Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5′ untranslated region

Available online at www.sciencedirect.com

Journal of Virological Methods 146 (2007) 375–388

Short communication

Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5 untranslated region Massimo Giangaspero ∗ , Ryˆo Harasawa Veterinary Microbiology, School of Veterinary Medicine, Faculty of Agriculture, Iwate University, 18-8 Ueda 3 Chome, Morioka 020-8550, Iwate, Japan Received 11 May 2007; received in revised form 12 July 2007; accepted 16 July 2007 Available online 23 August 2007

Abstract The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5 untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5 UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences. © 2007 Elsevier B.V. All rights reserved. Keywords: Palindromic nucleotide substitutions; Pestivirus; Taxonomy; 5 UTR

The palindromic nucleotide substitutions (PNS) genotyping method (Harasawa and Giangaspero, 1998) has been improved from the original concept limited to qualitative analysis of the three variable loci (V1, V2 and V3) in the 5 untranslated region (UTR) of Pestivirus RNA. Quantitative evaluation of genomic sequence divergence, in terms of palindromic nucleotide basepairings variations, has been applied for taxomical segregation of species, through the evaluation of 430 genomic sequences. Pestivirus sequences, of different geographical origin, from different host species or contaminants of biological products, have been obtained from databases (Table 1). The identification of species within the genus has been achieved according to highly conserved base-pairs in the three variable palindromic loci, based on a series of specific passages focussed on the quali-

∗

Corresponding author. Tel.: +81 19 621 6229; fax: +81 19 621 6229. E-mail address: [email protected] (M. Giangaspero).

0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.07.009

tative and quantitative analysis of the secondary structures of the strains within the genus. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5 UTR sequences of all the considered strains belonging to the genus (Table 2). Secondary structure sequences showing divergent base-pair combinations (irrespective of the number of strains) have been aligned for comparison. Prevalent base-pairs, present in 80% or more of the sequences, have been identified to determine most common secondary sequences within the genus. Strains showing sequence identity or incomplete sequence have been excluded. Due to 5 UTR incomplete sequence, eight strains (BVDV-1 95-4845A, 95-4845B, DeerGB1 and M98, BVDV-2 59386 and SCP, BDV BDncp and L83/L84) have been clustered according to qualitative evaluation and excluded from quantitative analysis. All the base-pairs composing each position have been identified according to their nucleotide pairing, and variants were quantified expressing the percentage within the genus. The

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Table 1 Pestivirus strains (n 430) evaluated according to the palindromic nucleotide substitution (PNS) method at the 5 untranslated region of RNA Species

Strain

Origin

Country

Accessiona

BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1

0192 1041/01 107/01 10-84 10846/91 10A/LC/97 1103/88 11207/98 114 817 1190V97 12 1248/01 125 85 128/88 133/02 1372/01 14-102 15-3 16-111 16484/93 17-112 1 77 17P 1891/99 1946/01 1/A/00 1/B/01 1/C/01 1R 1R93 2032/01 20-V661-2 2110C 2204/82 22146/81 2218/01 228/02 23-13 23-15 2318/01 2343/01 24/15 2430 95 252 84 2555/01 2586X 99 25H 26-V639 2703D 99 2708/01 2750A 99 28/1 2900/83 2/A/00 2B 2/B/01 2L91 2/VR/95 3114 93 318 3186V6 3187V 3251/01 3291-97-A

Contaminant Sheep Cattle Cattle Cattle Cattle Cattle Cattle Sheep Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Cattle Contaminant Cattle Cattle Cattle Cattle

Japan Spain Spain France Germany Italy Germany Germany UK Italy Japan Spain Ireland Germany Spain Spain France UK France Germany France Ireland Argentina Germany Spain Italy Italy Italy Argentina Argentina Spain France USA Germany Germany Spain Spain UK UK Spain Spain UK Ireland Ireland Spain Ireland Argentina France Ireland Spain Ireland UK Germany Italy Argentina Italy Japan Italy Ireland Argentina Italy Spain Spain Austria

D31799 AY159542 AY159523 AF298054 AJ304389 AJ293602 AJ304380 AJ304390 U65053 Vilˇcek et al. (2001) D26051 AY159545 AJ312909 AJ304379 AY159534 AY159552 Vilˇcek et al. (2001) Vilˇcek et al. (2001) AF298056 AJ304383 AF298057 AJ312927 AF244954 AJ304384 AY159539 Ciulli et al. (2002) Ciulli et al. (2002) Ciulli et al. (2002) AF244955 AF244956 AY159537 AF298058 L20921 AJ304377 AJ304376 AY159525 AY159554 Vilˇcek et al. (2001) AF298059 AY159543 AY159541 AF298060 AJ312930 AJ312925 AY159553 AJ312915 AF244965 Vilˇcek et al. (2001) AJ312913 AY159536 AJ312916 AF298061 AJ304375 Ciulli et al. (2002) AF244957 Ciulli et al. (2002) D31800 AJ293594 AJ312932 AF244958 AF298062 Vilˇcek et al. (2001) AY159550 Vilˇcek et al. (2001)

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1

3310/01 3336/00 3340/01 3417/00 3425/01 3478/00 3479-97-I 3499/00 3596/86 368/02 371 89 383 76 3/A/00 3/B/01 3P 3/VR/95 4050/00 4092/00 4163/00 4171/00 4283/00 42M 4325/01 438/02 4382/01 4629/01 4771 94 4796 94 4898 94 4998/89 4/B/01 4H 4/VR/95 5284/00 551/02 5551/84 561/01 5/B/01 65.2 66.1 66.3 66.5 66.6 68.883 6/B/01 720/02 7535 7546 7548 76865 7/B/01 80/1 cp 82 80/1 ncp 82 8087 99 819 85 832/01 86713 8/B/01 9189 9466/91 95-4845A 95-4845B 9-77 985 84 A

Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Sheep Sheep Sheep Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Deer Deer Cattle Cattle Cattle

Spain Spain Spain Spain Spain Spain Italy Spain Germany Spain Ireland Ireland Italy Italy Argentina Italy Spain Spain Spain Spain Spain Argentina Spain Spain Spain Spain Ireland Ireland Ireland Germany Italy Argentina Italy Spain Spain Germany Spain Italy Argentina Argentina Argentina Argentina Argentina Argentina Italy Spain Sweden Sweden Sweden Argentina Italy Ireland Ireland Ireland Ireland Spain Argentina Italy Belgium Germany New Zealand New Zealand France Ireland Austria

AY159532 AY159549 AY159547 AY159538 AY159521 AY159548 Vilˇcek et al. (2001) AY159546 AJ304388 AY159530 AJ312918 AJ312926 Ciulli et al. (2002) Ciulli et al. (2002) AF244968 AJ293595 AY159524 AY159533 AY159551 AY159519 AY159527 AF417999 AY159526 AY159540 AY159529 AY159528 AJ312920 AJ312910 AJ312931 AJ304385 Ciulli et al. (2002) AF244964 AJ293596 AY159544 AY159520 AJ304378 AY159531 Ciulli et al. (2002) AF244960 AF244953 AF244967 AF244961 AF244966 AF244962 Ciulli et al. (2002) AY159535 U65060 U65061 U65062 AF244969 Ciulli et al. (2002) AJ312929 AJ312914 AJ312921 AJ312924 AY159522 AF244970 Ciulli et al. (2002) ALIGN 000012 AJ304382 U80903 U80904 Vilˇcek et al. (2001) AJ312917 AF298064

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1

A014 A553 akT1 B1056 B551 98 BO2340/01 BR275 BRU*0615 BVR1199 C009T cb1 CD89 CP1872 CP1874 CP1885 CP1887 CP1940 CP1945 CRFK Culi1 Culi4 Culi6 CV-1 D D1120/1 D1432/P D771/1 D861 Deer DeerGB1 Draper Europa F FLK FS720 FU411 G H H503 H686 98 H851 98 HC725 HE726 HE728 HeLa HH i13 i297 i36P i393 i467 i53 i63 i66.2 i6.89 i720 i736 i89 Ind 446 Ind S 1222 Ind S 1166 Ind S 1455 Ind S 1168 Ind S 1170 Ind S 1171

Contaminant Sheep Cattle Sheep Cattle Cattle Cattle Contaminant Cattle Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Cattle Cattle Contaminant Cattle Sheep Sheep Sheep Sheep Deer Deer Cattle Human Cattle Contaminant Contaminant Contaminant Cattle Cattle Contaminant Cattle Cattle Contaminant Contaminant Contaminant Contaminant Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle

Japan UK Argentina UK Ireland Spain Brazil Japan Belgium Japan Argentina Belgium Belgium Belgium Belgium Belgium Belgium Belgium Japan Belgium Belgium Belgium Japan Austria UK UK UK UK UK New Zealand USA Belgium Austria Belgium Japan Japan Austria Austria Japan Ireland Ireland Japan Japan Japan Japan Japan Argentina Argentina Argentina Argentina Argentina Argentina Argentina Argentina Argentina Argentina Argentina Argentina India India India India India India India

D31801 U65025 Jones et al. (2001) U65029 AJ312911 AY159518 U94915 AB008837 ALIGN 000012 D31802 AF417998 ALIGN 000012 ALIGN 000012 ALIGN 000012 ALIGN 000012 ALIGN 000012 ALIGN 000012 ALIGN 000012 D50814 ALIGN 000012 ALIGN 000012 ALIGN 000012 D50815 Vilˇcek et al. (2001) U65032 U65033 U65030 U65031 Becher et al. (1997) U80902 L32880 AB000898 AF298065 ALIGN 000012 D31803 D31804 AF208066 Vilˇcek et al. (2001) AB008841 AJ312923 AJ312908 D31805 D31806 D31808 D50819 D50818 AF417989 AF417997 Jones et al. (2001) AF417992 AF417994 AF417987 AF417990 Jones et al. (2001) Jones et al. (2001) AF417988 AF417993 AF417991 AY279087 AY278459 AY278460 AY278461 AY279086 AY279526 AY279527

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1

Ind S 1181 Influenza2 IQ19A J JE K869 98 KA-91 KM KQ25A KQ25B Kyj L L1000 98 L256 L322 98 Lamspringe735 Lamspringe738 Ln 68 LQ28A M1515A/90 M169B/93 M245A/91 M346T/96 M388A/90 M557A/90 M98 Marloie Massimo1 Massimo2 Massimo4 MDBK MDCK MMR-K MOLT-4 Mumps MV98CB/95 NADL ncp2 NY-1 Oregon Osloss P PT810 Q1161/1 Q1161/2 Q713 R R1935/72 Renindeer2 S S21 S-ALT5/K Sanders SD-1 SE1015 SE5572 SE5726 SH9/11 Singer T TFB TFB2 TGAC TGAN TK-87-2

Cattle Contaminant Contaminant Cattle Contaminant Cattle Cattle Cattle Contaminant Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Contaminant Contaminant Contaminant Contaminant Contaminant Contaminant Contaminant Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Sheep Sheep Cattle Cattle Cattle Pig Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Roe deer Cattle Cattle Contaminant Cattle Cattle Cattle Cattle

India Switzerland Japan Austria Japan Ireland Japan Slovakia Japan Japan Slovakia Austria Ireland France Ireland Germany Germany Ireland Japan South Africa South Africa South Africa South Africa South Africa South Africa Switzerland Belgium Switzerland Italy Switzerland Japan Japan Japan Japan Japan South Africa USA Argentina USA USA USA Austria Germany UK UK Canada Austria Brazil Germany Austria Argentina South Africa USA USA Germany Germany Germany Germany USA Austria Argentina Argentina USA USA Japan

AY279528 AB010146 D31812 AF298067 D26611 AJ312912 AB019684 AF298068 D31809 D31810 Vilˇcek et al. (2001) AF298069 AJ312919 ALIGN 000012 AJ312922 AJ304391 AJ304392 AJ312928 D31811 U97429 U97430 U97436 U97440 U97442 U97449 AB014339 ALIGN 000012 AB008838 AB008839 AB008840 D50820 D50821 D26050 D50822 D26049 U97467 M31182 Jones et al. (2001) L32879 L32876 M96687 AF298070 Z79766 U65040 U65041 L32882 AF298071 U94916 Giangaspero et al. (unpublished) Vilˇcek et al. (2001) AF244963 U97474 L20928 M96751 Z79767 Z79770 Z79778 Frolich and Hofmann (1995) L32875 AF298072 AF244971 AF418000 Qi et al. (1993) Qi et al. (1993) AB019669

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-1 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2

TY CP/91 U U937 Vero Vkl VM W Weybridge Wi-38 WiDr YVD947 YVR2394 ZVD278 ZVR711 104/98 11/Mi/97 15-103 167 237 168 149 17011-96 173 157 175 375 17583-97 23025 34b 354 37Gr 4-5174 5521-95 59386 713-2 7937 890 97/730 AF112 AZ Spl B45-5 B50-5 B52-2 B5-4 B77-5 BD-78 BS-95-II BSE1239 BSE341 BSE921 C413 CD87 CPA CPAE EBTr Giessen-1 HE727 i33283 i4083 i61380 i628 IT-1732 Kosice Lees LV-96 MAD Spl MMR-T MN Fetus MP

Cattle Cattle Contaminant Contaminant Cattle Cattle Cattle Sheep Contaminant Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Sheep Sheep Cattle Sheep Sheep Cattle Cattle Cattle Contaminant Cattle Cattle Cattle Sheep Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Sheep Cattle Cattle Cattle Cattle Sheep Cattle Contaminant Contaminant Contaminant Cattle Contaminant Cattle Cattle Cattle Cattle Contaminant Cattle Sheep Cattle Cattle Contaminant Cattle Contaminant

Japan Austria Japan Japan Slovakia USA Austria UK Japan Japan Belgium Belgium Belgium Belgium Germany Italy France UK UK USA UK UK USA USA Argentina Argentina Austria France USA UK USA USA Canada New Zealand Germany USA Germany Germany Germany Germany Germany USA Italy Belgium Belgium Belgium USA Canada Japan Japan Japan Germany Japan Argentina Argentina Argentina Argentina Italy Slovakia UK Brazil USA Japan USA Belgium

AB042670 Vilˇcek et al. (2001) D50823 D50824 Vilˇcek et al. (2001) L20933 AF298073 U65024 D50825 D50826 ALIGN 000012 ALIGN 000012 ALIGN 000012 ALIGN 000012 AJ304381 AJ293603 AF298055 U65055 U65056 AF039179 U65058 U65059 AF039176 AF039172 AF244952 AF244959 Vilˇcek et al. (2003) AF298063 AF039174 U17146 AF039177 AF039175 L32886 AF026770 Beer et al. (2002) Ridpath et al. (1994) Beer et al. (2002) Beer et al. (2002) Beer et al. (2002) Beer et al. (2002) Beer et al. (2002) U18330 AJ288903 ALIGN 000012 ALIGN 000012 ALIGN 000012 AF002227 L32887 D50812 D50813 D50817 AF104030 D31807 AF417996 AF417995 AF417986 AF417985 AJ416018 Vilˇcek et al. (2002) U65051 AF410787 Ridpath et al. (1994) D26052 Ridpath et al. (1994) ALIGN 000012

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BVDV-2 BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV BDV CSFV CSFV CSFV CSFV

MS-1 Munich 1 Munich 2 Munich 3 ncp7 NY93 OY89 Parvo Q126 Rubella SCP Soldan SW90 SY-89 TC Shinozaki UVR420 V-FLL VS-123.4 VS-260 VS-63 WG4622 WVD829 135 661 137/4 170 337 2112/99 79248/01 80582/01 8320-22NZ 8320-31NZ 87877/01 90/8320/31 91/5809 A1263/2 A1870 A841/1 BD31 BD ncp Chamois1 Ch1Es D1586/2 G1305 G2048 JH2816 K1729/3 L83/L84 L991 Moredun cp Moredunncp Q1488/1 Q1488/6 Q1673/2 Rentier Rudolph T1789/1 T1802/1 V1414 V2377/12 V2536/2 V3196/1 Wisent Casimir X818 17-93 39 5440/99 Alfort

Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Contaminant Contaminant Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Contaminant Cattle Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Chamois Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Sheep Reindeer Sheep Sheep Sheep Sheep Sheep Sheep Wisent Sheep Pig Pig Sheep Pig

Japan Germany Germany Germany Argentina USA Japan Japan Canada Japan UK Brazil Japan Japan Japan Belgium Japan Brazil Brazil Brazil Netherlands Belgium UK UK UK Spain Spain Spain New Zealand New Zealand Spain UK UK UK UK UK USA USA Spain Japan UK UK UK UK UK Germany UK UK UK UK UK UK Germany UK UK UK UK UK UK Germany Australia Poland China Spain France

AB019688 Beer et al. (2002) Beer et al. (2002) Beer et al. (2002) Jones et al. (2001) AF039173 AB003621 D26614 L32890 D26048 U17148 U94914 AB003622 AB019689 AB04267 ALIGN 000012 AB019687 AF410790 AF410788 AF410789 ALIGN 000012 ALIGN 000012 U65054 U65052 U65057 AY159513 AY159515 AY159516 U65063 U65064 AY159517 AF026769 AF026768 U65027 U65028 U65026 U70263 De Moerlooze et al. (1993) AY738080 D50816 U65034 U65035 U65036 U65037 U65038 U17144 U65039 U65022 U65023 U66042 U65043 U65044 AB122086 U65045 U65046 U65047 U65048 U65049 U65050 AB122085 AF037405 L42413 AF407339 AY159514 J04358

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Table 1 (Continued ) Species

Strain

Origin

Country

Accessiona

CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV CSFV New taxon New taxon

Alfort 187 Alfort A19 Brescia C strain CAP cF114 Chiba-80 Eystrup Fukuoka/72 GPE (−) HCLV Hokkaido/66 Honduras Ibaraki/66 Ibaraki/81-115 Ibaraki/81-20 Ibaraki/81-38 Ibaraki/81-40 Kanagawa/74 KC LOM Miyazaki/81 Nakamura/66 Okinawa/86 Osaka/51 Osaka/71 Pader Saitama/81 Shimen Shizuoka/73 Switzerland 1/93 Switzerland 2/93 Switzerland 3/93/1 Switzerland 3/93/2 Switzerland 4/93 Vac A Venhorst VRI4762 Yamanashi/69 H138 Pronghorn

Pig Pig Pig Pig Pig Pig Pig Pig Pig Vaccine Pig Pig Pig Pig Pig Pig Pig Pig Pig Vaccine Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Giraffe Pronghhorn

France France Italy China Switzerland China Japan Germany Japan Japan China Japan Honduras Japan Japan Japan Japan Japan Japan Russia Japan Japan Japan Japan Japan Japan Garmany Japan China Japan Switzerland Switzerland Switzerland Switzerland Switzerland USA Netherlands Malaysia Japan Kenya USA

X87939 U90951 M31768 Z46258 X96550 AF333000 AB019659 AF326963 AB019150 AB019152 AF091507 AB019154 L42426 AB019156 AB019158 AB019160 AB019162 AB019164 AB019166 AF099102 AB019655 AB019168 AB019170 AB019172 AB019174 AB019176 AY072924 AB019178 AF092448 AB019180 AF045068 AF045069 AF045070 AF045071 AF045072 L42435 AF084049 L42437 AB019182 AB040131 Vilˇcek et al. (2005)

a

Reference is given if the sequence was not deposited.

Table 2 Characterization of palindromic positions in the genus Pestivirus Number of strains Number of strains showing incomplete sequence Number of strains showing identical sequence Number of types of base-pair combinations Highly conserved, homogeneous positions Prevalent positions Low variable positions

High variable positions LVP located in the loop structure

430 8 163 259 V1/4, 5, 10, 11; V2/8, V2 loop; V1/3, 5r, 6, 7, 13, 22, V2/3, 9, V3/3 (2 variants); V1/2, 8 (3 variants); V1/1, V3/1 (4 variants); V1/9, 21, V2/2 (5 variants); 1 type 3 V3/4 (3 variants); 3 type 4 V2/1, 7, V3/5 (4 variants); 4 type 5 V1/12, V2/4, 5, V3/2 (5 variants); 1 type 6 V1/15 (6 variants); 1 type 8 V1/14 (8 variants); 2 type 9 V2/6, V3/10 (9 variants) V1/18, V3/6 (10 variants); V1/16, 17, 20, V3/7 (12 variants); V3/8, 9 (14 variants); V1/19 (19 variants) V3/10 (9 variants)

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position of base-pairings has been defined by numbering from the bottom of the variable locus. Palindromic positions have been characterized according to changes in nucleotide base-pairs to determine relatedness and divergence among strains, identifying: highly conserved, homogeneous positions, genus-specific positions including base-pairs present in all of the strains of the genus (suitable for genus determination); prevalent position (PP) including base-pairs present in 80% or more of the strains of the genus (suitable for genus and species determination); low-variable position (LVP) including base-pairs present in less than 80% of the genus (suitable for species or genotype determination), LVP have been classified according to their homogeneity (the types have been named according to the number of variants: type 2 with two variants up to type 9 with nine variants), LVP located in the loop structure have been identified; highly variable position (HVP) including base-pairs present in less than 80% of the genus, also including 10 or more base-pair variants (not suitable for genotyping when hypervariable at species level, equivalent to isolate variability). In each position, the following have been identified: prevalent types of base-pairs representing the majority of the strains of the genus, divergent base-pairs not exceeding 20% among the strains, and base-pairs not exceeding 5% among strains and representing exceptions. The second step was quantitative, allowing the identification of genomic groups among Pestivirus strains according to secondary sequence structure base-pair combinations in the three palindromes and identifying strains showing multirelations (sequences showing low relatedness to different genomic groups with low divergence values) or borderlines (sequences showing qualitative similarities with a genomic group, but with high divergence values, candidates for reclustering as separate groups in the genus), and indicating divergence within groups and among groups quantifying the etherogeneity of a species and the genetic distance between species in terms of variation of base-pairs in the secondary structure (Table 3). The identified base-pair combinations (n 259: 177 out of BVDV-1 species isolates, 39 from BVDV-2, 23 from BDV, 18 from CSFV, and the Giraffe and Pronghorn isolates) have been clustered in 115 types according to stem LVP. Subsequently, types have been compared to obtain divergence values. Computing of divergence values was performed by alignment of the secondary structure sequences and comparison of each base-pairing. Base-pairings showing variation in one or two nucleotides scored 1 and those showing identity scored 0. The sum of the scores obtained throughout the entire palindromic sequence of the three variable loci V1, V2 and V3 provided the divergence value between the two sequences under consideration. Transitional mutation G*U—G–C or U*G—C–G have been considered exceptions in the genus, indicated in the figure G:Y or Y:G, thus corresponding to identity in the sequence. Highly conserved, homogeneous positions—genus sequence motifs, have not been included. Palindromic base-pair combinations have been clustered according to LVP located in the stems (LVP located in the loops were not utilised) to identify genomic groups (named types). Identified types included base-pair combinations (named subtypes). The different combinations have been ranked according

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to increasing divergence in the genus, expressed in the number of divergent base-pairs, with reference to most common base-pairs in the prevalent positions. A first verification determined the homogeneity among subtypes clustered in each type. Through a second verification, relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage. The resulting related types, showing no divergence, have been clustered in homogeneous groups representing cores of species. The resulting related types with homogeneous groups and which were partially divergent represented partially divergent components of species. Those showing relation with two or more homogeneous groups represented multirelated components of species and were ranked according to lower divergence. First verification—group homogeneity: Types identified by low-variable positions are evaluated to determine the homogeneity among subtypes (base-pair combinations) clustered in each type. Only types including at least two subtypes are evaluated. Divergence limit value is 13, corresponding to rounded 35% of total number of compared positions (n 39). Base-pair divergence values not exceeding limit value indicate homogeneity between base-pair combinations. Higher values indicate heterogeneity. Total comparisons (TC) is the number of comparisons among all subtypes within the type determined by LVP. Subtype comparisons (SC) is the number of comparisons between one subtype with other subtypes within the type. Values range indicates the variation of base-pair divergence values resulting from comparison of subtypes. Indicators of divergence between subtypes, within the types, are identified. The mean of basepair divergence is determined at the subtype level, including values indicating homogeneity (not exceeding 13) (low divergence values) as well as the values indicating heterogeneity (exceeding 13) (high divergence values) (subtype divergence mean—SDM), and the mean and percentage of the values indicating heterogeneity only (subtype relative divergence mean and %—SRDM, SRD%), resulting from comparison of each subtype with other subtypes within the type. The mean of base-pair divergence is determined at type level, including values indicating homogeneity (not exceeding 13) (low divergence values) as well as the values indicating heterogeneity (exceeding 13) (high divergence values) (type divergence mean—TDM), and the mean and percentage of the values indicating heterogeneity only (type relative divergence mean and %—TRDM, TRD%). Types including subtypes showing SRD% value exceeding 45 are considered heterogeneous, to be subjected to reallocation according to other positions. Second verification—heterogeneity of genomic groups: Genomic groups identified by LVP clustering are submitted to cross comparisons to verify their effective heterogeneity. Divergence values are computed by alignment of each subtype comparison, between different types, in order to identify homogeneous groups. Two such compared subtypes yielded results with an SRD% value lower than 45 and, divergent with a higher SRD% value. Two such compared types yielded results with a TRD% value lower than 45 and, divergent with a higher TRD% value.

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Table 3 Identified species within genus Pestivirus. Relations within and among species Species

Types—strains

BVDV1

Type 1 128/88; Type 2 CRFK; Type 3 832/01; Type 4 F, H; Type 5 23-13; Type 6 1946/01; Type 7 3425/01; Type 8 MDBK, U937; Type 9 Ind S 1166; Type 10 Ind S 1170; Type 11 80/1 cp 82; Type 12 17-112; Type 13 4382/01, 4629/01; Type 14 H686 98; Type 15 28/1; Type 16 26-V639; Type 17 2555/01; Type 18 8087 99; Type 19 985 84; Type 20 438/02; Type 21 4771 94; Type 22 SD-1; Type 23 3596/86; Type 24 TGAC; Type 25 9189; Type 26 7546, 7548; Type 27 YVR2394; Type 28 2343/01, 1041/01; Type 29 VM; Type 30 i63; Type 31 i736; Type 32 M1515A90; Type 33 KM, Kyj, G; Type 34 Massimo 4, CP1885; Type 35 3336/00, 5551/84; Type 36 Marloie, 3251/01, Lamspringe738; Type 37 L322 98, SE1015; Type 38 125 85, H851 98, 2703D 99, 2750A 99, 2586X 99, 4796 94, 80/1 ncp 82, 371 89; Type 39 Mumps, FS720; Type 40 2032/01, 2708/01, CP1872, 720/02; Type 41 3/VR/95, 4/VR/95, 2900/83, Reindeer2, PT810; Type 42 383 76, 819 85, 252 84, 1 77; Type 43 L1000 98, Q1161/1, 3187V, Q1161/2; Type 44 3114 93, TY CP/91, JE, 2L91, TK-87-2, BR275, KQ25A, KQ25B, No. 12, 0192, FU411, A014, C009T, HC725, HE726, HE728, IQ19A, LQ28A; Type 45 1/B/01, 2/B/01, 3/B/01, 4/B/01, 5/B/01, 6/B/01, 7/B/01, 8/B/01, 1/C/01, Europa, BRU*0615, Lamspringe 735, SE5726, Vkl, 16484/93, SE5572, SH9/11, 9466/91, 16-111, 3479-97-I; Type 46 akT1, Singer, Ln 68, BVR1199, 1R93, NADL, 66.5, i720; Type 47 Culi4, YVD947, 7535, 1/A/00, 2/A/00, 3/A/00, Ind S 1222, 2218/01; Type 48 Massimo 1, Massimo 2, 1891/99, 228/02, Culi 1, 10-84, 14-102, L256; Type 49 561/01, FLK, MOLT-4, WiDr, 2110C, 368/02, Q713, HeLa, Ind 446, Ind S 1168, Ind S 1171, Ind S 1181, Ind S 1455, 133/02, 3310/01, 4092/00, 1103/88, S21, 76865, Culi6, ZVD278, 10A/LC/97, 4325/01, Draper, 4171/00, BO2340/01, P, 4050/00, D, CP1887, 107/01, CP1945, 3417/00, Sanders, 551/02, TGAN, Vero, MDCK, CV-1, CD89; Type 50 MMR-K, 68.883, Weybridge, D771/1, M388A/90, D861, B1056, 114 817, 4898 94; Type 51 1190V97, i66.2, NY-1, 42M, U, T, 24/15, M346T96, Influenza2, i467, 86713, 3P, TFB, TFB2, i297, i89, i393, 2318/01, ZVR711, 1248/01, 3499/00; Type 52 i13, i53, 66.1, 66.6, Osloss; Type 53 20-V661-2, A553; Type 54 2204/82, 15-3; Type 55 HH, H503, 4H; Type 56 1R, ncp2, i6.89, i36P, 2430 95; Type 57 K869 98, B551 98; Type 58 KA91, 4283/00, CP1874, 3340/01; Type 59 2B, cb1, 17P; Type 60 D1120/1, D1432/P; Type 61 4998/89; Type 62 23-15; Type 63 CP1940; Type 64 10846/91; Type 65 5284/00; Type 66 3291-97-A; Type 67 11207/98; Type 68 3478/00; Type 69 66.3; Type 71 Wi-38; Type 74 A, L; Type 75 1372/01, 4163/00, 9-77; Type 76 Deer, S-ALT5/K, 2/VR/95, 22146/81, M557A/90, M245A/91; Type 77 Oregon, 318, 25H, 65.2, R193572, M169B/93 None Type 70 3186V6; Type 73 J, R, S, MV98CB95, W 0.33 6.43/9.54

Multirelated Borderlines Divergence within genus Divergence within species %/mean value BVDV2 BVDV-2A

BVDV-2B Multirelated Borderlines Divergence within genus Divergence within species %/mean value BDV

Multirelated Borderlines Divergence within genus Divergence within species %/mean value CSFV

Type 79 i628, i4083, i61380, Munich 1, 104/98, 4-5174, B52-2, Munich 2, CD87; Type 80 AZ Spl, MAD Spl, MN Fetus, Parvo, Kosice, 11/Mi/97, BSE341, NY93, 15-103, Q126, CPA, WVD829, Lees, BSE921, CPAE, EBTr, Rubella, MMR-T, HE727, 167 237, 168 149, 173 157, 175 375, 17583-97, 23025, 37Gr, 7937, V-FLL; Type 81 WG4622, IT-1732; Type 82 MP; Type 83 713-2; Type 95 5521-95, BSE1239, UVR420, 97/730; Type 99 Munich 3, Giessen-1, AF112, B45-5, B50-5, B5-4, BS-95-II, 17011-96, OY89, SW90, SY-89, TC Shinozaki, MS-1, B77-5; Type 100 C413, BD-78; Type 108 890 Type 78 VS-260, LV96; Type 84 Soldan; Type 85 34b, ncp7; Type 94 VS-123.4, VS-63; Type 106 i33283; Type 107 354 None (BVDV-2A vs. 2B Type 83 713-2; Type 95 5521-95, BSE1239, UVR420, 97/730; BVDV-2B vs. 2A Type 94 VS-123.4, VS-63) None 2.02 (BVDV-2A 1.96; BVDV-2B 2.25) 11.47/7.70 (BVDV-2A 0/4.96; BVDV-2B 0/7.28) Type 72 Chamois1; Type 86 Moredun cp; Type 87 X818; Type 88 L991; Type 96 Moredun ncp, T1789/1, Q1673/2, 8320-22NZ, 8320-31NZ, A1870, T1802/1, V3196/1, 90/8320/31, 91/5809; Type 101 JH2816, Ch1Es; Type 102 BD31; Type 103 A841/1, A1263/2, Q1488/1, Q1488/6; Type 104 V1414; Type 105 D1586/2; Type 109 Rentier Rudolph, Wisent Casimir; Type 111 135 661, 170 337, V2377/12, V2536/2, 137/4, G1305, G2048, K1729/3; Type 112 2112/99, 79248/01, 80582/01, 87877/01 None None 2.52 5.53/7.20 Type 89 Saitama/81; Type 90 5440/99; Type 91 17-93; Type 92 Fukuoka/72; Type 93 Honduras; Type 97 Alfort, Switzerland 1/93, Switzerland 4/93, Osaka/51, Osaka/71, Shizuoka/73, Pader, Chiba-80, Switzerland 2/93, Switzerland 3/93/1, Switzerland 3/93/2, Venhorst, VR14762; Type 98 39, C strain, CAP, cf114, Eystrup, KC, LOM, Miyazaki/81, Nakamura/66, Vac A, Yamanashi/69, Shimen, GPE (−), Hokkaido/66, Ibaraki/66, Ibaraki/81-115, Ibaraki/81-20, Ibaraki/81-38, Ibaraki/81-40, HCLV, Brescia, Alfort 187, Alfort A19; Type 110 Kanagawa/74; Type 113 Okinawa/86

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Table 3 Identified species within genus Pestivirus. Relations within and among species Species

Types—strains

Multirelated Borderlines Divergence within genus Divergence within species %/mean value

Type 110 Kanagawa/74 vs. BDV None 2.33 0/5.52

Pronghorn Multirelated Borderlines Divergence within genus Divergence within species %/mean value

Type 114 Pronghorn None None 4 0/0

Giraffe Multirelated Borderlines Divergence within genus Divergence within species %/mean value

Type 115 Giraffe None None 7 0/0

Divergence among species BVDV2 BVDV2-A BVDV2-B BDV CSFV Pronghorn Giraffe

100/19.52 100/19.77 100/18.54 100/19.72 100/19.29 100/19.43 100/26.00 BVDV1

Divergence %/divergence mean value

100/19.25 100/20.64 100/21.02 100/20.25 BVDV2

34.27/12.88 100/19.25 100/20.59 100/20.64 100/19.93 BVDV2-A

Identification of homogeneous groups: The identification of homogeneous groups is performed in a step-by-step manner. Cross comparisons between types within the genus are evaluated by computing the divergence percentage. The resulting related types, showing no divergence, are merged in homogeneous groups representing cores of species. Following the identification of a first group of related types, the remaining types are compared to identify the following homogeneous group, excluding the previous one. In case of presence in the genus of prevalent species, all types showing divergence % lower than 45 are selected for first identification of homogeneous group. Types showing divergence % 100, represent separate species. Evaluation of partial relations: Types showing partial divergence, with divergence % lower than 45 with homogeneous groups, are verified for possible clustering in the homogeneous groups. They are ranked according to lower divergence. The characteristics of the resulting group are identified by relatedness, divergence (SRD and TRD%), value range and mean. The resulting related and partially divergent types, showing SRD% lower than 45, from homogeneous groups represent partially divergent components of species. Groups of species-related types are evaluated for relatedness. Types grouped in species clusters resulting divergent among them are evaluated for partial relation. The characteristics of the resulting group are identified by relatedness, divergence (SRD and TRD%), value range and mean. Evaluation of multiple relations: Resulting related types, with or without partial divergence, showing no divergence or SRD% lower than 45, from two or more homogeneous groups represent multirelated components of species. They are ranked according

100/19.25 100/20.88 100/22.50 100/21.50 BVDV2-B

51.20/13.56 100/22.47 100/21.39 BDV

100/21.83 100/23.38 CSFV

100/22 Pronghorn

to lower divergence. Evaluation parameters are relatedness %, divergence % (SRD and TRD) and divergence value mean. Evaluation of borderlines: The resulting divergent and partially related types, showing SRD% higher than 45, from homogeneous groups but related only with one species represent borderlines, candidates for novel species clusters. Determination of genotypes, sub-genotypes and subgenotype variants: The procedure can also be applied to genotypes, sub-genotypes and sub-genotype variant determination (data not shown). Genotypes are identified according to base-pair combinations at the level of low-variable positions, and ranked according to increasing divergence in the species (mean value), with reference to prevalent base-pairs in prevalent positions. Sub-genotypes are identified according to base-pair combinations at the level of low-variable positions. Sub-genotypes are ranked according to increasing divergence of base-pair combinations in the genotype, with reference to prevalent base-pairs in the genotype. At sub-genotype level, the base-pair combinations are ranked according to increasing divergence in the genotype. Highly variable positions are excluded. Selection of genetic markers: The genetic markers useful for taxonomic purposes are identified at the genus and species level. The identification of markers follows the general criteria of characterization of palindromic positions. At genus level, base-pairings from highly conserved, homogeneous positions represent genus-specific PNS. Base-pairings from prevalent positions are evaluated for further genus marker selection. Basepairings from prevalent positions showing only exceptions, not related to any specific species, are considered genus-specific. At species level, base-pairings from genus prevalent positions, low-

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variable positions and, hypervariable positions are evaluated for species-specific PNS selection. Base-pairings are considered: (a) not determinative (ND) for species identification when not conserved and variable within the species, and/or shared by various species; (b) determinative for species identification (SD)—conserved positions species-specific, representing >80% within the species, shared with other species; (c) determinative for species identification (SD)—conserved positions species-specific, representing <80% within the species, shared with other species; (d) determinative for species identification at genus level (SDG)—highly conserved positions species-specific within the genus, representing >80% within the species. Prevalent base-pairs determinative for species identification may represent all the strains within the species or share the same posi-

tion with other base-pairings representing exceptions, shared or not with other species. The sharing of prevalent base-pairs determinative for species identification in one species may be related to prevalent base-pairs or only exceptions in the other species. According to those characteristics, PNS species markers have different importance. The most suitable PNS are SDG without exceptions, or with exceptions not shared with other species. They are identified only in CSFV, Pronghorn and Giraffe species. BVDV1, BVDV2 and BVD have to be characterised with SDG PNS with exceptions and SD PNS, which acquire significance only when combined. Species-specific PNS are selected according to the lower number of sharing species, the lower number of variants in the same position and the type of PNS.

Fig. 1. V1–V3 palindromic loci in the 5 UTR of the genus Pestivirus species. Base-pairings characteristic to the genus (PNS genus-specific) are shown in bold. The characteristic base-pairings of the species BVDV-1, BVDV 2, BDV, CSFV, and the new proposed taxons Giraffe and Pronghorn (PNS species-specific) are represented in bold and italic. Watson–Crick base-pairings are indicated by a dash (–); tolerated pairings in secondary structure are indicated by an asterisk (*); interchangeable base-pairings are indicated by a column (:). M = A or C; R = A or G; W = A or U; S = C or G; Y = C or U; K = G or U; Z = A or C or G; H = A or C or U; D = A or G or U; B = C or G or U; N = A or C or G or U.

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On the base of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn (Fig. 1). The PNS proved to offer a reliable alternative to classical taxonomical methods based on primary sequence structure. As secondary structures are essential for the survival of the viral population, variations in the implicated genomic regions might be related to biological characteristics, which in turn might correlate with classification. The relevant role of the 5 UTR in the translational, transcriptional and replicational mechanisms in pestiviruses implies that its secondary structures (e.g. palindromic structures) must be highly conserved, and that observed mutations are particularly meaningful. Preliminary studies on the analysis of the predicted secondary structure of the 5 UTR of RNA showed this procedure to be a simple and meaningful method for Pestivirus genotyping (Harasawa and Giangaspero, 1998), and for establishing probable relationships between secondary structure and tissue tropism (Weber et al., unpublished). Since these mutations are observed as conserved nucleotide changes, they may be considered as the occurrence of a micro-evolution step through selection in the phylogenetic history of the virus. In addition, the region is highly conserved among pestiviruses. On the basis of the above-mentioned considerations, the nucleotide substitutions at the level of the palindromic structures in the genomic 5 UTR may represent a useful genetic marker for taxonomical procedures in pestiviruses and theoretically for other RNA positive-strand viruses. The proposed keys for identification are (a) palindromic structures: three strictly conserved regions representing palindromic structures can be identified in the 5 UTR and (b) variable loci: stem–loop stable systems correspond to the palindromic structure regions and express nucleotide sequence variability in these regions. These variations are characteristic and well conserved among all the different species (genusspecific) or specific for a single species (species-specific). In terms of consensus motifs, shared by all the Pestivirus species, it is possible to identify typical base-pairs suitable for a simple procedure approach of species clustering, given by the evaluation of the predicted secondary structure. The palindromic structures are identifiable in linear sequences. However, it is easier to find them whilst observing the secondary structure. The evaluation of the secondary structure based on a qualitative observation of the nucleotide variations was supported and confirmed through the application of a quantitative approach. The determination of divergence between single strain sequences or genetic groups was easily obtained by comparing base-pairing from aligned secondary structure sequences. This provided clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. For example, within the BVDV-2 species, two main genogroups were identifiable, with strains showing common sequence characteristics to both groups (multirelated strains). They could be correctly allocated by quantitative analysis. Similarly, the relation between CSFV and BDV species appeared very clearly. Also in this case, ambiguous strain

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sequences could be clustered in the species showing the lowest divergence values. In conclusion, the proposed method provides results comparable with other taxonomical procedures based on 5 UTR primary structure evaluation, but it differs from them in that only the strategic and highly conserved genome regions in the 5 UTR, and therefore the most meaningful nucleotide sequences at this level, are considered. Thus accurate parameters for species identification in terms of nucleotide sequence homology are made available with great advantage for simplification of virological investigations. Pestivirus strains showing unexpected genomic sequences such as Giraffe (Plowright, 1969), Wisent Casimir (Becher et al., 1999), Chamois-1 (Arnal et al., 2004) and Pronghorn (Vilˇcek et al., 2005) have been easily characterized and clustered within the genus by the PNS method. Furthermore, species could be segregated by the PNS method purely on the base of genomic sequence characteristics, avoiding any reference to the origin of the animal host, and the application of quantitative analytical procedure allowed for a better determination of relation among species and genotypes. References Arnal, M., Fernandez-de-Luco, D., Riba, L., Maley, M., Gilray, J., Willoughby, K., Vilˇcek, S., Nettleton, P., 2004. A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). J. Gen. Virol. 85, 3653–3657. Becher, P., Orlich, M., Kosmidou, M., Baroth, M., Thiel, J.-H., 1999. Genetic diversity of pestiviruses: identification of novel groups and implications for classification. Virology 262, 64–71. Becher, P., Orlich, M., Shannon, A.D., Horner, G., Konig, M., Thiel, H.-J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366. Beer, M., Wolf, G., Kaaden, O.R., 2002. Phylogenetic analysis of the 5 untranslated region of German BVDV type II isolates. J. Vet. Med. B 49, 43–47. Ciulli, S., Battilani, M., Scagliarini, A., Ostanello, F., Prosperi, S., 2002. Identificazione e caratterizzazione di ceppi di BVDV isolati da soggetti immunotolleranti nella provincia di Bologna. Veterinaria Italiana 38 (45/46), 12–18. De Moerlooze, L., Lecomte, C., Brown-Shimmer, S., Schmetz, D., Guiot, C., Vandenbergh, D., Allaer, D., Rossius, M., Chappuis, G., Dina, D., Renard, A., Martial, J.A., 1993. Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5 untranslated region. J. Gen. Virol. 74, 1433–1438. Frolich, K., Hofmann, M., 1995. Isolation of bovine viral diarrhea virus-like pestiviruses from roe deer (Capreolus capreolus). J. Wildlife Dis., 243–246. Harasawa, R., Giangaspero, M., 1998. A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5 -untranslated region. J. Virol. Methods 70, 225–230. Jones, L.R., Zandomeni, R., Weber, E.L., 2001. Genetic typing of bovine viral diarrhea virus isolates from Argentina. Vet. Microbiol. 81 (4), 367–375. Plowright, W., 1969. Other virus diseases in relation to the JP15 programme. In: Proceedings of the 1st Technical Review Meeting, Joint Campaign Against Rinderpest, Phase IV, Mogadiscio, Organization of African Unity, Kenya, pp. 19–23. Ridpath, J.F., Bolin, S.R., Dubovi, E.J., 1994. Segregation of bovine viral diarrhoea virus into genotypes. Virology 205, 66–74. Qi, F., Gustad, T., Lewis, T.L., Berry, E.S., 1993. The nucleotide sequence of the 5 -untranslated region of bovine viral diarrhoea virus: its use as a probe in rapid detection of bovine viral diarrhoea viruses and border disease viruses. Mol. Cell. Probes 7, 349–356. Vilˇcek, O.E., Durkovic, B., Bobakova, M., Sharp, G., Paton, D.J., 2002. Identification of bovine viral diarrhoea virus 2 in cattle in Slovakia. Vet. Rec. 151, 150–152.

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diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch. Virol. 146, 99–115. Vilˇcek, S., Ridpath, J.F., Van Campen, H., Cavender, J.L., Warg, J., 2005. Characterization of a novel pestivirus originating from a Pronghorn antilop. Virus Res. 108, 187–193.