND...I.R V.L..R.Y...ND...I.....N.HTS.T.H..AKS.R...V...V.L...K...D I...A...I.G I...R...M...D.A...I.G A.MK.S...S..RK...ND.A.P..K...I.R T.MK.A...S..RK...ND.A.P..K...I.E. FDLViii iiiiiiiiiiii71 VVVLFNRDGMSSISQWRTEDCAARRLGNLSKFAWDAVTKGRMDPCRLAFKMVTELGNDVAIRAEILTVVW AnaPV(1110RN043) iii47...tp..h...e...vi.r...i... AnaPV(1110RN047) iii47...tp..h...e...vi.r...i... AnaPV(1201RN001) iii47...tp..h...e...vi.r...i... AnaPV(1201RN003) iii47...tp..h...e...vi.r...i... AnaPV(1203RN009) iii47...tp..h...e...vi.r...i... Biti-CA98 iiiiiiiiii68..i...tp..h...e...iir...t... Dasy-GER00 iiiiiiiii47..i...tp..h...e...iir...t... Xeno-USA99 iiiiiiiii47...h...e...i...v...t... Gono-GER85 iiiiiiiii71...h...v...k... PanGut-GER09 IIIIIII47...IN.MK.RH...E...I...SMI..V...MI.. HoBuc-HUN09 IIIIIIIi47...AA.MI.QH.R...K...NAIV.V...MI.. Orth-GER05 iiiiiiiii47 M...VN.TI.QH...K...K...R...S.MIV.V...I. Igu-GER00 iiiiiiiiii47 M...VN.TI.QH.G...K...K...R...S.MIV.V...I. FDLV i iiiiiiiiiiiii141 LITGWSTIPRTLHKDLWSSAIYRRLSL- AnaPV(1110RN043) iii117...s..q...- AnaPV(1110RN047) iii117...s..q...- AnaPV(1201RN001) iii117...s..q...- AnaPV(1201RN003) iii117...s..q...- AnaPV(1203RN009) iii117...s..q...- Biti-CA98 IIIIIIIiii138...S..Q...- Dasy-GER00 iiiiiiiii117...s..q...- Xeno-USA99 iiiiiiiii117...s..q...- Gono-GER85 iiiiiiiii141...t...- PanGut-GER09 IIIIIII117...S..K...N...K...E HoBuc-HUN09 iiiiiiii117...s..kn..n...- Orth-GER05 iiiiiiiii117...s..kg..n..r..s...p-- Igu-GER00 iiiiiiiiii117...s..kg..n..r..s...p-- B. FDLVii iiiiiiiiiiiiii MVMAIPQHLHRTTPSRAMLIKSRNLAKRSRTKRRNLNRCNSRIGAKSKKATLQ AnaPV(1110RN043) iiii M.VVLP.PPH...IR.R...DPVT AnaPV(1110RN047)iiiii M.VVLP.PPH...IR.R...DPVT AnaPV(1201RN001)iiiii M.VVLP.PPH...IR.R...DPVT AnaPV(1201RN003)iiiii M.VVLP.PPH...IR.R...DPVT AnaPV(1203RN009)iiiii M.VVLP.PPH...IR.R...DPVT HeV iiiiiiiiiiiiiiii1 MDLGGEEISQNLLLEVMSIW.A..FT.RTT.MKISW.MRMSLPKAQVRLSLTLLLRTMI.STRRK 65
2 FIG S1 Multiple alignments of (A) U protein and (B) putative SB protein in the genomes of FDLV, AnaPV and other reptilian paramyxoviruses. Biti-CA98, Reptilian paramyxovirus (AAS ); Dasy-GER00, Snake paramyxovirus (ACT ); Xeno-USA99, Lizard paramyxovirus (ACT ); Gono-GER85 Reptilian paramyxovirus (AAS ); PanGut- GER09 Snake paramyxovirus (ADT ); HoBuc-HUN09, Snake paramyxovirus (AFQ ); Orth-GER05, Snake paramyxovirus (ACT ); Igu-GER00, Lizard paramyxovirus (ACT ).
3 A B C D FIG S2 Panel A, Normal RK13 cells. Panel B, CPEs of AnaPV on RK13 cells. Panel C, Normal Vero cells. Panel D, CPEs of AnaPV on Vero cells.
4 Col-2c AsaPV Orth-GER05 Snake ATCC-VR-1409 paramyxovirus (AAK ) Crot-GER03 Pyth-GER01 CeraCe-98 Reptilian paramyxovirus (AAK ) Igu-GER00 Pyt THer-GER99 HoBuc FDLV PanGut Reptilian paramyxovirus (AAK ) Gono-GER85 Xeno-USA99 Snake ATCC-VR-1408 paramyxovirus (AAK ) CrotX RN RN RN RN RN047 ElaGut-91 Reptilian paramyxovirus (AAK ) Reptilian paramyxovirus (AAK ) Dasy-GER00 Pyt-2 (ADC ) Pyt-2 (ADC ) Pyt-2 (ADC ) 0.05 FIG S3 Phylogenetic analysis of amino acid sequences of partial L protein of AnaPV and other snake and reptilian paramyxoviruses. The trees were constructed by maximum likelihood method with bootstrap values calculated from 1,000 trees and rooted on midpoint. The scale bars indicates the branch length that corresponds to 0.05 substitutions per site. Five strains from AnaPV were named as 1201RN001, 1110RN043, 1110RN047, 1201RN003, and 1203RN009. AsaPV, Atlantic salmon paramyxovirus strain ASPV-Ro (EF ); HoBuc-HUN09, Snake paramyxovirus (AFQ ); PanGut-GER09, Snake paramyxovirus
5 (ADT ); Col-2c, Snake paramyxovirus(adc ); Pyt-6, Snake paramyxovirus (ADC ); Dasy-GER00, Snake paramyxovirus (ACT ); Crot-GER03, Snake paramyxovirus (ACT ); Orth-GER05, Snake paramyxovirus (ACT ); Pyth-GER01, Snake paramyxovirus (ACT ); THer-GER99, Tortoise paramyxovirus (ACT ); Igu-GER00, Lizard paramyxovirus (ACT ); Xeno-USA99, Lizard paramyxovirus (ACT ); ElaGut-91, Reptilian paramyxovirus (AF ); Gono-GER85, Reptilian paramyxovirus (AAS ); CrotX1-96, Reptilian paramyxovirus (AF ); CeraCe-98, Reptilian paramyxovirus (AF )
6 TABLE S1 Replication of AnaPV in 27 cell lines of different origins Cell line Viral load, log 10 copies/ml, mean±sd Organism and anatomic site Abbreviation Source Day 0 Day 1 P Day 3 P Day 5 P Human Respiratory tract Embryonic lung fibroblasts HFL In-house development 5.81± ±0.01 < ± ± Lung adenocarcinoma Calu-3 ATCC no. HTB ± ± ±0.02 < ± Lung adenocarcinoma A549 ATCC no. CCL ± ± ±0.03 < ± Laryngeal epidermoid carcinoma Hep-2 ATCC no. CCL ± ± ±0.03 < ± Gastrointestinal tract Colorectal adenocarcinoma Caco-2 ATCC no. HTB ± ± ±0.10 < ± Ileocecal colorectal adenocarcinoma HRT-18G ATCC no. CRL ± ± ± ± Liver Hepatocellular carcinoma Huh-7 JCRB0403, JCRB cell bank 6.45± ±0.01 < ± ± of Okayama University Genitourinary tract Cervical adenocarcinoma HeLa ATCC no. CCL ± ± ± ± Fetal kidney HEK In-house development 6.88± ± ± ± Neuromuscular cells Neuron-committed teratocarcinoma NT2 ATCC no. CRL ± ± ± ± Rhabdomyosarcoma RD ATCC no. CCL ± ±0.05 < ± ± Central nervous system SF268 Kindly provided by Prof. S. 6.70± ± ±0.04 < ± R. Shih Immune cell Malignant histiocytoma His-1 In-house development 6.50± ± ± ± Non-human Mammals Madin-Darby canine kidney MDCK ATCC no. CCL ± ± ± ±0.04 <0.001
7 Bovine kidney MDBK ATCC no. CCL ± ±0.01 < ±0.01 < ± Rabbit kidney RK13 ATCC no. CCL ± ±0.01 < ± ±0.02 <0.001 Primary mouse embryonic fibroblasts 3T3 ATCC no. CCL ± ± ±0.03 < ±0.03 <0.001 Camel skin Dubca ATCC no. CRL ± ± ± ± Rhesus monkey kidney LLC-MK2 ATCC no. CCL ± ± ± ± African green monkey kidney Vero ATCC no. CCL ± ± ± ± African green monkey kidney VeroE6 ATCC no. CRL ± ± ± ± (clone of Vero-76) Feline kidney CRFK ATCC no. CCL ± ± ± ± Porcine kidney PK-15 ATCC no. CCL ± ± ± ± Rattus norvegicus kidney RK3E ATCC no. CRL ± ± ±0.05 < ± Rattus norvegicus kidney RMC ATCC no. CRL ± ± ± ± Chicken Chicken fibroblasts DF-1 ATCC no. CRL ± ± ± ± Insect Aedes albopictus C6/36 ATCC no. CRL ± ± ± ±
8 TABLE S2 Comparison of genome characteristics between AnaPV and other members in Paramyxovirinae Genus Virus Genome features Pairwise percent amino acid identities of AnaPV proteins with analogous proteins in Paramyxovirinae genome size G+C content N U P V I M F HN L Ferlavirus FDLV 15,378 bp 43.13% Aquaparamyxovirus AsaPV 16,965 bp 45.88% 18.5 NA NA Morbillivirus CDV 15,690 bp 42.98% 27.9 NA 19.5 NA NA MeV 15,894 bp 47.43% 26.7 NA NA PPRV 15,948 bp 48.10% 28.0 NA NA Respirovirus BPIV-3 15,456 bp 35.96% 26.7 NA 18.6 NA NA HPIV-1 15,600 bp 37.24% 25.4 NA 18.0 NA NA HPIV-3 15,462 bp 34.52% 25.6 NA 17.0 NA NA SeV 15,384 bp 46.09% 26.3 NA NA Rubulavirus MuV 15,384 bp 42.49% 22.7 NA HPIV4a 17,052 bp 36.23% 22.8 NA NA HPIV4b 17,304 bp 36.40% 23.7 NA NA PoRV 15,180 bp 46.70% 24.4 NA NA Henipavirus HeV 15,882 bp 48.00% 29.7 NA NA NiV 18,246 bp 38.17% 28.9 NA NA CedPV 18,162 bp 37.20% 27.6 NA 16.5 NA NA Avulavirus APMV6 16,236 bp 46.35% 23.6 NA 20.5 NA NA NDV 15,186 bp 46.18% 25.5 NA NA GooPV 15,192 bp 46.55% 27.1 NA 19.2 NA NA Unclassified BeiPV 19,212 bp 42.78% 26.7 NA NA Paramyxovirinae JPV 18,954 bp 40.95% 27.3 NA NA TlmPV 19,152 bp 39.62% 27.7 NA NA NA, not available
9 TABLE S3 Molecular features and predicted gene products of AnaPV Genome region mrna features, nt Deduced protein Total Hexamer Intergenic MW, Coding length 5'UTR ORF 3'UTR phase regions, nt Size, aa kda pi frame Leader (ATT) N 1, , CCT U CTT P/V/I (P) 1, , CTT P/V/I (V) 1, CTT P/V/I (I) 4, CTT M 1, , CTT F 1, , CTT HN 1, , CCT L 6, , (CTT) 2, Trailer
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