Research 102 (2004) 207–213

New Calicivirus isolated from walrus

Lilia Ganova-Raeva a,∗, Alvin W. Smith b, Howard Fields a, Yury Khudyakov a

a Division of Viral Hepatitis, Centers for Disease Control and Prevention, National Center for Infectious Diseases, 1600 Clifton Road NE, MS A-33 Atlanta, GA 30333, USA b Laboratory of Caliciviral Studies, College of Veterinary Medicine, Oregon State University, Corvallis, OR USA Received 3 October 2003; received in revised form 30 January 2004; accepted 30 January 2004

Abstract

The sequence and organization of a new member of was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652–7778, encoded a putative protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family. © 2004 Elsevier B.V. All rights reserved.

Keywords: Vesivirus; Capsid; Walrus; SSH

1. Introduction (Smith et al., 1998), respiratory disorders, conjunctivitis, pneumonia, vesiculation and diarrhea in felines (Geissler Caliciviridae, former members of Picornaviridae (Berke et al., 1997), hemorrhagic disease in rabbits and hares ac- et al., 1997; Berke and Matson, 2000), are now a separate companied by massive liver necrosis (Lamarque et al., 1997; family of with linear, single stranded positive RNA Meyers et al., 1991), gastroenteritis and pediatric acute gas- genome containing a poly(A)-tail at the 3-end. They are troenteritis in humans (Fankhauser et al., 1998; Green et al., divided into four genera: Vesivirus, Lagovirus, 1995). An interesting case was described in a laboratory and Sapovirus (Berke and Matson, 2000; Buchen-Osmond, worker who developed vesiculation and fever after infection 2003; Green et al., 2000). Caliciviruses have broad host with SMSV (Smith et al., 1998) that causes a disease with range. First observed in swine and domestic cats, they were similar symptoms in marine mammals. found in reptiles, rodents, felines, canines, birds, marine All four genera have very similar genomic organization. mammals (Smith et al., 1998), chimpanzees and humans In Vesivirus and Norovirus open reading frame (ORF1) en- (Berke et al., 1997; Fankhauser et al., 1998; Green et al., codes a nonstructural polyprotein composed of helicase, pro- 1995; Lamarque et al., 1997; Liu et al., 1999; Seal and Neill, tease and polymerase, ORF2 encodes the capsid protein and 1995). They have been shown to cross species (Lamarque ORF3 encodes a protein with putative nucleic acid bind- et al., 1997; Smith et al., 1998) and are known to cause var- ing function. The nonstructural polyprotein and the capsid ious diseases: vesicular exanthema, encephalitis, myocardi- protein are encoded by one large ORF in Lagovirus and tis, fever, diarrhea and abortion in pigs (Guo et al., 1999; Sapovirus. The size of a caliciviral genome varies from about Neill et al., 1998), flipper vesiculation in marine mammals 7400–8300 nucleotides (nt). In this study, we report the cloning and sequencing of the genome of a new calicivirus isolated from frozen walrus fe- ∗ Corresponding author. Tel.: +1-404-639-1158; fax: +1-404-639-1563. ces gathered from the ice shelf of the Chukchi Sea in 1977 E-mail address: [email protected] (L. Ganova-Raeva). (Smith et al., 1983). In this work we used isolate 7420, that

0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.01.033 208 L. Ganova-Raeva et al. / Virus Research 102 (2004) 207–213 was successfully grown in cell culture, which is unique fea- reverse transcription-polymerase chain reaction (RT-PCR) ture for caliciviruses, even though only at titer of about 102. on marine caliciviruses (Reid et al., 1999) did not am- The isolate was found to infect swine casing liver pathol- plify specific product using cDNA generated from isolate ogy, but lost infectivity after passaging (A. Smith, personal 7420. To recover and clone the viral genetic material we communication). used Suppression Subtractive Hybridization (SSH). Total RNA was extracted by TRIZOL reagent (Roche, Alameda, CA). One hundred microliters VMK cells inoculated with 2. Materials and methods isolate 7420 were used to create tester and 100 ␮l VMK cells, to create a driver. The RNA was reverse transcribed The virus was isolated and three-times plaque purified in and amplified by the SMART PCR cDNA synthesis kit Vero monkey kidney (VMK) cells after primary inoculation (Clontech Laboratories, Inc., Palo Alto, CA). The SMART with walrus fecal material (Smith et al., 1983). Primers II primer was used as described in the kit but instead of Hel1/Hel2 and 1F/1R considered suitable for diagnostic the oligo-dT, a new primer was designed. It contained a

Table 1 Genome amplification primers designed for this study Name Product size (bp) Sequence First-generation A17F 685 GCTATGGCTCAAACGCTCTCAAAA A702R GAGTAGTCAACATTAGCCGGGTGT F2249F 330 GTATTCTAAGGAGTACGTATTGGAT F2970R ATGACTCGAGGATGATGGTTC H3474F 643 TTGGCCGTGGTGGCGTGAA H4117R GAGCCCTTTGTAGGATATTGTTGG I3930F 752 GCGACTGCGGTCTCCCATA I4682R TGAAGGCAGCGGCACAACT K4976F 689 GGTGGGTTGCCTTCGGGTAT K5665R TGGCTAATTCTCAAACACCT L5339F 732 ATAATTCGGCAATTCTACTACATCAA L6071R GGATCCCAAGTAGAGCCAAGTT Second-generation 2120FE 738 GAAACACCAGTCAAACCAAC C165RE AGGTCTTATCGTAAACGGTGTGAA C744FE 700 GTGCGCTACGGAATTGGATGGAT DE412RE GACGATGGCGTTTTTGTGGGTGAC DE1049FE 520 GCTCGGGCTTGCACTTCCACAC FG1274RE TCCTCGAGAGCCTTGACCACAG Third-generation AB533F 1607 ACGACCCCGGCTTTTCTGTTTTT AB598F 1542 CAACCTGGCGGCTCACT CCFE86 765 GGATGAATTAGATGATGATTGG CCFI111 740 GATGATCCTTTTAATTGTTGCTTTGCT 2120RI 1607/1542 GTTGGTTTGACTGGTGTTTC 6068FE 571 CCACCACTACTACGCCAC 6071FI 516 ACTCGGATCTACTTGGGATCC 6380GAPF 653/1277 TACTCAACRTGGTCTGGCGG 6566GAPR 571/516/765/740 GGRATKGTGAAGATCACAGGTTC 7085RI 653 CCATCCATCTGGTAGCCCAGG 7087FI 434/506/730 GGGCTACCAGATGGATGGCC 7512RI 434 CCTTGTGTAAAGGCTTCTGGG 7572RE 506/1277 GATGCTTTGTCAAAGTCCCAA 7818R 730 GCTAGCTGCACTCCCTAGAAGG 7490FE 434 AAGTTGTGAGGATTGCAAC 8351RI 861 TAATTCTACAGTCTACTA 8374RE 884 CCTAATGCAACCTACCAATT GenBank nucleotide sequences used for primer design: AF091736, SMU15301, SMU15302, VEU76874, SMU52089, M87481, M87482, U76874, U76887, U76885, U76883, U76881, U76879, U76888, Z24757, AF231353, Y15427, Y15441, Y15442, AJ006019, AF109468, AF109467, AB032758, AB022679, P36285, U52005, P36284, X99445, AF283778, L40021, X86667, AF182760, M86379, AF109465, AF258618, Z49271, U54983, Z29514, X87608, M67473, AJ011099, AF145896, X86557, AF182760, AF093797, M87661. F: forward primer, R: reverse primer. The primer designation corresponds to the approximate location of annealing in the genome. Different primer generations were designed after successful rounds of amplification and sequencing. Different product sizes listed reflect use of different forward/reverse primers. L. Ganova-Raeva et al. / Virus Research 102 (2004) 207–213 209 stretch of seven 5-nitro-indol (Loakes and Brown, 1994) ilarity to SMSV was less than 83% at the 5-end of the residues at the 3-end (7N primer). This primer was intro- genome and a fragment of about 800nt close to the 3-end duced to utilize total RNA. The cDNA PCR product was (position 6900–7700nt) had only 60% homology to other used without further modifications in the PCR select cDNA Vesivirus sequences. An open reading frame search revealed subtraction protocol (SSH) as described (CLONTECH Lab- three major ORFs. ORF1 at position 4–5646 nt, ORF2 at oratories, Inc., Palo Alto, CA) (Diatchenko et al., 1996). position 5652–7778 nt and ORF3 at position 7775–8107 nt. The subtracted products from the tester and driver were Multiple ATG codons were found in different phases within cloned with pTAdvantage vector in Escherichia coli Top the 5-end region of OFR1 and ORF2, a feature typical for 10F electro-competent cells from Clontech (Clontech Lab- Vesivirus. oratories, Inc., Palo Alto, CA). Randomly selected clones ORF1 of WCV was 86% identical to ORF1 of SMSV-4. from the tester and the driver were isolated and probed ORF1 translated into protein with an estimated molecu- by hybridization with biotinilated driver cDNA at 65 ◦C lar weight of 209 kD that corresponded to the polyprotein overnight. All non-hybridizing clones were sequenced by found in the related calicivirus species. It was comprised the dye-termination method on ABI PRIZMTM 377 DNA of a helicase, as identified by similarity to the Pfam00910 Sequencer (Applied Biosystems, Foster City, CA). The helicase motif; a thiol-protease, as identified by similarity obtained sequences were submitted for BLAST search to the corresponding proteins in other caliciviruses; and an (NCBI, http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and RNA-dependant RNA-polymerase, as identified by simi- were found to have 83–87% homology to vesiviral se- larity to the Pfam 00680 RNA dep RNA-pol motif. The quences in GenBank encoding the capsid, the 5-end and the polyprotein was 90% identical and 93% similar to the cor- 3-end of the polymerase gene. The enrichment for clones responding polyprotein of the VESV type-strain A48. The of interest by SSH was estimated to be 5.3 × 103. nucleotide sequence at positions 2204–2312nt was unique Based on these findings we collected from GenBank for WCV. It had no significant homology to any known 40 additional vesiviral nucleotide sequences. They were DNA sequence and was reproducible by PCR. This unique aligned and used to design primer sets for the amplifica- sequence translated into protein that resembled very closely tion of the entire putative genome of the new virus (listed (85% identical and 94% similar) the corresponding protein in the caption for Table 1). The software used for the of SMSV serotype 4. This observation suggested a specific primer design was MegAlign and PrimerSelect (Dnastar codon usage in this area so it can be used as a target for Inc., Madison, WI). All primers were tested and all ob- designing WCV-specific primers or oligonucleotide probes. tained PCR products were cloned, sequenced and used to ORF2 of WCV (nucleotides 5652–7778) was 83% ho- create second and third generation of primers to recover mologous to a group of sea lion caliciviruses representing the entire genome. PCR conditions for amplification were serotypes-17, -7 and -1. ORF2 of WCV translated into a 708 as follows: 95 ◦C/120, 10 cycles of 95 ◦C/20,45◦C/20, amino acid-long capsid protein precursor. The best sequence 72 ◦C/60–90 (depending on the expected fragment size), match for the putative WCV capsid protein was the SMSV-1 30 cycles of 95 ◦C/20,52◦C/20,72◦C/60–90, one cycle capsid (80% identity, 88% similarity). The WCV capsid was of 72 ◦C/360. Additional primers, internal to the larger only 68% identical to the type strain VESV A48 capsid and fragments, were prepared to enable sequencing of the com- only 28% identical to the human Manchester and Sapporo plete viral genome. To identify the 3- and 5-ends of the or the Lagovirus capsids. The region at position viral genome two rounds of linear amplification were per- 6919–6984 nt also was unique for WCV. It did not resem- formed using nested primers. The extreme 3-end of the ble closely any other GenBank entry and translated into an genome was not sequenced because we did not obtain a amino acid sequence that was 76% similar to the capsid DNA fragment of sufficient sequence quality. Each nu- protein of the Bovine Calicivirus-Bos 1. This area can also cleotide position was identified by sequencing at least four be used as a target for designing WCV-specific primers or times using both strands. All sequences were analyzed and oligonucleotide probes. It represented positions 435–487 of assembled using SeqMan, SeqEd, MegAlign (DNAStar, the capsid consensus for caliciviruses and coincided with Lasergene Software) and BLAST. The GenBank Accession the variable P2 domain as described further in Fig. 2. number for the new genome is NC 004541 (AF321298). The peculiar folding of the capsid protein gave rise to Phylogenetic analysis was performed using the GCG Wis- the name of this viral family. The crystal structure has consin Package (Accelerys, Inc., San Diego, CA). been determined for the Norwalk calicivirus (Prasad and Matson, 1994). An N-terminal region is followed by S (“shell”) region of eight-stranded antiparallel beta sandwich 3. Results and discussion required for the correct assembly of the T = 3 icosahedral particle. A “hinge” leads from S into P (“protruding”) region Analysis of the WCV genome sequence showed that comprised of P1and P2 sub domains. P1 is represented by the new virus genome was up to 87% homologous to conserved blocks of residues 226–278 and residues 406–520 SMSV-serotype 1. The most conserved part was contained interrupted by a hypervariable block P2. The P domain within the region encoding the RNA-polymerase. The sim- is important for particle assembly. P2 is involved in cell 210 L. Ganova-Raeva et al. / Virus Research 102 (2004) 207–213

FCV274 FCV276 FCVV83 FCVV77 FCV KCD FCVF9 FCV CFI-68 FIV FCVdog213 FCV255 FCV182CVS5A FCV LLK FCV Urbana FCV KS40 FCV NADC FCVJapanF4 FCV2280 SMSV4 VESV A48 SMSV17 VESV-like Pan1 SMSV1 WCV Hu_caa60262 Hu_aab60927 Hu_SLV_Lyon Hu_SLV_Stockholm Hu_SLV_Mex Hu_aab51208 Hu_SLV_Lyon598 Hu_cac80626 SLV_London Hu_SLV_Mex340 EBHSV-GD EBHSV-BS89 RHDV-BS89 RHDV-Rainham NLV_Thistlehall90UK Chiba Chiba407 Hu_NLVValetta Hu_NLVKoblenz Hu_NLVMusgrove NLVL_Southhampton Norwalk NLV_Aichi124-89 NorwalkSRSV-KY89-89J DesertShield NLV_Stav95Nor Hu_NLVWinchester NLV_BS5 Hu_NLVSindelsham Chitta Hu_NLVHawaii NLVL_SnowMountain Hu_NLVBham minireoToronto Hu_NLVAukland swine NLVL_GII Camberwell Hu_NLVBerlin 139.5

120 100 80 60 40 20 0 Nucleotide Substitutions (x100)

Fig. 1. Phylogenetic tree of 61 complete capsid proteins from various caliciviruses representing all genera in the family (Norovirus, Lagovirus, Vesivirus and Sapovirus). The tree was generated by Clustal W slow and accurate method using Gonnet residue weight table, gap penalty of 11 and gap ex- tension penalty of 0.2. The alignment generated for this tree was used for the consensus shown in Fig. 2. GenBank accessions of the used caliciviral capsid proteins, from Genus Norovirus: AAA16285-DSV395, AAA59229-NorwalkSRSV-KY89-89J, AF093797-NLV BS5, AAD37377-NLV Stav95Nor, BAA83413-NLV Aichi 124-89, CAB89102-NLV Thistlehall90UK, AAA92984-NLVL Southhampton, AAB61685-NLVL SnowMountain, AAB50466- Norwalk, AAK50355-NLVl GII, AAB00437-Hu NLVAukland, BAB83516-swine, BAA84716-Chitta, AAA18930-minireoToronto, CAB89092-Hu NLVBham, AAB97768-Hu NLVHawaii, CAB89095-Hu NLVMusgrove, AAL18878-Hu NLVBerlin, CAB89096-Hu NLVSindlesham, CAB89090-Hu NLV Winchester, CAB89097-Hu NLVValetta, AAD33961-Camberwell, CAC80626-Hu cac80626, AAB60927-Hu aab60927, CAA60262-Manchester; Lago- virus: CAA93445-EBVH BS89, CAA66639-EBVH GD, CAA06806-RHDV-Rainham, S55399-RHDV-BS89; Vesivirus: CAA67811-FCV KS40, AAA74416-FCV LLK, JQ2354-FCV NADC, AAA79324-FCV Urbana, AAC16027-FCV182CVS5A, CAA67808-FCV2280, AAA74413-FCV255, AAB87460-FCV274, AAF86354-FCVdog213, P27406-FCVF9, P27405-FCV JapanF4, AAC16028-FCV V77, AAB87459-FCV V83, P36284-SMSV1, P36285-SMSV4, AAC61759-VESV-like Pan1, AF321298-WCV, AAC57040-SMSV17, AAB87081-FCV276, JQ2356-FCV KCD, P27404-FCV CFI-68 FIV, AAC13889-VESV A48; Sapovirus: BAB18267-Chiba, BAA82106-Chiba407, AAK72048-Hu NLVKoblenz, CAC41375-Hu SLV Lyon598, CAC41372-Hu SLV Lyon, AAL31559-Hu SLV Mex, AAL31557-Hu SLV Mex340, AAF05772-Hu SLV Stockholm, AAG01042-HuCV Potsdam, AAC40584-SLV London. L. Ganova-Raeva et al. / Virus Research 102 (2004) 207–213 211 by dots. Conserved regions described in the text are . Only few representative sequences from each genus are Fig. 1 . Vesivirus identify conserved regions as previously described for f – a Fig. 2. Proposed domains (pre-capsid, N, S, P1, P2, P1, C) based on consensus capsid sequence of 61 proteins as listed in the caption for shaded. Lower case letters shown in this alignment. Bold letters represent complete conservation within the consensus. Amino acids identical to the consensus are represented 212 L. Ganova-Raeva et al. / Virus Research 102 (2004) 207–213 binding and contains the determinant of strain specificity. this motif represents the hinge connecting the S and the The P domains contribute to the stability and size of the following P1 regions just like in the Norwalk virus. P1 is viral capsid (Bertolotti-Ciarlet et al., 2002). P is followed composed of two fairly conserved domains, separated by by C-terminal region (residues 521–530). A cryo-EM 3D a hyper variable region P2. The central conserved motif computer reconstruction is available for the Primate Cali- (described as region E for Vesivirus) we found in P2 of civirus Pan-1 (Prasad and Matson, 1994) and six regions the consensus as NnTsfkftpilGSlq at positions 530–544. of conservation labeled A through F have been previously The P2 domain of the Norwalk virus has been identified identified for Vesivirus within the capsid protein precursor as involved in determining host specificity and containing (Neill et al., 1998; Tohya et al., 1997). Region A has been the antigenic determinants for FCV, RHDV (Geissler et al., described as the part of the pre-capsid protein and cleaved 1997; Milton et al., 1992; Seal and Neill, 1995; Tohya off at a consensus site before final capsid assembly. The et al., 1997) and the human caliciviruses (Fankhauser et al., rather large hypervariable region E has a central conserved 1998; Green et al., 1995; Hardy et al., 1996; Lew et al., motif NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R) found in all 1994; Liu et al., 1999; Prasad et al., 1999). P2 is physically vesiviruses that has been used for typing purposes by PCR exposed on the surface of the virus coat. Using these con- and has important functional role (Neill et al., 1998). sensus characteristics, the described 3D-structures (Prasad The amino-acid sequences of these capsid proteins from et al., 1999; Prasad and Matson, 1994) and the previously Norovirus and Vesivirus differ significantly. They only share observed functional regions as guidelines (Geissler et al., 27% similarity in a region that corresponds to residues 1997; Green et al., 1995; Hardy et al., 1996; Matson et al., 13-222 for the Norwalk virus and residues 154–360 from 1996; Meyers et al., 1991; Milton et al., 1992; Seal and the Vesivirus Pan-1 capsid protein precursor. The overall Neill, 1995; Tohya et al., 1997) we propose to re-assign the differences, however, do not appear to significantly affect functional regions of all Caliciviridae capsids to follow the the final three-dimensional structure and in both viruses the pattern N-S-P1-P2-P1-C as shown in Fig. 2. assembled units (about 180 units by molecular weight) cre- Overlapping the capsid reading frame was a third small ate similar order of icosahedral five-fold and three-fold-axes ORF that encoded a small basic protein with putative DNA that form calices on the surface (Prasad et al., 1999; Prasad binding function. The WCV ORF3 protein was 88% identical and Matson, 1994). and 94% similar to the corresponding protein from SMSV-1. These observations led us to compile and examine an In conclusion, the WCV genomic primary structure dis- alignment of 61 amino acid sequences of capsid precursors closed in the present study shares many genetic features with from all caliciviral genera to identify patterns of protein con- Caliciviridae. Similar to other caliciviruses the WCV genome servation. Fig. 1 represents the phylogenetic relationships contains three ORFs encoding for a nonstructural polypro- of the newly described WCV within Caliciviridae based on tein, a capsid and a small basic protein with a putative nu- complete capsid protein sequences. The divergence of these cleic acid binding function. To our best knowledge, this work proteins evaluated in pair distances varied from 0.7 to 374 describes for the first time the isolation and genome charac- and their similarities varied from 11 to 99%. This alignment teristics of a new member of Caliciviridae, genus Vesivirus, also generated the consensus capsid sequence as shown in isolated from walrus and proposes a uniform approach to Fig. 2. This consensus shows that the amino-terminal region studying and understanding the functions of the domains of could be cleaved off the pre-capsid in all but and the capsid proteins of all caliciviruses. lagoviruses. This feature agrees with previous studies on the potential cleavage sites of the capsid precursors (Milton et al., 1992; Neill et al., 1998; Seal and Neill, 1995; Tohya et al., 1997; Prasad and Matson, 1994). The consen- References sus shows that the region overlapping N, S and P1 is the most conserved among all caliciviral capsids. It contains a Berke, T., Golding, B., Jiang, X., Cubitt, D.W., Wolfaardt, M., Smith, A.W., Matson, D.O., 1997. Phylogenetic analysis of the Caliciviruses. segment of about 50 amino acids that is 68–85% conserved J. Med. 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