JVI Accepted Manuscript Posted Online 18 November 2015 J. Virol. doi:10.1128/JVI.02197-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Mechanism of cell culture adaptation of an enteric calicivirus, porcine sapovirus Cowden strain

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Zhongyan Lu a, Masaru Yokoyama b, Ning Chen a,c, Tomoichiro Oka a,d, Kwonil Jung a, Kyeong-

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Ok Chang e, Thavamathi Annamalai a, Qiuhong Wang a#, Linda J. Saif a#

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Food Animal Health Research Program, Ohio Agricultural Research and Development Center,

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Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The

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Ohio State University, Wooster, OH, USAa; Pathogen Genomics Center, National Institute of

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Infectious Diseases, Tokyo, Japanb; Boehringer Ingelheim (China) Investment Co., Ltd.

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Shanghai, Chinac; Department of Virology II, National Institute of Infectious Disease, Tokyo,

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Japand; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,

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Kansas State University, Manhattan, KS, USAe

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Running Head: Mutations in VP1 make porcine sapovirus grow in vitro

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#Address correspondence to Dr. Qiuhong Wang, [email protected] or Dr. Linda J. Saif,

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[email protected].

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Word count text: 6272

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Word count abstract: 241

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Abstract Porcine sapovirus (PoSaV) Cowden strain is one of only a few culturable enteric

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caliciviruses. Compared to wild-type (WT) PoSaV Cowden strain, the tissue culture-adapted

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(TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA

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polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse genetics system, we

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identified that four (178, 289, 324, and 328) amino acid substitutions in VP1, but not the

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substitutions in the RdRp region, were critical for the cell culture adaptation of PoSaV Cowden

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strain. The other two substitutions in VP1 (291 and 295) reduced virus replication in vitro. Three

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dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-

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dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324,

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and 328 were located at the protruding subdomain 2 (P2) of VP1, which may influence virus

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binding to the cellular receptors; and residue 295 was located at the interface of two monomeric

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VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutations at

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residues 291 or 295 from that of TC to WT reduced virus replication in vitro, it enhanced viral

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replication in vivo, and the revertants induced higher serum and mucosal antibody responses

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compared to TC PoSaV Cowden strain. Our findings have revealed the molecular basis for

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PoSaV adaptation to cell culture. They may provide new, critical information for the cell culture

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adaptation of other PoSaV strains and human SaVs or noroviruses.

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Importance Tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable

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enteric caliciviruses. We discovered that four amino acid substitutions in VP1 (178, 289, 324,

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and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (291 and

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295) reduced virus replication in vitro, but enhanced virus replication and induced higher serum

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and mucosal antibody responses in gnotobiotic pigs, compared with the tissue culture-adapted

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strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly

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and oligomerization, residues 289, 291, 324, and 328 may influence virus binding to the cellular

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receptors, and residue 295 may influence VP1 dimerization. Our findings will provide new

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information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

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Introduction

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Caliciviruses, in the family Caliciviridae, are small, icosahedral, and non-enveloped

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viruses of 27 to 35 nm in diameter, which have a positive sense, single-stranded RNA genome of

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6.5 to 8.3 kb (1, 2). Caliciviruses have been classified into five genera (Norovirus, Sapovirus,

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Vesivirus, Lagovirus and Nebovirus) and several proposed genera (3, 4). Among them,

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noroviruses (NoVs) and sapoviruses (SaVs) are the leading causes of gastroenteritis in humans

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of all ages. SaVs are often associated with sporadic, self-limiting gastroenteritis, of which the

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severity is reportedly milder than NoVs (5, 6). However, SaVs also cause outbreaks worldwide

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(7-10) and deaths associated with SaV infection have been reported in long-term care facilities

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(11).

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Because most enteric caliciviruses are unculturable, research on pathogenesis and

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immunity, as well as the development of antivirals, has been hampered. Porcine SaV (PoSaV)

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Cowden strain, previously known as porcine enteric calicivirus (PEC), belongs to genogroup III

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(GIII) SaV, and is one of only a few culturable enteric caliciviruses (2, 12, 13). PoSaV Cowden

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strain was adapted to a porcine kidney cell line (LLC-PK) after passage of the virus in

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gnotobiotic (Gn) pigs, followed by 20 passages in primary porcine kidney cells in the presence of

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an intestinal content preparation from uninfected Gn pigs (12, 14).

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Gn pigs have been established as a relevant animal model, because of the similarity of

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anatomy, genetics, physiology, and immunity with humans (15-17). PoSaV naturally infects pigs

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and causes mild to moderate gastroenteritis in Gn pigs (14, 18, 19), thus mimicking the SaV

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diarrhea reported in humans and providing an animal model suitable for studies of replication

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and pathogenesis of enteric caliciviruses.

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The genome of PoSaV is composed of two open reading frames (ORFs). The ORF1

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encodes a polyprotein that is processed into several nonstructural proteins (NSs) and the major

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structural protein VP1 by a viral protease. The ORF2 encodes a small structural protein VP2

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(20). The VP1 is divided into two domains: a shell (S) domain (amino acid positions 3-216) and

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a protruding (P) domain (amino acid positions 217-544) (21). The P domain is further divided

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into P1 (amino acid positions 217-272 and 425-544) and P2 (amino acid positions 273-424)

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subdomains (21, 22).

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Reverse genetics systems are an important tool to rescue unculturable viruses and to

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study virus replication mechanisms. A reverse genetics system pCV4A that was constructed for

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PoSaV Cowden strain contained the full-length genomic cDNA of the tissue culture-adapted

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(TC) PoSaV Cowden strain, directly downstream from the T7 RNA polymerase promoter (20).

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Infectious TC PoSaV particles were rescued after transfection of LLC-PK cells with the in vitro

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transcribed and capped PoSaV genomic RNA (20).

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In this study, we investigated the genetic basis of cell culture adaptation of PoSaV

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Cowden strain by comparative sequence analyses of the genomes of different passages of wild-

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type (WT) and TC PoSaVs, and generation of a series of PoSaV mutants using the reverse

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genetics system. We further investigated the differences in replication both in vivo and in vitro,

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as well as their putative structural differences. To our knowledge, our studies are the first to

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identify which amino acid residues are critical for the cell culture adaptation of a SaV. This study

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provides new information on cell culture adaptation of SaVs that may be applicable to other

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human SaVs or to NoVs.

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Materials and Methods

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Cells and viruses.

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The LLC-PK cell line (#CL-101) and a human embryonic kidney cell line, HEK 293T/17 (#CRL-11268), were obtained from the American Type Culture Collection (ATCC). The LLC-

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PK cells were passaged and maintained as previously described (20, 23). The HEK 293T/17 cells

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and a baby hamster kidney cell line (BHK-T7) stably expressing T7 RNA polymerase were

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cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, NY, USA) with

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10% fetal bovine serum (FBS, Thermo Scientific, MA, USA), 1% nonessential amino acids

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(NEAA, Invitrogen, NY, USA), and 1% Antibiotic-Antimycotic (Invitrogen, NY, USA).

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Two passage levels of WT PoSaV Cowden strain (Gn pig passage level 5, I-1113, and

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#13, R418) from the small or large intestinal contents (SICs/LICs) of Gn pigs were used for

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sequencing. The TC PoSaV was propagated in LLC-PK cells (TC PoSaV-2010, cell culture

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passage level 30) with 50µM glycochenodeoxycholic acid (GCDCA) (Sigma-Aldrich, MO,

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USA) as previously described (24).

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Sequence analyses.

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The genomes of TC PoSaV-2010 (passage level 30, GenBank accession no. KT922088)

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and WT PoSaV I-1113 (Gn pig passage level 5, GenBank accession no. KT922087), and the

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VP1 region of WT PoSaV R418 (Gn pig passage level 13, GenBank accession no. KT945132)

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were sequenced by the primer walking method based on the TC PoSaV genome (GenBank

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accession no. AF182760) as previously described (25). The 5’- and 3’-ends were determined

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using 5’-rapid amplification of cDNA ends (RACE) and 3’-RACE methods. Sequence editing

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and assembly were performed using the Lasergene software package (v10) (DNASTAR Inc.,

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WI, USA). Multiple sequence alignment was done by ClustalW using DNA Data Bank of Japan

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(DDBJ) (http://www.ddbj.nig.ac.jp).

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Generation of full length cDNA clones of PoSaV WT and TC chimeric genomes, mutants,

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and revertant mutant strains

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The plasmid pCV4A containing the full-length cDNA of TC PoSaV (TC PoSaV-2005,

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cell culture passage level 27) was provided by Dr. Kyeong-Ok Chang at Kansas State University

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(20). The primers for the generation of these chimeric clones are listed in the Table 1. The

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genomic organization and mapping of the mutations are illustrated (Figure 1). The TC-WTVP1

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was generated by replacing partial VP1 fragment (nucleotide position 5227-6060, amino acid

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position 30-308) of pCV4A with the corresponding sequence fragment of WT PoSaV Cowden

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strain. Briefly, the WT PoSaV Cowden VP1 fragment containing two ApaI restriction enzyme

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sites (nucleotide position 5227-5232 and 6055-6060) was reverse transcribed using SuperScript

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III reverse transcriptase (Life Technologies, NY, USA), and amplified by PCR with primers

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ApaI-F and ApaI-R using PrimeStar HS high-fidelity DNA polymerase (Clontech Laboratories,

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Inc., CA, USA). The amplicons were digested by ApaI restriction enzyme and cloned into ApaI-

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digested pCV4A plasmid.

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Full-length cDNA clones of TC-WTRdRp, TCVP1-S178C, TCVP1-H289Y, TCVP1-D291N,

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TCVP1-R295K, TCVP1-I324MM324I and TCVP1-G328EE328G were generated based on the

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pCV4A backbone by using QuikChange II XL site-directed mutagenesis kit (Agilent

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Technologies, TX, USA) following the manufacturer’s instructions (Figure 1). For example, TC-

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WTRdRp was generated when the two RNA-dependent RNA polymerase (RdRp) amino acid

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residues at 1252 and 1379 of pCV4A were mutated from TC to WT (H1252Y and K1379R).

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Three full-length cDNA clones of chimeric genomes (TC-WTVP1-C178S, TC-WTVP1-Y289H, 7

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and TC-WTVP1-C178S&Y289H) were generated based on the backbone TC-WTVP1, whose

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amino acid positions 324 and 328 in VP1 were TC type. Full-length cDNA clones of TCVP1-

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I324M and TCVP1-G328E were generated by digesting the pCV4A with EcoRI restriction

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enzyme (nucleotide position 4582-6877) and replacing with I324M or G328E engineered PCR

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products. Using TCVP1-I324M as an example, two fragments were PCR amplified with primers

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EcoRI-F and 6111-R, and 6111-F and EcoRI-R using pCV4A as template, respectively. PCR

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product containing the I324M mutation site was assembled by overlap PCR with primers EcoRI-

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F and EcoRI-R using the two fragments as templates. The overlap PCR products containing

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I324M mutation site were EcoRI restriction enzyme digested and inserted into EcoRI restriction

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enzyme digested pCV4A. The recombinant plasmid was transformed and amplified in competent

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10-beta E.coli (New England BioLabs Inc., MA, USA).

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In vitro transcription and capping of viral genomic RNA

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In vitro transcription and capping of viral genomic RNA were performed following the

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manufacturer’s instructions. Briefly, the reverse genetics plasmid DNA was extracted from

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E.coli, linearized by NotI restriction enzyme digestion, and purified by phenol-chloroform

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extraction twice. Subsequently, genomic RNA was transcribed in vitro from the linearized

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plasmid by using MEGAscript T7 transcription kit (Life Technologies, NY, USA). The reaction

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mixture was treated with DNase and the RNA was purified with an RNeasy mini kit (QIAGEN,

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CA, USA) and analyzed by agarose gel (1%) electrophoresis under denaturing conditions with

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formaldehyde. The transcribed RNA was capped using ScriptCap m7G capping system

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(Cellscript, WI, USA) followed by RNA purification using the RNeasy mini kit. The RNA

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transcripts were suspended in RNase-free water to a final concentration of 500ng/µl for

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transfection. 8

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Transfection of BHK-T7 cells or HEK 293T/17 cells to rescue infectious viruses The purified RNA transcripts were transfected into one-day-old HEK 293T/17 or BHK-

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T7 cells (~50-70% confluent) in 24-well cell culture plates with Lipofectamine 2000 (Invitrogen,

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NY, USA). Briefly, one-day-old HEK 293T/17 or BHK-T7 cells were washed with OPTI-MEM

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I (Invitrogen, NY, USA). ~1.5µg capped RNA and 4µl Lipofectamine 2000 were diluted in 50µl

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OPTI-MEM I separately, and incubated at room temperature for 5 min. Then the RNA and

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Lipofectamine 2000 solution were mixed (total volume of 100µl) and incubated at room

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temperature for 20 min before adding to HEK 293T/17 or BHK-T7 cell monolayers. After 6

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hours of incubation at 37℃, the supernatant was replaced with DMEM growth medium (10%

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FBS, 1% NEAA, and 1% Antibiotic-Antimycotic). After 1 day of incubation at 37℃, HEK

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293T/17 or BHK-T7 cell lysates were harvested by freezing and thawing once followed by

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centrifugation at 2095 × g for 5 min to remove cell debris. The cell lysates were used to inoculate

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LLC-PK cells to generate virus pools.

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Recovery of progeny virus in LLC-PK cells

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The LLC-PK cell monolayers in 6-well plates were washed with MEM and inoculated

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with the HEK 293T/17 or BHK-T7 cell lysates in the presence of 50 μM GCDCA. Cytopathic

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effects (CPEs) were monitored daily. The infected cells were incubated for up to 6 days post-

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inoculation, before harvesting the first passage of each mutant. Each mutation was confirmed by

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reverse transcription (RT) PCR with primer sets covering the mutated region followed by

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sequence analysis.

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Plaque assays

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10-fold serial-diluted samples were inoculated into wells of 6-well cell culture plates. After incubation at 37°C for 1.5 hrs with rocking, the inoculum was removed and the cell 9

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monolayer was overlaid with 0.85% low-melting temperature agarose (Sigma-Aldrich, MO,

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USA) in MEM supplemented with 50 µM GCDCA (20). After plaques formed (5 days post-

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inoculation), cell monolayers were stained with 1 mL of 0.03% neutral red-PBS solution for 30

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min at 37°C. The solution was removed and the plaques were counted and observed under a

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microscope. The plaque sizes were quantified using Icy bioimage program (26).

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Growth kinetics test for progeny mutants in LLC-PK cells

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A growth kinetics curve of each mutant was determined by collecting cell lysates at

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different post-inoculation time points. LLC-PK cells in 6-well plates were incubated with each

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mutant virus at 0.01 multiplicity of infection (MOI) for 1 hr. The inoculum was removed and the

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plate was washed once before adding maintenance MEM in the presence of 50 μM GCDCA.

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Supernatants and cell lysates were collected at 24, 48, 72, and 96 hours post-inoculation (hpi)

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after three cycles of freezing and thawing. Virus infectivity titers were determined in LLC-PK

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cells as 50% tissue culture infectious dose (TCID50) by immunohistochemistry (IHC) staining

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using 96-well plates as described below (23).

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IF and IHC staining for the detection of VP1 proteins in cell culture

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IF staining was performed for the detection of VP1 protein in mutant infected cells (20),

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while IHC staining was performed for virus infectivity titration (23). Briefly, cell monolayers

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were fixed with 10% neutral formalin buffer at room temperature for 30 min, then the fixed cells

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were permeabilized with 1% Triton X-100 in PBS at room temperature for 10 min. Gn pig

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hyperimmune serum to WT PoSaV Cowden strain was used as primary antibody (18).

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Fluorescein isothiocyanate (FITC) -conjugated goat anti-swine IgG (H+L) serum (KPL, MD,

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USA) or horseradish peroxidase (HRP)-conjugated goat anti-swine IgG (H+L) serum (KPL, MD,

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USA) were used as secondary antibodies. The IF signal was observed using fluorescent 10

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microscopy IX70 (Olympus, PA, USA). For IHC, cells were stained with substrate 3-amino-9-

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ethylcarbazole (AEC) (Sigma-Aldrich, MO, USA) at room temperature for at least 2 hrs and

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observed using light microscopy.

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Three dimensional (3D) structural analyses of the VP1 proteins of WT and TC PoSaV

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The 3D structure of a SaV VP1 protein is not available in the database. The VP1 protein

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of WT PoSaV shares higher sequence identity (38%) with that of FCV (PDB code 3M8L) than

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those of San Miguel sea lion virus (SMSV, 34%, PDB code 2GH8) and the recombinant

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Norwalk virus (rNV, 29%, PDB code 1IHM). When the sequence identity is 30-50%, the

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obtained model tends to have about 90% of the main chain modeled with 1.5 Å root means

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square error (27). Therefore, the VP1 dimer structural models of WT and TC PoSaV were

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constructed based on the crystal structure of the FCV VP1 protein at a resolution of 3.40Å by

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homology modeling method using ‘MOE-Align’ and ‘MOE-Homology’ in the Molecular

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Operating Environment (MOE, ver. 2014-09, Chemical Computing Group Inc., Quebec,

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Canada). Twenty-five intermediate models were obtained from one homology modeling in the

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MOE, among which the intermediate models with the best scores were selected according to the

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scoring function Generalized Born/Volume Integral (GB/VI). The final 3D models were

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thermodynamically simulated by energy minimization using AMBER10: EHT force field

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combined with the GB model of aqueous solvation implemented in the MOE (28-30). Physically

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unacceptable local structures of the optimized 3D models were further refined on the basis of

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evaluation using the Ramachandran plot in the MOE. The structures of WT PoSaV VP1 dimers

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were generated from the monomeric structures by MOE on the basis of the assembly information

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of the FCV VP1 crystal structures. The quality of the models was assessed using the 3D-structure

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evaluation program Verify3D (31). 11

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Prediction of the effects of point mutations on the stability of PoSaV The change in the stability of WT PoSaV VP1 protein by each mutation was analyzed

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using the Protein Design application in the Molecular Operating Environment (MOE, 2014-09).

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The structure of the WT PoSaV VP1 was constructed as described earlier. The single point

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mutations on the VP1 were generated individually, and ensembles of protein conformations were

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generated using the LowMode MD module with Boltzmann distribution in the MOE to calculate

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average stability. The stability scores in the structures refined by energy minimization were

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obtained using the stability scoring function of the Protein Design application in the MOE.

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Gn pigs and experimental design

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Gn pigs were derived and maintained as previously described (18, 32). A total of 36 Gn

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pigs were assigned to five groups (Table 2) and inoculated orally with: 1) TC PoSaV (cell culture

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passage level 30, n=9), 2) TCVP1-R295K (n=7), 3) TCVP1-D291N (n=9), 4) WT PoSaV (PS799,

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Gn pig passage level 13, n=7), or 5) MEM (n=4), respectively. The TC PoSaV and TCVP1-

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D291N, TCVP1-R295K mutants were harvested from cell culture and concentrated to ~7.0 log10

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TCID50/mL (equivalent to real-time quantitative RT-PCR (RT-qPCR) titer ~11.5 log10 genome

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equivalents (GE)/mL) by ultracentrifugation at 126,000 × g, for 1.5 hrs at 4℃. The WT PoSaV

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Cowden strain inoculated in Gn pigs was filtered through 0.22 μm-pore size filters prior to

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inoculation. Each pig was inoculated orally with 5 mL inoculum containing the TC PoSaV or

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mutant PoSaVs (~7.0 log10 TCID50/mL), or with 5mL ~11.5 log10 GE/mL RT-qPCR titer of WT

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PoSaV.

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All inoculated Gn pigs were observed daily for clinical signs. Their feces were scored as

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follows: 0, normal; 1, pasty; 2, semi-liquid; and 3, liquid, with fecal scores ≥2 indicating diarrhea

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(18). Rectal swabs were collected daily for virus shedding titration. Blood was collected for 12

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serum from each Gn pig before inoculation, and at post-inoculation day (PID) 1, 3, 6, 9, 16, 23,

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27, and at euthanasia. Two to three Gn pigs of each group were euthanized at acute phase

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infection: on the next day after an increase of fecal viral RNA titer, or on the day following the

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onset of clinical signs. The remaining pigs were euthanized after fecal viral shedding was no

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longer detected at termination of the experiment. Animal care and use for these studies was

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approved by the Institutional Animal Care and Use Committee (IACUC) at The OSU. Mucosal

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antibody samples were collected at necropsy by scraping the mucosa from the ileum and

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centrifuging at 4℃, 2095 × g for 20 min.

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Histopathologic examination

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At necropsy, blood, SIC and LIC were collected from each Gn pig. Fresh duodenum,

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proximal-, mid-, and distal-jejunum, ileum, colon, cecum, liver, spleen, lung, and kidney

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specimens were collected and immersed immediately in 10% neutral buffered formalin (NBF).

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10% NBF fixed tissues were trimmed, embedded in paraffin, sectioned at 4 μm, stained with

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Harris’s hematoxylin and alcoholic eosin Y solution (H&E, Sigma-Aldrich, MO, USA), and

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examined for histopathology microscopically.

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Duodenum, proximal-, mid-, and distal-jejunum, ileum, colon and cecum specimens were

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collected in duplicate, immersed in sucrose solution [130mM Na2HPO4, 30mM KH2PO4, 10%

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(w/v) sucrose, and 0.01% sodium azide, pH 7.2] on ice, embedded in Optimum Cutting

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Temperature (O.C.T.) compound (Sakura, PA, USA) and stored at -20℃ overnight, then

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sectioned at 4-7 μm in a cryostat microtome. To detect the PoSaV antigen in tissues, frozen

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sections were fixed with acetone for 20 min at -20℃ followed by washing with PBS twice for 5

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min each. Tissue sections were then blocked with 5% normal goat serum in 0.01M PBS-0.05% 13

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Tween 20 (PBST, pH 7.2) for 20 min. After blocking, the tissue sections were incubated with

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hyperimmune guinea pig serum to PoSaV VLPs (33) at 4℃ overnight, followed by incubating

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with Alexa Fluor 488 conjugate goat anti-guinea pig IgG (H+L) serum (Life Technologies, NY,

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USA) at room temperature for 1 hr. Thereafter, tissue sections were counterstained with DAPI

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for nuclei and examined using fluorescent microscopy.

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Detection of PoSaV RNA in rectal swabs (RSs)

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RSs were collected, suspended into 4 mL of MEM, and centrifuged at 2095 × g for 30

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min. The supernatant was collected as 10% fecal suspension and stored at -20℃ until RNA

286

extraction. The total RNA was extracted from 50 μl RS suspensions using MagMax RNA

287

extraction kit (Life Technologies, NY, USA) following manufacturer’s instructions. Virus titer

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was determined using One-step TaqMan SaV specific RT-qPCR as described (23).

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Detection of PoSaV-specific antibodies in serum and mucosal samples by enzyme-linked

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immunosorbent assay (ELISA)

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A recombinant baculovirus expressing PoSaV VP1 was generated as previously

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described and used to infect Sf9 cells for VLP production (33). Briefly, the purified PoSaV VLPs

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were used as antigens to coat NUNC 96-well plates (MaxSorp surface, Thermo Scientific, MA,

294

USA) at 4℃ overnight at a final concentration of 2 μg/mL (100 ng/well) in 0.05M carbonate

295

buffer, pH 9.6. The plates were blocked with 2% non-fat dry milk (NFDM) in PBST at 37℃ for

296

1 hr. After washing three times with PBST, serum samples were four-fold serially diluted in

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PBST containing 2% NFDM and added to the wells. The plates were incubated at 37℃ for 1 hr

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and washed with PBST three times. HRP-conjugated goat anti-swine IgG (H+L) serum (KPL,

299

MD, USA) diluted 1: 3000, or HRP-conjugated goat anti-swine IgA serum (AbD Serotec, NC, 14

300

USA) diluted 1:5000 in PBST containing 2% NFDM were added to wells followed by incubation

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at 37℃ for 1 hr. After washing the plates three times with PBST, the substrate 3, 3’, 5, 5’-

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tetramethylbenzidine (TMB, KPL, MD, USA) was added to each well for color development at

303

room temperature. An equal volume of 1M phosphoric acid was added to terminate the reactions

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after 5 min incubation at room temperature. The absorbance at 450 nm were measured using a

305

spectrometer (SpectraMax 430 PC, Molecular Devices, LLC., CA, USA). The antibody titer was

306

determined as the reciprocal of the highest serum dilution with an absorbance value greater than

307

or equal to the mean absorbance of a series of negative control serum samples plus 3 times of

308

standard deviation (SD) of the negative controls (33).

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Virus neutralization (VN) test

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Serum samples were tested for VN antibodies to PoSaV by a 50% cell culture infectivity

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reduction test. 100 TCID50/well TC PoSaV was incubated with an equivalent volume of 4-fold

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serially diluted serum samples at 37℃ for 1 hr before applying to cell monolayers. Quadruplicate

313

wells were used for each serum dilution. The non-neutralized PoSaV was detected on the LLC-

314

PK cells by IHC staining as described above. The VN antibody titers were determined using

315

Reed–Muench method (34) and expressed as the reciprocal of the highest serum dilution that

316

inhibited PoSaV infection in 50% wells.

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Statistical analysis

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One-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test was

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used to assess differences in plaque size, mean duration of virus shedding and log-transformed

320

titers (including antibody titer, VN antibody titer, viral RNA titer) among groups. One-way

321

ANOVA was used to assess villus height to crypt depth ratios (VH:CD) and the mean numbers

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of antigen-positive cells per villus. A significance level of P < 0.05 was used for all comparisons. 15

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Results

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Consistent mutations occurred in the RdRp and VP1 regions at different passages of TC

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PoSaV compared to WT PoSaV.

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To investigate which genes were critical for PoSaV adaptation to cell culture, the genome

328

of the TC PoSaV at passage level 30 in LLC-PK cells was sequenced in this study, and compared

329

with those of WT PoSaV at pig passage levels 5 and 13, TC PoSaV at cell culture passage level

330

20 (25) and the infectious clone pCV4A carrying the cDNA of TC PoSaV-2005 (cell culture

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passage level 27) (20). Two and six conserved amino acid mutations were observed in the RdRp

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(1252 and 1379) and VP1 (178, 289, 291, 295, 324 and 328) regions of TC PoSaV (Figure 1 and

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Table 3), respectively.

334

The VP1 region was critical for cell culture-adaptation of PoSaV.

335

To examine whether the RdRp or the VP1 region was critical for PoSaV adaptation to

336

cells, we engineered two chimeric genomes (Figure 1) based on the previously established

337

PoSaV reverse genetics system, pCV4A: 1) TC-WTRdRp, whose 1252 and 1379 amino acid

338

residues of the polyprotein (RdRp region) were mutated from TC to WT phenotype (H1252Y

339

and K1379R); 2) TC-WTVP1, whose partial VP1 region (5227-6060 nt, 30-308 amino acids

340

excluding amino acid residues 324 and 328) was replaced with the corresponding WT fragment.

341

The capped genomic RNA transcripts were transfected into BHK-T7. After infecting LLC-PK

342

cells with BHK-T7 cell lysates, the PoSaV VP1 proteins were detected exclusively by

343

immunofluorescent assay (IFA) in the LLC-PK cells inoculated with TC-WTRdRp but not TC-

344

WTVP1 transfected products. We further compared the growth kinetics and plaque sizes of TC-

345

WTRdRp virus to those of TC-pCV4A virus in LLC-PK cells (Figure 2). TC-WTRdRp had similar 16

346

plaque sizes (0.168±0.052 mm2) as TC-pCV4A (0.146±0.066 mm2) and similar growth

347

kinetics as TC-pCV4A in LLC-PK cells, showing increasing titers between 0-72 hpi with similar

348

peak titers (7.1±0.2 log10 TCID50/mL for TC-pCV4A and 7.0±0.0 log10 TCID50/mL for TC-

349

WTRdRp). These results suggested that the VP1 region, but not the RdRp region, was critical for

350

cell culture-adaptation of PoSaV.

351

Four (178, 289, 324 and 328) of the six amino acid residues in the VP1 region were essential

352

for PoSaV adaptation to LLC-PK cells.

353

To address which of the individual mutations in the VP1 region was essential for PoSaV

354

adaptation, each of the sites was mutated to WT sequence individually (Figure 1): TCVP1-S178C,

355

TCVP1-H289Y, TCVP1-D291N, and TCVP1-R295K, TCVP1-I324M and TCVP1-G328E. Mutants

356

TCVP1-D291N and TCVP1-R295K replicated in LLC-PK cells. No infectious virus was rescued

357

from LLC-PK cells infected with the transfection lysates of TCVP1-S178C, TCVP1-H289Y,

358

TCVP1-I324M or TCVP1-G328E infectious clones. Furthermore, when the point mutations of TC-

359

WTVP1 were mutated back to TC sequences, the double mutant (TC-WTVP1-C178S&Y289H

360

carrying TC amino acids at 324 and 328 residues) instead of the individual point mutants (TC-

361

WTVP1-C178S, TC-WTVP1-Y289H), was rescued in the LLC-PK cells. Infectious virus was

362

rescued from back mutations TCVP1-I324MM324I and TCVP1-G328EE328G infectious

363

clones. These results indicate that the four amino acid residues at sites 178, 289, 324 and 328 in

364

the VP1 region are essential for PoSaV adaptation to LLC-PK cells.

365

Single amino acid substitutions in the VP1 region altered PoSaV growth kinetics in vitro.

366

To examine whether amino acid substitutions D291N, R295K and TC-WTVP1-

367

C178S&Y289H can alter viral replication in LLC-PK cell cultures, the growth kinetics and

368

plaque sizes of TCVP1-D291N, TCVP1-R295K and TC-WTVP1-C178S&Y289H to those of TC17

369

pCV4A in LLC-PK cells were compared (Figure 2). The TCVP1-D291N (0.041±0.011 mm2) and

370

TCVP1-R295K (0.047±0.016 mm2) viruses, especially the TC-WTVP1-C178S&Y289H (0.018±

371

0.003 mm2) virus, formed significantly smaller (P < 0.05) plaques than the TC-pCV4A (0.146±

372

0.066 mm2) and TC-WTRdRp (0.168±0.052 mm2) (Figure 2A). The infectious titers of TCVP1-

373

D291N, TCVP1-R295K and TC-WTVP1-C178S&Y289H increased post-inoculation. However, the

374

peak titers of TC-pCV4A (7.1±0.2 log10 TCID50/mL) and TC-WTRdRp (7.0±0.0 log10

375

TCID50/mL) were the highest, followed by TCVP1-R295K (6.6±0.0 log10 TCID50/mL), TCVP1-

376

D291N (6.4±0.0 log10 TCID50/mL), and TC-WTVP1-C178S&Y289H (4.9±0.1 log10

377

TCID50/mL) (Figure 2B). This data led us to conclude that the amino acid substitutions D291N

378

and R295K in the VP1 region reduced PoSaV replication in LLC-PK cells.

379

Comparative structural analyses of the VP1 proteins of TC and WT PoSaV predicted the

380

location and potential function of amino acid residues.

381

The 3D structural models of both TC and WT PoSaV Cowden matched the template

382

structure (FCV VP1, PDB code 3M8L) (Figure 3A). Subsequently, 3D structural analysis was

383

performed to examine whether the residue changes in VP1 between WT and TC PoSaVs resulted

384

in structural changes. Amino acid residue 178 was located in the S domain near the dimer-dimer

385

interface (Figure 3B). C178 of WT was exposed, but S178 of TC was hidden. The amino acid

386

residues 289, 291, 324 and 328 in the WT PoSaV were located at the P2 subdomain region,

387

while the amino acid 295 residue was located at the interface of two monomeric VP1 proteins

388

forming a dimer at the P2 subdomain (Figure 3B).

389 390

To investigate whether the mutations at residues 178, 289, 291, 295, 324 and 328 influenced the stability of WT PoSaV, we analyzed the changes in thermodynamic stability by 18

391

the mutations using the Protein Design application in the MOE. The positive number indicated

392

decreased stability, while the negative number indicated increased stability. The changes in △

393

△ G by each point mutation C178S, Y289H, N291D, K295R, M324I, and E328G were

394

0.54±0.04, 4.51±0.02, 1.80±0.24, -1.43±0.31, -0.42±0.15 and 1.86±0.37 kcal/mol, respectively

395

(Figure 4). Mutations C178S, Y289H, N291D and E328G decreased the stability, whereas

396

K295R and M324I, especially K295R, compensated the changes.

397

Single amino acid substitutions in the VP1 region altered PoSaV replication in pigs.

398

To investigate whether the amino acid substitutions from TC to WT in the VP1 could

399

restore the virulence of the WT PoSaV in vivo, we performed pathogenesis studies in Gn pigs.

400

Among the experimental groups, the WT PoSaV inoculated Gn pigs had significantly longer

401

viral RNA shedding duration (30.3±3.8 days) and higher peak viral RNA titer (10.8±0.4 log10

402

GE/g) than the other three inoculated Gn pig groups. Relative to the mutants, the TCVP1-D291N

403

inoculated Gn pigs had longer virus RNA shedding duration (23.2±3.4 days) and higher peak

404

RNA titer (8.6±0.8 log10 GE/g) than those of TCVP1-R295K or TC PoSaV Cowden strain

405

inoculated Gn pigs as shown (Figure 5 and Table 4).

406

Clinical signs and histopathological lesions were observed exclusively in the WT PoSaV

407

infected Gn pigs.

408

Moderate diarrhea (fecal score=2) was observed in three of seven (43%) WT PoSaV

409

inoculated Gn pigs. Diarrhea developed by PID 3 to 12 and persisted for 2 to 16 days. No

410

diarrhea or other clinical signs were observed in TC PoSaV Cowden, TCVP1-D291N, TCVP1-

411

R295K, or mock inoculated Gn pigs.

412 413

Microscopically, histopathological lesions were not observed in organs from the TC PoSaV-, TCVP1-D291N-, TCVP1-R295K-inoculated or control Gn pigs. WT PoSaV-inoculated Gn 19

414

pigs euthanized at acute phase infection exhibited moderate to severe, diffuse, and atrophic

415

enteritis, demonstrating shortened and blunt villi from duodenum to mid-jejunum of the small

416

intestine (Figure 6A, 6B and 6C). Duodenal, proximal and mid-jejunal tissues showed a

417

moderate, diffuse villous atrophy (Figure 6A, 6B and 6C). VH:CD of duodenal and mid-jejunal

418

tissues were 3.54±1.06 and 3.98±1.84, respectively, significantly lower than those (5.42±2.26

419

and 5.95±2.25, respectively) of control pigs (Table 5). Interestingly, the VH:CD ratio of jejunal

420

tissues of the TCVP1-D291N inoculated Gn pigs was statistically lower than those of for the

421

TCVP1-R295K, TC PoSaV Cowden inoculated, and control Gn pigs (Table 5), indicating more

422

severe villous atrophy in the jejunum of TCVP1-D291N-inoculated Gn pigs compared with

423

TCVP1-R295K-, TC PoSaV-inoculated and control Gn pigs.

424

Virus VP1 antigens were detected in the frozen tissues of WT PoSaV-inoculated Gn pigs

425

by immunofluorescent (IF) staining. Most antigen-positive cells were distributed in the mature

426

enterocytes lining the intestinal villi of mid-jejunum, and to a lesser extent, in duodenum and

427

proximal jejunum (Figure 6). Antigen-positive cells were rarely detected in distal jejunum and

428

ileum. The mean numbers of antigen-positive cells per villus differed significantly among

429

different regions of the small intestine with most of the positive epithelial cells observed in

430

duodenum to mid-jejunum with an increasing trend from duodenum (1.18±0.55) to proximal

431

jejunum (2.45±0.42) and to mid jejunum (3.78±1.30) (Figure 7). Antigen-positive cells were not

432

observed in any tissue sections of TC PoSaV-, TCVP1-D291N-, or TCVP1-R295K-inoculated Gn

433

pigs.

434

IgG, IgA, and VN antibody titers in pig serum samples were titrated (Figure 8A, 8B and

435

8C). From PIDs 6 to 27, the Gn pigs inoculated with WT had the highest IgG, IgA and VN

436

serum antibody titers. The Gn pigs inoculated with TCVP1-D291N had higher serum IgG, IgA 20

437

and VN antibody titers than those inoculated with TC or TCVP1-R295K PoSaV at PIDs 6 to 27.

438

The Gn pigs inoculated with WT also had the highest IgA mucosal antibody titers at PID 27

439

(Figure 8D). The Gn pigs inoculated with TCVP1-D291N and TC PoSaV had significantly higher

440

IgA mucosal antibody titers than those inoculated with TCVP1-R295K (One-way ANOVA

441

followed by Duncan’s multiple range test on log4 transferred titers, P < 0.05).

442 443

Discussion

444

The genomes of WT (Gn pig passage level 5) and TC PoSaV Cowden strain (cell culture

445

passage level 20) were reported in 1999 (25). It was also reported that WT PoSaV Cowden strain

446

grew in porcine primary kidney cell culture after thirteen passages in Gn pigs, but only with

447

mock Gn pig intestinal contents in the medium (14). Therefore, we selected one Gn pig intestinal

448

sample from both the fifth and the thirteenth passage levels of WT PoSaV for genomic sequence

449

analysis. Besides the reported six amino acid substitutions between WT and TC PoSaVs (25), we

450

identified two additional substitutions at amino acid residues 324 and 328 of VP1. We compared

451

seven genomes of different passages of WT and TC PoSaV Cowden strain (Table 3). Compared

452

to WT PoSaV Cowden strain, eight of 19 amino acid mutations were conserved among all TC

453

PoSaV genomes. The differences between the published WT PoSaV Cowden genome and our

454

results are likely due to the intestinal content samples from different pigs and the different

455

passage levels tested. Because the two newly identified sites (324 and 328 in the VP1 region)

456

were conserved in the newly sequenced fifth and thirteenth Gn pig passages of WT PoSaV, both

457

sites were also included in this study. The establishment of the reverse genetics system pCV4A

458

for PoSaV Cowden strain provided an important tool to study the molecular mechanisms for

459

PoSaV cell culture adaptation (20). In this study, we generated a series of PoSaV mutants to test 21

460

which of the eight amino acids were critical for cell culture adaptation. In the previous report,

461

one-step in vitro transcription and capping was used to generate infectious PoSaV genomic RNA

462

for transfection (20). Infectious PoSaV Cowden virions were successfully rescued from LLC-PK

463

cells using the one-step method (20, 35). However, for unknown reasons, we failed to rescue

464

infectious virus. Because infectious MNV was rescued from RAW 264.7 cells using a two-step

465

in vitro transcription followed by capping (36), we tried the two-step method and rescued

466

infectious PoSaV from LLC-PK cells. The genome-linked virus protein (VPg) is encoded by

467

NS5 gene, and is covalently linked to the 5’ end of the SaV genomic RNA (20, 37, 38). In our

468

system, the cap structure analog, m7G(5’)ppp(5’)G, was added to the 5’ end of the SaV genomic

469

RNA transcripts to simulate SaV VPg (36, 39).

470

Among the mutants, TCVP1-S178C, TCVP1-H289Y, TCVP1-I324M, and TCVP1-G328E

471

could not be recovered in 4-5 repeats of the experiment. Whether viral particles were formed was

472

unknown. However, the HEK293T/17 cells transfected with in vitro transcribed and capped

473

RNA of TCVP1-S178C, TCVP1-H289Y, TCVP1-I324M, and TCVP1-G328E showed several VP1-

474

positive cells by IHC staining using hyperimmune serum against the VLPs of PoSaV Cowden

475

strain (data not shown). This suggests that the defect may occur at any step post VP1 expression,

476

such as virion assembly and binding to the receptors on LLC-PK cells or entry into the LLC-PK

477

cells. TCVP1-D291N, TCVP1-R295K and TC-WTVP1-C178S&Y289H were culturable in the LLC-

478

PK cell line, although the titer of TC-WTVP1-C178S&Y289H strain was 2 log10 lower compared

479

with the other culturable strains. In the in vitro study, both TCVP1-D291N and TCVP1-R295K

480

mutants showed reduced replication compared with the TC PoSaV. However, in our Gn pig

481

study, both TCVP1-D291N and TCVP1-R295K mutants had relatively higher peak viral RNA

482

titers, longer fecal viral RNA shedding duration, and higher serum and mucosal antibody 22

483

responses than TC PoSaV, but lower than those of WT PoSaV. This indicates that the replication

484

efficiency of PoSaV mutants were discordant in vitro and in vivo. In vitro, peak viral infectivity

485

titers (highest to lowest) were TC PoSaV > TCVP1-R295K > TCVP1-D291N > TC-WTVP1-

486

C178S&Y289H; in vivo, peak viral RNA titers (highest to lowest) were WT PoSaV > TCVP1-

487

D291N > TCVP1-R295K > TC PoSaV. Because the critical mutation sites are all located in VP1,

488

probably different receptors were used by PoSaV Cowden strain in vivo to infect small intestinal

489

epithelial cells in pigs and in vitro to infect porcine kidney LLC-PK cells.

490

Three passages of MNV1 in the macrophage cell line RAW 264.7 resulted in a total of

491

three amino acid substitutions, which included V716I and H845R in the 3A-like protease (NS4)

492

region and E296K in the P2 subdomain of VP1. Only V716I and E296K were suspected to be

493

related to decreased virulence in mice and increased titer in RAW 264.7 cells (40, 41). Using

494

reverse genetics, the K296E but not I716V back mutation restored the virulence of the MNV1

495

revertant in mice (41). Therefore, a single amino acid substitution in the P2 subdomain of VP1 of

496

a norovirus may affect its virulence in the host and the replication efficacy in cell culture. A

497

previous study concluded that TC PoSaV Cowden strain was attenuated in vivo compared with

498

the WT PoSaV Cowden strain after oral inoculation of Gn pigs with TC PoSaV Cowden strain

499

cell culture supernatant (cell culture passage #20) or a filtrate of WT PoSaV Cowden strain (18).

500

In our study, we concentrated the TC PoSaV Cowden strain, TCVP1-D291N and TCVP1-R295K,

501

which were 1 log10 higher doses than in the previous study. However, clinical signs were

502

observed exclusively in the WT PoSaV-infected pigs, but not in the TC PoSaV Cowden strain,

503

TCVP1-D291N or TCVP1-R295K infected Gn pigs. These results indicated that even at 1 log10

504

higher inoculation dose, TC PoSaV Cowden strain did not cause diarrhea in Gn pigs. Although

505

revertants TCVP1-D291N and TCVP1-R295K did not cause diarrhea in Gn pigs, they replicated 23

506

more efficiently and induced relatively higher immune responses compared with TC PoSaV,

507

suggesting that the two amino acid substitutions in the P2 subdomain of VP1 affected PoSaV

508

virulence in the host and could be better candidates than TC PoSaV Cowden strain for PoSaV

509

vaccine development.

510

The first calicivirus structure was reported in 1994 for a primate calicivirus (42).

511

Currently, structures of virus-like particles (VLPs) or virion particles of caliciviruses have been

512

determined for human NoV GI.1 and GII.10 (43, 44), GV MNV (45, 46), San Miguel sea lion

513

virus (47), Tulane virus (48), rabbit hemorrhagic disease virus (49), and feline calicivirus (FCV)

514

(50). Therefore, structures are available in all classified genera of the family Caliciviridae,

515

except for the genera Sapovirus and Nebovirus. Chen, et al, reported that sapovirus showed more

516

structural similarity to vesivirus (22). FCV VP1 protein has the closest phylogenetic relatedness

517

(37% amino acid identity) to PoSaV Cowden strain among the existing calicivirus VP1

518

structures. To understand the location of the mutation sites based on structural predictions, we

519

performed 3D structure modelling and analysis of the VP1 protein of PoSaV Cowden strain

520

using FCV VP1 as template. In a previous study, the complete removal of the P domain of

521

recombinant Norwalk virus-like particles resulted in the formation of smooth particles, which

522

demonstrated that S domain is sufficient for assembly of the capsid (51). Since the P2 subdomain

523

of caliciviruses is the most protruding part of VP1 and is highly variable, it has been considered

524

responsible for binding to the host receptors (52). A recent study indicated that the a2,3- and

525

a2,6-linked sialic acids on O-linked glycoproteins are receptors on LLC-PK cells for PoSaV

526

Cowden strain (53). In our study, based on their locations in the structural model, amino acid

527

position 178 was located in S domain, which may influence VP1 oligomerization, virion

528

assembly, and stability. Amino acid positions 289, 291, 324, and 328 were located in the P2 24

529

subdomain, which affect the binding to the receptors on LLC-PK cells and may impair virus

530

replication. Amino acid position 295 was located in the P2 subdomain at the interface of two

531

monomeric VP1 proteins, which may influence VP1 dimerization.

532

The changes in thermodynamic stability by the mutations indicate whether or not the

533

protein structure can be maintained. The overall change in thermodynamic stability is the sum of

534

each estimated value. Decreasing the stability is a disadvantage to maintain the structure,

535

whereas increasing the stability is an advantage. Therefore, the mutations that decrease the

536

stability are critical for the function, e.g., a cell culture adaptation, rather than the structure.

537

Then, the mutations that increase the stability play a role in the compensation for the decreasing

538

stability. In this study, the thermodynamic stability analysis indicated that C178S, Y289H,

539

N291D and E328G decrease the stability, while K295R and M324I increase the stability. These

540

results suggest that C178S, Y289H, N291D and E328G would provide essential functions for

541

cell culture adaptation, whereas K295R and M324I would compensate for decreased stability of

542

C178S, Y289H, N291D and E328G.

543

We compared 12 WT GIII PoSaV VP1 protein sequences available in GenBank

544

(http://www.ncbi.nlm.nih.gov/nucleotide/). We found that C178 in the S domain is conserved

545

among all the 12 WT GIII PoSaVs [WT PoSaV Cowden (GenBank accession no. KT922087),

546

SaV1-CHN (ACP43737), HW20-KOR (ADN84680), ID3-HUN (ABD38714), LL14-US

547

(AAR37376), MM280-US (AAX32888), JJ259-US (AAX37311), QW270-US (AAX37314),

548

PES-VENEZ (AAY88248), PoS6-HUN (ACS68238), PoS9-HUN (ACS68240), s20-JAP

549

(BAE94661)], suggesting that C178S may be the most a critical mutation during PoSaV Cowden

550

strain tissue culture-adaptation.

25

551

In this study, WT PoSaV antigen was observed in the epithelial cells of Gn pig small

552

intestine from duodenum to mid-jejunum, but rarely in distal jejunum or ileum. We confirmed

553

the region of PoSaV infection in Gn pigs as reported previously (18). Also, as previously

554

reported, morphologic alterations in duodenal and jejunal villi were observed in WT PoSaV

555

Cowden infected Gn pigs (18, 54). IF staining of small and large intestinal impression smears,

556

as well as villus length measurement also confirmed that the small intestine was the major

557

infection site (18, 54). Our data supports previous studies of the pathogenesis of WT PoSaV

558

Cowden strain in Gn pigs.

559

This study demonstrated that cell culture adaptation of PoSaV Cowden strain is due to the

560

amino acid substitutions in the VP1 region. The single revertant mutation (TC to WT) at certain

561

positions 291 and 295 in the VP1 region reduced virus replication in vitro, but partially regained

562

PoSaV replication efficiency in vivo. The genetic basis delineated for cell culture adaptation of

563

PoSaV may provide new critical information for the rescue of other uncultivable PoSaVs and

564

human SaVs. In future studies, we plan to construct reverse genetics systems for selected PoSaV

565

and human SaV strains, by introducing site-directed mutations at structurally corresponding

566

positions in the VP1 protein to rescue such unculturable SaVs.

567 568 569

Funding Information This work was supported by grants from the National Institute of Allergy and Infectious

570

Diseases, National Institutes of Health [R01 AI49742-04 to Linda J. Saif, R21 AI081009-2 to

571

Qiuhong Wang (PI) and Linda J. Saif (co-PI), R01 AI056351 to Ralph Baric, and U01AI08001

572

to Kyeong-Ok Chang]. ZL was supported by a scholarship provided by the China Scholarship

573

Council. The funders had no role in study design, data collection and interpretation, or the 26

574

decision to submit the work for publication. Salaries and research support were provided by state

575

and federal funds provided to the Ohio Agricultural Research and Development Center

576

(OARDC), The Ohio State University.

577 578 579

Acknowledgements We acknowledge Dr. Juliette Hanson, Mr. Jeff Ogg, Mr. Andrew Wright, Ms. Ronna

580

Wood, and Ms. Megan Strother for assistance with animal care; Dr. Chun-Ming Lin, Ms.

581

Xiaohong Wang, and Ms. Susan Sommer-Wagner for technical assistance.

27

582

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583

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Wang Q, Zhang Z, Saif LJ. 2012. Stability of and attachment to lettuce by a culturable

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Yunus MA, Chung LM, Chaudhry Y, Bailey D, Goodfellow I. 2010. Development of

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719

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721

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723

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724 725

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729

Cho KO. 2014. Both alpha2,3- and alpha2,6-linked sialic acids on O-linked

730

glycoproteins act as functional receptors for porcine Sapovirus. PLoS Pathog

731

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732 733

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Flynn WT, Saif LJ, Moorhead PD. 1988. Pathogenesis of porcine enteric caliciviruslike virus in four-day-old gnotobiotic pigs. Am J Vet Res 49:819-825.

734 735

34

736

Figure legends

737

Figure 1. Diagrams of the constructions of the TC and WT PoSaV and the mutants derived

738

from the pCV4A backbone.

739

The eight amino acid residues at positions 1252 and 1379 in the ORF1 polyprotein and at

740

positions 178, 289, 291, 295, 324 and 328 in the VP1 that differed between WT (red) and TC

741

(green) PoSaV are indicated in bold and italic in each mutant. The mutants TCVP1-D291N and

742

TCVP1-R295K were tested in Gn pigs with WT and TC as controls. The mutant TC-WTVP1-

743

C178S&Y289H was not tested (NT) due to the 2 log10 lower virus infectivity titer that could not

744

be equalized by further concentration. Replication of the mutants in cell culture or in Gn pigs is

745

noted.

746 747

Figure 2. Growth kinetics and representative plaques of TC-pCV4A (TC) and the

748

culturable mutants, TC-WTRdRp, TCVP1-D291N (291), TCVP1-R295K (295) and TC-WTVP1-

749

C178S&Y289H (Double) in LLC-PK cells.

750

(A) The plaque sizes of mutants TCVP1-D291N, TCVP1-R295K, and TC-WTVP1-C178S&Y289H

751

were smaller than those of TC-pCV4A and TC-WTRdRp in LLC-PK cells. (B) LLC-PK cells were

752

inoculated with each virus at 0.01 multiplicity of infection (MOI). Cell lysates were collected at

753

24, 48, 72 and 96 hours post-inoculation (hpi) for the titration of infectious virus. TC-pCV4A

754

and TC-WTRdRp replicated in LLC-PK cells to significantly higher titers (72 hpi) than the

755

culturable mutants (One-way analysis of variance (ANOVA) followed by Duncan’s multiple

756

range test on log10 transferred titers, P < 0.05).

757

35

758

Figure 3. Superimposition of the modeled structures of PoSaV Cowden and the template

759

FCV structure and the 3D models of WT and TC PoSaV VP1.

760

(A) Superimposition of the modeled structures (WT PoSaV Cowden on the left and TC PoSaV

761

Cowden on the right) and the template structure (FCV VP1, PDB code 3M8L) by homology

762

modeling. The blue ribbon structure denotes WT PoSaV Cowden VP1, the cyan ribbon structure

763

denotes TC PoSaV Cowden, and the magenta ribbon structure denotes the template structure

764

FCV VP1. (B) Amino acid residue 178 was located at the dimer-dimer interface in the S domain;

765

residues 289, 291, 324 and 328 were located in the P2 subdomain; residue 295 was located on

766

the interface of two monomeric VP1 proteins in P2 subdomain.

767 768

Figure 4. The changes in △△ G for each point mutation C178S, Y289H, N291D, K295R,

769

M324I, and E328G.

770

Mutations C178S, Y289H, N291D and E328G decreased the thermodynamic stability, whereas

771

K295R and M324I compensated the changes.

772 773

Figure 5. The viral RNA shedding in geometric mean titer (GMT) at various post-

774

inoculation days (PIDs) of TCVP1-D291N and TCVP1-R295K compared with TC-pCV4A

775

and WT PoSaV Cowden strain in Gn pigs.

776

Gn pigs were inoculated with the corresponding virus inoculum. Rectal swabs (RSs) were

777

collected daily for viral RNA shedding titration. The WT PoSaV Cowden strain had the highest

778

peak titer and longest duration of viral RNA shedding among the experimental groups. The TC

779

PoSaV Cowden strain had the lowest peak titer and shortest shedding duration among the

36

780

experimental groups. Viral RNA shedding peak titers and durations of TCVP1-D291N and TCVP1-

781

R295K inoculated Gn pigs were intermediate between WT and TC groups.

782 783

Figure 6. Histopathological examination of small intestinal samples of WT PoSaV Cowden

784

or mock (NC) infected Gn pigs.

785

Hematoxylin and eosin (H&E) (left) and immunofluorescent (IF) (right) staining for samples

786

collected from different regions in the small intestine. (A) to (C) WT PoSaV Cowden inoculated

787

Gn pigs at PID 5, showing fusion, shortening (arrows), or blunting (arrowhead) of villi in

788

duodenum, proximal jejunum, and mid-jejunum, respectively. (D) to (F) mock inoculated Gn

789

pigs at PID 5, showing normal villi in duodenum, proximal jejunum, and mid-jejunum. Intestinal

790

samples were collected in duplicate for IF staining and for H&E staining. Bar, 50 μm.

791 792

Figure 7. The distribution of antigen positive cells per villus in different regions of the small

793

intestine of WT PoSaV-inoculated pigs.

794

Mean antigen positive cells per villus were significantly different among duodenum, proximal

795

jejunum, and mid-jejunum of small intestine (One-way ANOVA, P < 0.05).

796 797

Figure 8. Serum and mucosal antibody responses in Gn pigs.

798

(A) GMT of IgG antibodies to corresponding PoSaV strains or mock (NC) in serum samples of

799

Gn pigs. (B) GMT of IgA antibodies to corresponding PoSaV strains or mock (NC) in serum

800

samples of Gn pigs. (C) GMT of viral neutralizing antibodies to corresponding PoSaV strains or

801

mock (NC) in serum samples of Gn pigs. (D) GMT of IgA antibodies to corresponding PoSaV

802

strains in ileal mucosal samples of Gn pigs. Serum samples were collected from each Gn pig 37

803

before inoculation and at PID 1, 3, 6, 9, 16, 23, 27. Ileal mucosal samples were collected at PID

804

5 and PID 27. Data points marked with different letters at each day differed significantly (One-

805

way ANOVA followed by Duncan’s multiple range test on log10 transferred titers, P < 0.05).

806

38

807

Table 1. Primers for the generation of the chimeric PoSaV clones. Primer name ApaI-F ApaI-R TC-3763-CTF TC-3763-CTR TC-4145-AGF TC-4145-AGR TC-5671-ATF TC-5671-ATR TC-6004-CTF TC-6004-CTR TC-6010-GAF TC-6010-GAR TC-6023-GAF TC-6023-GAR WT-5671-TAF WT-5671-TAR WT-6004-TCF WT-6004-TCR EcoRI-F EcoRI-R 6111-F 6111-R 6122-F 6122-R 6111-6122-F 6111-6122-R QCback-F QCback-R

Sequence 5′- GAGTCCAGACCAGTCCAGCCAGC 5′- TGGGTAGTGGTTGATGATGTTG 5′-CGTGAATGACCCAAGGTACCCCTTCTCACAACA 5′-TGTTGTGAGAAGGGGTACCTTGGGTCATTCACG 5′-AGAAAAGAATGACCAAGGCAAAAGACGCCTGCTGTG 5′-CACAGCAGGCGTCTTTTGCCTTGGTCATTCTTTTCT 5′-TTGGTGGGGCTATAGCATGTTTGGCACTTTACGTG 5′-CACGTAAAGTGCCAAACATGCTATAGCCCCACCAA 5′-CCCGTGTCAATGGAAAGTACACTGACAACACAGGT 5′-ACCTGTGTTGTCAGTGTACTTTCCATTGACACGGG 5′-CCCGTGTCAATGGAAAGCACACTAACAACACAGGTA 5′-TACCTGTGTTGTTAGTGTGCTTTCCATTGACACGGG 5′-AGCACACTGACAACACAGGTAAGGCAGTGTTTCA 5′-TGAAACACTGCCTTACCTGTGTTGTCAGTGTGCT 5′-TTGGTGGGGCTATAGCAAGTTTGGCACTTTACGTG 5′-CACGTAAAGTGCCAAACTTGCTATAGCCCCACCAA 5′-CCCGTGTCAATGGAAAGCACACTAACAACACAGGT 5′-ACCTGTGTTGTTAGTGTGCTTTCCATTGACACGGG 5’-GAGGCCTACGAGGAATTCAAG 5’-GAGCCTGATTAAAAGAATTCATAATA 5’-CAACAATGTTCAACACAGGAAC 5’- GTTGAACATTGTTGATGCAGC 5’-CAACACAGAAACTGCCGTAAATG 5’- GGCAGTTTCTGTGTTGAATATTG 5’- CAATGTTCAACACAGAAACTGCC 5’- CAGTTTCTGTGTTGAACATTGTTG 5’- GGCTGCATCAACAATATTCAACACAGGAACTGCC 5’- GGCAGTTCCTGTGTTGAATATTGTTGATGCAGCC

Location in PEC genome 5203-5225 6090-6069 3747-3779 4122-4157 5654-5688 5987-6021 5987-6022 6002-6035 5654-5688 5987-6021 4570-4590 6891-6866 6104-6125 6117-6097 6114-6138 6129-6107 6107-6129 6127-6104 6096-6129

Orientation Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

808 39

809

810 811

Table 2. Experimental design for inoculation of Gn pigs with PoSaV Cowden TC, WT, or mutants. Group No. a

Inoculum (orally)

No. of Gn pigs

1

TC Cowden strain

9

2

mutant TCVP1-R295K

7

3

mutant TCVP1-D291N

9

WT Cowden strain 4 PS799 5 MEM a All pigs were 4 to 7 days of age at inoculation. b

7 4

Titer (GE/mL) b 11.5 log10 GE/mL in MEM, 5mL/pig 11.5 log10 GE/mL in MEM, 5mL/pig 11.5 log10 GE/mL in MEM, 5mL/pig 11.5 log10 GE/mL in MEM, 5mL/pig MEM, 5 mL/pig,

No. of Gn pigs euthanized at acute infection phase (PID 5-7) 3 2 3 3 2

RT-qPCR titer 11.5 log10 GE/mL is equivalent to ~7.0 log10 TCID50/mL by infectivity assay.

40

812

Table 3. Summary of amino acid substitutions in the genomes of different passages of WT or TC PoSaV Cowden strain. Gene

NS1

WT-PoSaV in Gn pigs Passage 5 Passage 13 Passage 13 (I-1113a) (R418a) (PS499a)

Amino acid position

Nucleotide position

17

59

TTT

TTT

TTT

TTT

18 24

62 80

GAC CCA

GGC(G18D) CCA

GGC CCA

GGC CCA

29

94

GCG

GCG

GCG

GCG

356

1075

ATT

ATT

ATT

ATT

367

1109

AAG

AAG

AAG

AAG

733 1252 1379 1392

2207 3763 4145 4185

GGC TAC AGA ATA

75

5362

ACA

ACA

ACA

ACA

178 289 291 295 324 328

5671 6004 6010 6023 6111 6122

TGT TAC AAC AAG ATA GGA

TGT TAC AAC AAG ATG GAA

TGT TAC AAC AAG ATG GAA

TGT TAC AAC AAG ATG GAA

27

6851

CAT

CAT

CAT

CAT

35

6873

AAT

AAT

AAT

AAT

Passage 5 (25)

NS3 NS4 NS7

VP1

GAC /GGC GGC TAC TAC AGA AGA ATA ATG /ATA

GGC TAC AGA ATA

ORF2

TC-PoSaV in LLC-PK cells Passage 20 Passage 27 Passage 30 (25) TCT TTT TTT (S17F) GAC GGC GGC CCA CCA CTA (L24P) ACG GCG GCG (T29A) GTT ATT ATT (V356I) AGG AAG AAG (R367K) GGC(D733G) GGC GGC CAC CAC(Y1252H) CAC AAA(R1379K) AAA AAA ATA(M1392I) ATA ATA GCA ACA ACA (A1785T) AGT AGT AGT (C178S) CAC CAC CAC (Y289H) GAC GAC GAC (N291D) AGG (K295R) AGG AGG ATA (M324I) ATA ATA GGA (E328G) GGA GGA CAA CAT CAT (E27H) GAT AAT AAT (D35N) 41

813

a

Sample ID based on the labeling system in the lab.

814

42

815

Table 4. Comparative viral RNA shedding parameters of PoSaV Cowden TC, WT, and mutants.

816

Duration days Peak titer log10 PID of peak titer f (SD) GE/mLδ (SD)δ TC 6 1-4 19.8 (2.6)c 7.7 (0.4)c 3-14 295 5 1-4 20.8 (3.4)c 7.9 (1.0)c 5-15 d d 291 6 1-6 23.2 (3.4) 8.6 (0.8) 3-19 WT 4 1-3 30.3 (3.8)e 10.8 (0.4)e 6-10 NC 2 a TC, 295, 291, WT and NC refer to TC PoSaV Cowden strain, TCVP1-R295K, TCVP1-D291N, WT PoSaV Cowden strain, and

817

negative control, respectively.

818

b

819

qPCR.

820

δ

821

f

Groups a

No. Gn pigs

Onset (PID) b

The onset of RNA shedding [post-inoculation days (PIDs)] refers to the PIDs that fecal viral RNA was first detected by RT-

Superscript denotes significant differences among the groups (One-way ANOVA followed by Duncan's multiple-range test).

The PID of peak titer refers to the PID that has the highest viral RNA titer by RT-qPCR.

43

822

Table 5. Ratios of villus length/crypt depth in different regions of small intestines of Gn pigs. Groups a

823

Regions in the small intestine [Avg. (SD)]δ Duodenum Jejunum Ileum TC 5.21(0.86)b 5.93 (1.60)b 4.68 (0.52) b b 295 5.97 (1.15) 6.44 (0.78) 5.26 (0.66) 291 5.32 (0.94)b 5.09 (1.47)c 5.20 (0.76) WT 3.54 (1.06)c 3.98 (1.84)d 5.23 (1.20) NC 5.42 (2.26)b 5.95 (2.25)b 6.57 (0.33) a TC, 295, 291, WT and NC refer to TC PoSaV Cowden strain, TCVP1-R295K, TCVP1-D291N, WT PoSaV Cowden strain, and

824

negative control, respectively.

825

δ

Superscripts denote significant differences among the groups not sharing the same superscript (One-way ANOVA).

44

ORF1

ORF2

10

6774 NS1-7 (1711 aa)

VP1 (544 aa)

Sapovirus Cowden

VP2 (165 aa)

6771 ORF1 position 1252

1379

Y

R

WT

In vitro replication in LLC-PK 178 289 291 295 324 328 VP1 position

C

RdRp H

TC

K

TC-WTRdRp

R

S

TC-WTVP1 (324 and 328 in TC type)

K

TCVP1-S178C

K

H

K

TC-WTVP1-C178S&Y289H

K

H D R

I

G

H D R

Yes

Yes

Yes

NT

No

NT

No

NT

No

NT

Yes

NT

Yes

Yes

Yes

Yes

No

NT

Yes

NT

G

YD R I

VP1 S HN K I

RdRp

Yes

G

I

VP1 S

No

G

VP1 C

RdRp H

G

C YNK I

RdRp

TCVP1-H289Y

I

VP1

RdRp H

H D R

In vivo replication in Gn pigs

M E

VP1 S

RdRp H

Y N K

VP1

RdRp Y

7320 (A)n

7265

G

VP1

(324 and 328 in TC type) H

TCVP1-D291N

S

RdRp H

TCVP1-R295K

K

K

H

K

S

G

H D

K

I

G

H D R

M G

VP1 S

RdRp

TCVP1-I324M-M324I

I

VP1

RdRp

TCVP1-I324M

HN R

VP1 S

RdRp H

Back mutation

K

H D R

I G

VP1

(same as TC) H

TCVP1-G328E Back mutation

(same as TC)

S

RdRp H

TCVP1-G328E-E328G

K

K

RdRp

H D R

I

E

VP1 S

H D R

VP1

No I

NT

G

Yes

NT

Fig. 2B

Log10 transferred infectious titers (TCID50/ml)

Growth kinetics in LLC-PK cells 8 6 4 2 0 0

24 TC

295

48 72 Hours post inoculation 291

Double

TC-WTRDRP

96

WT PoSaV Cowden

Fig. 3A

TC PoSaV Cowden

Top view

Side view

WT PoSaV Cowden

TC PoSaV Cowden

FCV (PDB code 3M8L)

FCV (PDB code 3M8L)

Fig. 3B

WT PoSaV Cowden H289Y D291N G328E

TC PoSaV Cowden I324 G328

R295K

H289 D291

P2 subdomain R295

Side view

P1 subdomain C178

C178S

S domain

H289Y

Top view

H289

G328

G328E

I324

I324M I324M G328E

H289Y

I324 G328

H289

PoSaV RNA shedding titer in per gram feces PoSaV shedding GMT (log10 GE/ml) by RT-qPCR

12

10

8

6

4

2

0 0

2

4

6

8

10

12

14

TC

16

295

18 PID

20

291

22

WT

24

26

28

30

32

34

36

Distribution of IHC-positive cells in small intestine of WT PoSaV infected Gn pigs C

Numbers of positive cells/villus

6

5

4

B 3

2

A

1

0 Duodenum

Proximal jejunum

Regions of small intestine

Mid-jejunum

B

B A

A

1024

B C

B

256

C

B

64

C

B

A

C

C

16

C

4

C

CB

1 0

1

3

6

9

16

23

27

PID TC

C

295

291

WT

A

A B

B

A

A

B B

B

1024 256

C

B

C

64 A

16 B CC

1 0

1

3

6

9

16

PID TC

295

291

WT

NC

23

27

A

A

4096

A

A

1024

B

B

256

B

B

B B

64

C

C

A

16 B

4

BB

1 0

1

3

6

9

16

23

PID TC

4096

4

PoSaV IgA antibody in serum 16384

NC

VN antibody GMT titer to PoSaV in serum VN antibody GMT titer to PoSaV in serum

A

A

A

IgA PoSaV antibody GMT in serum

IgG PoSaV antibody GMT in serum

PoSaV IgG antibody in serum 4096

D

295

291

WT

NC

IgA PoSaV antibody in ileal mucosa IgA PoSaV antibody GMT in ileal mucosa

A

4096 A

1024 256

B

B

64 16 4 1 5

27

PID TC

295

291

WT

NC

27