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Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the

Porcine Sapovirus Cowden Strain
Zhongyan Lu,a Masaru Yokoyama,b Ning Chen,a,c Tomoichiro Oka,a,d Kwonil Jung,a Kyeong-Ok Chang,e Thavamathi Annamalai,a
Qiuhong Wang,a Linda J. Saifa
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural, & Environmental Sciences, Department of
Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USAa; Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japanb;
Boehringer Ingelheim (China) Investment Co., Ltd., Shanghai, Chinac; Department of Virology II, National Institute of Infectious Disease, Tokyo, Japand; Department of
Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USAe

ABSTRACT

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The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-
type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-
dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that
4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical
for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced
virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dim-
er-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protrud-
ing subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the inter-
face of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291
or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and
the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain.
Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical informa-
tion for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.

IMPORTANCE
The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered
that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two sub-
stitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-
level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Struc-
tural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324,
and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will
provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

C aliciviruses, in the family Caliciviridae, are small, icosahedral,

and nonenveloped viruses with a diameter of 27 to 35 nm,
which have a positive-sense, single-stranded RNA genome of 6.5
to 8.3 kb (1, 2). Caliciviruses have been classified into five genera
(Norovirus, Sapovirus, Vesivirus, Lagovirus, and Nebovirus) and
several proposed genera (3, 4). Among them, noroviruses (NoVs)
and sapoviruses (SaVs) are the leading causes of gastroenteritis in
humans of all ages. SaVs are often associated with sporadic, self-
limiting gastroenteritis, the severity of which is reportedly milder
than that of NoVs (5, 6). However, SaVs also cause outbreaks
worldwide (7–10), and deaths associated with SaV infection have
been reported for long-term-care facilities (11).
Because most enteric caliciviruses are unculturable, re-
search on pathogenesis and immunity, as well as the develop-
ment of antivirals, has been hampered. The porcine SaV
(PoSaV) Cowden strain, previously known as porcine enteric
calicivirus (PEC), belongs to genogroup III (GIII) of the SaVs
and is one of only a few culturable enteric caliciviruses (2, 12,
13). The PoSaV Cowden strain was adapted to a porcine kidney
cell line (LLC-PK) after passage of the virus in gnotobiotic
(Gn) pigs, followed by 20 passages in primary porcine kidney
cells in the presence of an intestinal content preparation from
uninfected Gn pigs (12, 14).
Gn pigs have been established as a relevant animal model be-
cause of the similarity of anatomy, genetics, physiology, and im-
munity with humans (15–17). PoSaV naturally infects pigs and
causes mild-to-moderate gastroenteritis in Gn pigs (14, 18, 19),
thus mimicking SaV diarrhea in humans and providing an animal
model suitable for studies of the replication and pathogenesis of
enteric caliciviruses.
Received 1 September 2015 Accepted 8 November 2015
Accepted manuscript posted online 18 November 2015
Citation Lu Z, Yokoyama M, Chen N, Oka T, Jung K, Chang K-O, Annamalai T,
Wang Q, Saif LJ. 2016. Mechanism of cell culture adaptation of an enteric
calicivirus, the porcine sapovirus Cowden strain. J Virol 90:1345–1358.
doi:10.1128/JVI.02197-15.
Editor: S. Schultz-Cherry
Address correspondence to Qiuhong Wang, wang.655@osu.edu, or Linda J. Saif,
saif.2@osu.edu.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

February 2016 Volume 90 Number 3 Journal of Virology jvi.asm.org 1345

Lu et al.
The genome of PoSaV is composed of two open reading
frames (ORFs). ORF1 encodes a polyprotein that is processed
into several nonstructural proteins (NSs) and the major struc-
tural protein VP1 by a viral protease. ORF2 encodes a small
structural protein, VP2 (20). VP1 is divided into two domains:
a shell (S) domain (amino acid positions 3 to 216) and a pro-
truding (P) domain (amino acid positions 217 to 544) (21).
The P domain is further divided into the P1 (amino acid posi-
tions 217 to 272 and 425 to 544) and P2 (amino acid positions
273 to 424) subdomains (21, 22).
Reverse-genetics systems are important tools to rescue uncul-
turable viruses and to study virus replication mechanisms. A re-
verse-genetics system, pCV4A, that was constructed for the PoSaV
Cowden strain contained the full-length genomic cDNA of the
tissue culture-adapted (TC) PoSaV Cowden strain, directly down-
stream from the T7 RNA polymerase promoter (20). Infectious
TC PoSaV particles were rescued after transfection of LLC-PK
cells with the in vitro-transcribed and -capped PoSaV genomic
RNA (20).
In this study, we investigated the genetic basis of cell culture
adaptation of the PoSaV Cowden strain by comparative sequence
analyses of the genomes of different passages of wild-type (WT)
and TC PoSaVs and the generation of a series of PoSaV mutants
using the reverse-genetics system. We further investigated their
differences in replication both in vivo and in vitro as well as their
putative structural differences. To our knowledge, our studies are
the first to identify which amino acid residues are critical for the
cell culture adaptation of a SaV. This study provides new informa-
tion on cell culture adaptation of SaVs that may be applicable to
other human SaVs or to NoVs.
MATERIALS AND METHODS
Cells and viruses. The LLC-PK cell line (ATCC CL-101) and a human
embryonic kidney cell line, HEK 293T/17 (ATCC CRL-11268), were ob-
tained from the American Type Culture Collection (ATCC). LLC-PK cells
were passaged and maintained as previously described (20, 23). HEK
293T/17 cells and a baby hamster kidney cell line (BHK-T7) stably ex-
pressing T7 RNA polymerase were cultured in Dulbecco’s modified Ea-
gle’s medium (DMEM; Life Technologies, NY, USA) with 10% fetal bo-
vine serum (FBS; Thermo Scientific, MA, USA), 1% nonessential amino
acids (NEAA; Invitrogen, NY, USA), and 1% antibiotic-antimycotic (In-
vitrogen, NY, USA).
Two passage levels of the WT PoSaV Cowden strain (Gn pig passage
level 5 [I-1113] and level 13 [R418]) from the small intestinal contents
(SICs) or large intestinal contents (LICs) of Gn pigs were used for se-
quencing. TC PoSaV was propagated in LLC-PK cells (TC PoSaV-2010;
cell culture passage level 30) with 50 ␮M glycochenodeoxycholic acid
(GCDCA; Sigma-Aldrich, MO, USA) as previously described (24).
Sequence analyses. The genomes of TC PoSaV-2010 (passage level 30)
and WT PoSaV I-1113 (Gn pig passage level 5) and the VP1 region of WT
PoSaV R418 (Gn pig passage level 13) were sequenced by the primer
walking method based on the TC PoSaV genome (GenBank accession no.
AF182760), as previously described (25). The 5= and 3= ends were deter-
mined by using 5=-rapid amplification of cDNA ends (RACE) and 3=-
RACE methods. Sequence editing and assembly were performed by using
the Lasergene software package (v10; DNASTAR Inc., WI, USA). Multi-
ple-sequence alignment was done with ClustalW using the DNA Data
Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp).
Generation of full-length cDNA clones of PoSaV WT and TC chime-
ric genomes, mutants, and revertant mutant strains. Plasmid pCV4A
containing the full-length cDNA of TC PoSaV (TC PoSaV-2005; cell cul-
ture passage level 27) was provided by Kyeong-Ok Chang (20). The prim-
ers for the generation of these chimeric clones are listed in Table 1. The
1346 jvi.asm.org Journal of Virology
genomic organization and mapping of the mutations are illustrated (Fig.
1). TC-WTVP1 was generated by replacing a partial VP1 fragment (nucle-
otide [nt] positions 5227 to 6060 and amino acid positions 30 to 308) of
pCV4A with the corresponding sequence fragment of the WT PoSaV
Cowden strain. Briefly, the WT PoSaV Cowden VP1 fragment containing
two ApaI restriction enzyme sites (nucleotide positions 5227 to 5232 and
6055 to 6060) was reverse transcribed by using SuperScript III reverse
transcriptase (Life Technologies, NY, USA) and amplified by PCR with
primers ApaI-F and ApaI-R using PrimeStar HS high-fidelity DNA poly-
merase (Clontech Laboratories Inc., CA, USA). The amplicons were di-
gested by the ApaI restriction enzyme and cloned into the ApaI-digested
pCV4A plasmid.
Full-length cDNA clones of TC-WTRdRp, VP1 of the TC strain with
an S-to-C mutation at position 178 (TCVP1-S178C), TCVP1-H289Y,
TCVP1-D291N, TCVP1-R295K, TCVP1-I324M¡M324I, and TCVP1-
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G328E¡E328G were generated based on the pCV4A backbone by using
a QuikChange II XL site-directed mutagenesis kit (Agilent Technologies,
TX, USA) according to the manufacturer’s instructions (Fig. 1). For ex-
ample, TC-WTRdRp was generated when the two RNA-dependent RNA
polymerase (RdRp) amino acid residues at positions 1252 and 1379 of
pCV4A were mutated from the TC to the WT residues (H1252Y and
K1379R). Three full-length cDNA clones of chimeric genomes (TC-
WTVP1-C178S, TC-WTVP1-Y289H, and TC-WTVP1-C178S&Y289H)
were generated based on the TC-WTVP1 backbone, whose residues at
amino acid positions 324 and 328 in VP1 were of the TC type. Full-length
cDNA clones of TCVP1-I324M and TCVP1-G328E were generated by di-
gestion of pCV4A with the EcoRI restriction enzyme (nucleotide posi-
tions 4582 to 6877) and replacement with I324M or G328E engineered
PCR products. Using TCVP1-I324M as an example, two fragments were
PCR amplified with primers EcoRI-F and 6111-R and with primers
6111-F and EcoRI-R, respectively, using pCV4A as the template. The PCR
product containing the I324M mutation was assembled by overlap PCR
with primers EcoRI-F and EcoRI-R, using the two fragments as the tem-
plates. The overlap PCR products containing the I324M mutation were
digested with the EcoRI restriction enzyme and inserted into EcoRI re-
striction enzyme-digested pCV4A. The recombinant plasmid was trans-
formed and amplified in competent Escherichia coli 10-beta cells (New
England BioLabs Inc., MA, USA).
In vitro transcription and capping of viral genomic RNA. In vitro
transcription and capping of viral genomic RNA were performed accord-
ing to the manufacturer’s instructions. Briefly, the reverse-genetics plas-
mid DNA was extracted from E. coli, linearized by NotI restriction enzyme
digestion, and purified by phenol-chloroform extraction twice. Subse-
quently, genomic RNA was transcribed in vitro from the linearized plas-
mid by using a MEGAscript T7 transcription kit (Life Technologies, NY,
USA). The reaction mixture was treated with DNase, and the RNA was
purified with an RNeasy minikit (Qiagen, CA, USA) and analyzed by
agarose gel (1%) electrophoresis under denaturing conditions with form-
aldehyde. The transcribed RNA was capped by using the ScriptCap m7G
capping system (Cellscript, WI, USA), followed by RNA purification us-
ing the RNeasy minikit. The RNA transcripts were suspended in RNase-
free water to a final concentration of 500 ng/␮l for transfection.
Transfection of BHK-T7 cells or HEK 293T/17 cells to rescue infec-
tious viruses. The purified RNA transcripts were transfected into 1-day-
old HEK 293T/17 or BHK-T7 cells (⬃50 to 70% confluent) in 24-well cell
culture plates with Lipofectamine 2000 (Invitrogen, NY, USA). Briefly,
1-day-old HEK 293T/17 or BHK-T7 cells were washed with Opti-MEM I
(Invitrogen, NY, USA). Approximately 1.5 ␮g capped RNA and 4 ␮l Li-
pofectamine 2000 were diluted in 50 ␮l Opti-MEM I separately and incu-
bated at room temperature for 5 min. The RNA and the Lipofectamine
2000 solution were then mixed (total volume of 100 ␮l) and incubated at
room temperature for 20 min before being added to HEK 293T/17 or
BHK-T7 cell monolayers. After 6 h of incubation at 37°C, the supernatant
was replaced with DMEM (10% FBS, 1% NEAA, and 1% antibiotic-anti-
mycotic). After 1 day of incubation at 37°C, HEK 293T/17 or BHK-T7 cell
February 2016 Volume 90 Number 3
 Mutations in VP1 Make Porcine Sapovirus Grow In Vitro

TABLE 1 Primers for generation of chimeric PoSaV clones

Primer Sequencea Location in PEC genome (positions) Orientation
ApaI-F 5=-GAGTCCAGACCAGTCCAGCCAGC 5203–5225 Forward
ApaI-R 5=-TGGGTAGTGGTTGATGATGTTG 6090–6069 Reverse
TC-3763-CTF 5=-CGTGAATGACCCAAGGTACCCCTTCTCACAACA 3747–3779 Forward
TC-3763-CTR 5=-TGTTGTGAGAAGGGGTACCTTGGGTCATTCACG 3779–3747 Reverse
TC-4145-AGF 5=-AGAAAAGAATGACCAAGGCAAAAGACGCCTGCTGTG 4122–4157 Forward
TC-4145-AGR 5=-CACAGCAGGCGTCTTTTGCCTTGGTCATTCTTTTCT 4157–4122 Reverse
TC-5671-ATF 5=-TTGGTGGGGCTATAGCATGTTTGGCACTTTACGTG 5654–5688 Forward
TC-5671-ATR 5=-CACGTAAAGTGCCAAACATGCTATAGCCCCACCAA 5688–5654 Reverse
TC-6004-CTF 5=-CCCGTGTCAATGGAAAGTACACTGACAACACAGGT 5987–6021 Forward
TC-6004-CTR 5=-ACCTGTGTTGTCAGTGTACTTTCCATTGACACGGG 6021–5987 Reverse
TC-6010-GAF 5=-CCCGTGTCAATGGAAAGCACACTAACAACACAGGTA 5987–6022 Forward
TC-6010-GAR 5=-TACCTGTGTTGTTAGTGTGCTTTCCATTGACACGGG 6022–5987 Reverse

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TC-6023-GAF 5=-AGCACACTGACAACACAGGTAAGGCAGTGTTTCA 6002–6035 Forward
TC-6023-GAR 5=-TGAAACACTGCCTTACCTGTGTTGTCAGTGTGCT 6035–6002 Reverse
WT-5671-TAF 5=-TTGGTGGGGCTATAGCAAGTTTGGCACTTTACGTG 5654–5688 Forward
WT-5671-TAR 5=-CACGTAAAGTGCCAAACTTGCTATAGCCCCACCAA 5688–5654 Reverse
WT-6004-TCF 5=-CCCGTGTCAATGGAAAGCACACTAACAACACAGGT 5987–6021 Forward
WT-6004-TCR 5=-ACCTGTGTTGTTAGTGTGCTTTCCATTGACACGGG 6021–5987 Reverse
EcoRI-F 5=-GAGGCCTACGAGGAATTCAAG 4570–4590 Forward
EcoRI-R 5=-GAGCCTGATTAAAAGAATTCATAATA 6891–6866 Reverse
6111-F 5=-CAACAATGTTCAACACAGGAAC 6104–6125 Forward
6111-R 5=-GTTGAACATTGTTGATGCAGC 6117–6097 Reverse
6122-F 5=-CAACACAGAAACTGCCGTAAATG 6114–6138 Forward
6122-R 5=-GGCAGTTTCTGTGTTGAATATTG 6129–6107 Reverse
6111-6122-F 5=-CAATGTTCAACACAGAAACTGCC 6107–6129 Forward
6111-6122-R 5=-CAGTTTCTGTGTTGAACATTGTTG 6127–6104 Reverse
QCback-F 5=-GGCTGCATCAACAATATTCAACACAGGAACTGCC 6096–6129 Forward
QCback-R 5=-GGCAGTTCCTGTGTTGAATATTGTTGATGCAGCC 6129–6096 Reverse
a
Boldface and underlined nucleotides are the mutation sites; underlined sequences are EcoRI digestion sites.

lysates were harvested by freezing and thawing once, followed by centrif-
ugation at 2,095 ⫻ g for 5 min to remove cell debris. The cell lysates were
used to inoculate LLC-PK cells to generate virus pools.
Recovery of progeny virus in LLC-PK cells. The LLC-PK cell mono-
layers in 6-well plates were washed with minimum essential medium
(MEM) and inoculated with HEK 293T/17 or BHK-T7 cell lysates in the
presence of 50 ␮M GCDCA. Cytopathic effects (CPEs) were monitored
daily. Infected cells were incubated for up to 6 days postinoculation, be-
fore harvesting of the first passage of each mutant. Each mutation was
confirmed by reverse transcription-PCR (RT-PCR) with primer sets cov-
ering the mutated region, followed by sequence analysis.
Plaque assays. Tenfold serially diluted samples were inoculated into
wells of 6-well cell culture plates. After incubation at 37°C for 1.5 h with
rocking, the inoculum was removed, and the cell monolayer was overlaid
with 0.85% low-melting-temperature agarose (Sigma-Aldrich, MO, USA)
in MEM supplemented with 50 ␮M GCDCA (20). After plaques formed
(5 days postinoculation), cell monolayers were stained with 1 ml of a
0.03% neutral red–phosphate-buffered saline (PBS) solution for 30 min at
37°C. The solution was removed, and the plaques were counted and ob-
served under a microscope. The plaque sizes were quantified by using the
Icy bioimage program (26).
Growth kinetics test for progeny mutants in LLC-PK cells. A growth
kinetics curve for each mutant was determined by collecting cell lysates at
different postinoculation time points. LLC-PK cells in 6-well plates were
incubated with each mutant virus at a multiplicity of infection (MOI) of
0.01 for 1 h. The inoculum was removed, and the plate was washed once
before the addition of maintenance MEM in the presence of 50 ␮M
GCDCA. Supernatants and cell lysates were collected at 24, 48, 72, and 96
h postinoculation (hpi) after three cycles of freezing and thawing. Virus
infectivity titers were determined in LLC-PK cells as the 50% tissue cul-
ture infectious dose (TCID50) by immunohistochemistry (IHC) staining
using 96-well plates, as described below (23).
IF and IHC staining for detection of VP1 proteins in cell culture.
Immunofluorescence (IF) staining was performed for the detection of
VP1 protein in mutant-infected cells (20), while IHC staining was per-
formed for virus infectivity titration (23). Briefly, cell monolayers were
fixed with 10% neutral formalin buffer at room temperature for 30 min,
and the fixed cells were then permeabilized with 1% Triton X-100 in PBS
at room temperature for 10 min. Gn pig hyperimmune serum to the WT
PoSaV Cowden strain was used as a primary antibody (18). Fluorescein
isothiocyanate (FITC)-conjugated goat anti-swine IgG(H⫹L) serum
(KPL, MD, USA) or horseradish peroxidase (HRP)-conjugated goat anti-
swine IgG(H⫹L) serum (KPL, MD, USA) was used as a secondary anti-
body. The IF signal was observed by using an IX70 fluorescence micro-
scope (Olympus, PA, USA). For IHC, cells were stained with the substrate
3-amino-9-ethylcarbazole (AEC) (Sigma-Aldrich, MO, USA) at room
temperature for at least 2 h and observed by using light microscopy.
Three-dimensional structural analyses of the VP1 proteins of WT
and TC PoSaVs. The three-dimensional (3D) structure of a SaV VP1
protein is not available in the database. The VP1 protein of WT PoSaV
shares higher sequence identity (38%) with that of feline calicivirus (FCV)
(PDB accession no. 3M8L) than with those of San Miguel sea lion virus
(SMSV) (34%) (PDB accession no. 2GH8) and recombinant Norwalk
virus (rNV) (29%) (PDB accession no. 1IHM). When the sequence iden-
tity is 30 to 50%, the obtained model tends to have ⬃90% of the main
chain modeled with a 1.5-Å root mean square error (27). Therefore, the
VP1 dimer structural models of WT and TC PoSaVs were constructed
based on the crystal structure of the FCV VP1 protein at a resolution of
3.40 Å by the homology modeling method using “MOE-Align” and
“MOE-Homology” in the Molecular Operating Environment (MOE)
(version 2014-09; Chemical Computing Group Inc., Quebec, Canada).
Twenty-five intermediate models were obtained from one homology
modeling with the MOE, among which the intermediate models with the
best scores were selected according to the generalized Born (GB)/volume

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FIG 1 Diagrams of constructions of TC and WT PoSaVs and the mutants derived from the pCV4A backbone. The 8 amino acid (aa) residues, at positions 1252
and 1379 in the ORF1 polyprotein and at positions 178, 289, 291, 295, 324, and 328 in VP1, that differed between the WT (red) and TC (green) PoSaVs are
indicated in boldface and italic type for each mutant. The TCVP1-D291N and TCVP1-R295K mutants were tested in Gn pigs with WT and TC strains as controls.
The TC-WTVP1-C178S&Y289H mutant was not tested (NT) due to the 2-log10-lower virus infectivity titer that could not be equalized by further concentration.
Replication of the mutants in cell culture or in Gn pigs is noted.

1348 jvi.asm.org Journal of Virology February 2016 Volume 90 Number 3

 Mutations in VP1 Make Porcine Sapovirus Grow In Vitro

TABLE 2 Experimental design for inoculation of Gn pigs with WT or TC PoSaV Cowden or mutants
No. of Gn pigs euthanized at acute
Groupa Oral inoculum No. of Gn pigs Titer (log10 GE/ml in MEM [5 ml/pig])b infection phase (PIDs 5–7)
1 TC Cowden strain 9 11.5 3
2 TCVP1-R295K mutant 7 11.5 2
3 TCVP1-D291N mutant 9 11.5 3
4 WT Cowden strain PS799 7 11.5 3
5 MEM 4 2
a
All pigs were 4 to 7 days of age at inoculation.
b
A RT-qPCR titer of 11.5 log10 GE/ml is equivalent to ⬃7.0 log10 TCID50/ml by an infectivity assay.

integral (VI) scoring function. The final 3D models were thermodynam- Gn pigs and experimental design. Gn pigs were derived and main-
ically simulated by energy minimization using the AMBER10 extended tained as previously described (18, 32). A total of 36 Gn pigs were assigned

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Huckel theory (EHT) force field combined with the GB model of aqueous
solvation implemented in the MOE (28–30). Physically unacceptable local
structures of the optimized 3D models were further refined on the basis of
evaluation using the Ramachandran plot in the MOE. The structures of
WT PoSaV VP1 dimers were generated from the monomeric structures by
the MOE on the basis of the assembly information for the FCV VP1 crystal
structures. The quality of the models was assessed by using the 3D struc-
ture evaluation program Verify3D (31).
Prediction of effects of point mutations on stability of PoSaV. The
change in the stability of the WT PoSaV VP1 protein by each mutation was
analyzed by using the Protein Design application in the MOE (version
2014-09). The structure of WT PoSaV VP1 was constructed as described
above. The single point mutations in VP1 were generated individually,
and ensembles of protein conformations were generated by using the
LowMode molecular dynamics (MD) module with Boltzmann distribu-
tion in the MOE to calculate average stability. The stability scores in the
structures refined by energy minimization were obtained by using the
stability scoring function of the Protein Design application in the MOE.
to five groups (Table 2) and inoculated orally with (i) TC PoSaV (cell
culture passage level 30) (n ⫽ 9), (ii) TCVP1-R295K (n ⫽ 7), (iii) TCVP1-
D291N (n ⫽ 9), (iv) WT PoSaV (PS799) (Gn pig passage level 13) (n ⫽ 7),
or (v) MEM (n ⫽ 4). TC PoSaV and the TCVP1-D291N and TCVP1-R295K
mutants were harvested from cell culture and concentrated to ⬃7.0 log10
TCID50/ml (equivalent to a real-time quantitative RT-PCR [RT-qPCR]
titer of ⬃11.5 log10 genome equivalents [GE]/ml) by ultracentrifugation
at 126,000 ⫻ g for 1.5 h at 4°C. The WT PoSaV Cowden strain inoculated
into Gn pigs was filtered through 0.22-␮m-pore-size filters prior to inoc-
ulation. Each pig was inoculated orally with a 5-ml inoculum containing
TC PoSaV or mutant PoSaVs (⬃7.0 log10 TCID50/ml) or containing WT
PoSaV with a RT-qPCR titer of ⬃11.5 log10 GE/ml.
All inoculated Gn pigs were observed daily for clinical signs. Their
feces were scored as 0 for normal, 1 for pasty, 2 for semiliquid, and 3 for
liquid, with fecal scores of ⱖ2 indicating diarrhea (18). Rectal swabs (RSs)
were collected daily for titration of virus shedding. Blood was collected for
serum from each Gn pig before inoculation; at postinoculation days
(PIDs) 1, 3, 6, 9, 16, 23, and 27; and at euthanasia. Two to three Gn pigs of

TABLE 3 Summary of amino acid substitutions in the genomes from different passages of the WT or TC PoSaV Cowden strain
WT-PoSaV sequence in Gn pigs at passage: TC-PoSaV sequence in LLC-PK cells at passage:
Amino acid Nucleotide
Gene positionc position 5 b a
5 (I-1113 ) 13 (R418 ) a a
13 (PS499 ) 20b 27 30
NS1 17 59 TTT TTT TTT TTT TTT TCT (S17F) TTT
18 62 GAC GGC (G18D) GGC GGC GAC GGC GGC
24 80 CCA CCA CCA CCA CCA CCA CTA (L24P)
29 94 GCG GCG GCG GCG GCG GCG ACG (T29A)

NS3 356 1075 ATT ATT ATT ATT ATT GTT (V356I) ATT
367 1109 AAG AAG AAG AAG AAG AGG (R367K) AAG

NS4 733 2207 GGC GAC/GGC GGC GGC GGC (D733G) GGC GGC

NS7 1252 3763 TAC TAC TAC TAC CAC (Y1252H) CAC CAC
1379 4145 AGA AGA AGA AGA AAA (R1379K) AAA AAA
1392 4185 ATA ATA ATG/ATA ATA ATA(M1392I) ATA ATA

VP1 75 5362 ACA ACA ACA ACA ACA ACA GCA (A1785T)
178 5671 TGT TGT TGT TGT AGT (C178S) AGT AGT
289 6004 TAC TAC TAC TAC CAC (Y289H) CAC CAC
291 6010 AAC AAC AAC AAC GAC (N291D) GAC GAC
295 6023 AAG AAG AAG AAG AGG (K295R) AGG AGG
324 6111 ATA ATG ATG ATG ATA (M324I) ATA ATA
328 6122 GGA GAA GAA GAA GGA (E328G) GGA GGA

ORF2 27 6851 CAT CAT CAT CAT CAT CAT CAA (E27H)
35 6873 AAT AAT AAT AAT AAT AAT GAT (D35N)
a
Sample identification based on the labeling system in the laboratory.
b
See reference 25.
c
Boldface numbers refer to consensus amino acid substitutions among different passages of TC compared with WT PoSaV.

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FIG 2 Growth kinetics and representative plaques of TC-pCV4A (TC) and the culturable mutants TC-WTRdRp, TCVP1-D291N (291), TCVP1-R295K (295), and
TC-WTVP1-C178S&Y289H (Double) in LLC-PK cells. (A) The plaque sizes of the TCVP1-D291N, TCVP1-R295K, and TC-WTVP1-C178S&Y289H mutants are
smaller than those of TC-pCV4Aand TC-WTRdRp in LLC-PK cells. (B) LLC-PK cells were inoculated with each virus at a MOI of 0.01. Cell lysates were collected
at 24, 48, 72, and 96 hpi for titration of infectious virus. TC-pCV4A and TC-WTRdRp replicated in LLC-PK cells to significantly higher titers (72 hpi) than did the
culturable mutants (P ⬍ 0.05 by one-way ANOVA followed by Duncan’s multiple-range test on log10-transformed titers).

each group were euthanized at the acute phase of infection: on the next
day after an increase of the fecal viral RNA titer or on the day following the
onset of clinical signs. The remaining pigs were euthanized after fecal virus
shedding was no longer detected at the termination of the experiment.
Animal care and use for these studies were approved by the Institutional
Animal Care and Use Committee (IACUC) at The Ohio State University.
Mucosal antibody samples were collected at necropsy by scraping the
mucosa from the ileum and centrifuging the sample at 2,095 ⫻ g for 20
min at 4°C.
Histopathological examination. At necropsy, blood, SICs, and LICs
were collected from each Gn pig. Fresh duodenum; proximal, mid-, and
distal jejunum; ileum; colon; cecum; liver; spleen; lung; and kidney spec-
imens were collected and immersed immediately in 10% neutral buffered
formalin (NBF). Tissues fixed in 10% NBF were trimmed, embedded in
paraffin, sectioned at 4 ␮m, stained with Harris’ hematoxylin and alco-
holic eosin Y solution (H&E; Sigma-Aldrich, MO, USA), and examined
for histopathology microscopically.
Duodenum; proximal, mid-, and distal jejunum; ileum; colon; and
cecum specimens were collected in duplicate, immersed in a sucrose so-
lution (130 mM Na2HPO4, 30 mM KH2PO4, 10% [wt/vol] sucrose, and
0.01% sodium azide [pH 7.2]) on ice, embedded in a optimum-cutting-
temperature (OCT) compound (Sakura, PA, USA), stored at ⫺20°C over-
night, and then sectioned at 4 to 7 ␮m in a cryostat microtome. To detect
the PoSaV antigen in tissues, frozen sections were fixed with acetone for 20
min at ⫺20°C, followed by washing with PBS twice for 5 min each. Tissue
sections were then blocked with 5% normal goat serum in 0.01 M PBS–
0.05% Tween 20 (PBST) (pH 7.2) for 20 min. After blocking, the tissue
sections were incubated with hyperimmune guinea pig serum to PoSaV
virus-like particles (VLPs) (33) at 4°C overnight, followed by incubation
with Alexa Fluor 488-conjugated goat anti-guinea pig IgG(H⫹L) serum
(Life Technologies, NY, USA) at room temperature for 1 h. Thereafter,
tissue sections were counterstained with 4=,6-diamidino-2-phenylindole
(DAPI) for nuclei and examined by using fluorescence microscopy.
Detection of PoSaV RNA in rectal swabs. RSs were collected, sus-
pended in 4 ml of MEM, and centrifuged at 2,095 ⫻ g for 30 min. The
supernatant was collected as a 10% fecal suspension and stored at ⫺20°C
until RNA extraction. Total RNA was extracted from 50-␮l RS suspen-
sions by using a MagMax RNA extraction kit (Life Technologies, NY,
USA) according to the manufacturer’s instructions. The virus titer was
determined by using One-Step TaqMan SaV-specific RT-qPCR as de-
scribed previously (23).
Detection of PoSaV-specific antibodies in serum and mucosal sam-
ples by an enzyme-linked immunosorbent assay (ELISA). A recombi-
nant baculovirus expressing PoSaV VP1 was generated as previously de-
scribed and used to infect Sf9 cells for VLP production (33). Briefly,
purified PoSaV VLPs were used as antigens to coat Nunc 96-well plates
(MaxSorp surface; Thermo Scientific, MA, USA) at 4°C overnight at a
final concentration of 2 ␮g/ml (100 ng/well) in 0.05 M carbonate buffer
(pH 9.6). The plates were blocked with 2% nonfat dry milk (NFDM) in
PBST at 37°C for 1 h. After being washed three times with PBST, serum
samples were 4-fold serially diluted in PBST containing 2% NFDM and
added to the wells. The plates were incubated at 37°C for 1 h and washed
with PBST three times. HRP-conjugated goat anti-swine IgG(H⫹L) se-
rum (KPL, MD, USA) diluted 1:3,000 or HRP-conjugated goat anti-swine
IgA serum (AbD Serotec, NC, USA) diluted 1:5,000 in PBST containing
2% NFDM was added to wells, followed by incubation at 37°C for 1 h.

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FIG 3 Superimposition of the modeled structures of PoSaV Cowden and the template FCV structure and the 3D models of WT and TC PoSaV VP1s. (A) Superim-
position of the modeled structures (WT PoSaV Cowden on the left and TC PoSaV Cowden on the right) and the template structure (FCV VP1 [PDB accession no.
3M8L]) by homology modeling. The blue ribbon structure denotes WT PoSaV Cowden VP1, the cyan ribbon structure denotes TC PoSaV Cowden VP1, and the
magenta ribbon structure denotes the template structure of FCV VP1. (B) Amino acid residue 178 was located at the dimer-dimer interface in the S domain; residues 289,
291, 324, and 328 were located in the P2 region; and residue 295 was located on the interface of two monomeric VP1 proteins in the P2 region.

After the plates were washed three times with PBST, the substrate
3,3=,5,5=-tetramethylbenzidine (TMB; KPL, MD, USA) was added to each
well for color development at room temperature. An equal volume of 1 M
phosphoric acid was added to terminate the reactions after 5 min of incu-
bation at room temperature. The absorbance at 450 nm was measured by
using a spectrometer (SpectraMax 430 PC; Molecular Devices LLC, CA,
USA). The antibody titer was determined as the reciprocal of the highest
serum dilution with an absorbance value greater than or equal to the mean
absorbance of a series of negative-control serum samples plus 3 times the
standard deviation (SD) of the negative controls (33).
Virus neutralization test. Serum samples were tested for virus neu-
tralization (VN) antibodies to PoSaV by a 50% cell culture infectivity
reduction test. A total of 100 TCID50/well of TC PoSaV was incubated
with an equivalent volume of 4-fold serially diluted serum samples at 37°C
for 1 h before application to cell monolayers. Quadruplicate wells were
used for each serum dilution. Nonneutralized PoSaV was detected on
LLC-PK cells by IHC staining as described above. The VN antibody titers
were determined by using the Reed-Muench method (34) and expressed
as the reciprocal of the highest serum dilution that inhibited PoSaV infec-
tion in 50% of wells.
Statistical analysis. One-way analysis of variance (ANOVA) fol-
lowed by Duncan’s multiple-range test was used to assess differences
in plaque sizes, mean durations of virus shedding, and log-trans-
formed titers (including antibody titer, VN antibody titer, and viral
RNA titer) among groups. One-way ANOVA was used to assess ratios
of villus height to crypt depth (VH/CD ratios) and the mean numbers
of antigen-positive cells per villus. A significance level of a P value of
⬍0.05 was used for all comparisons.
Nucleotide sequence accession numbers. The genomes of WT PoSaV
I-1113 (Gn pig passage level 5) and TC PoSaV-2010 (passage level 30)
were deposited in GenBank under accession numbers KT922087 and
KT922088, respectively. The VP1 region of WT PoSaV R418 (Gn pig
passage level 13) was deposited in GenBank under accession number
KT945132.

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Lu et al.
RESULTS
Consistent mutations occur in the RdRp and VP1 regions at dif-
ferent passages of TC PoSaV compared to WT PoSaV. To inves-
tigate which genes were critical for PoSaV adaptation to cell cul-
ture, the genome of TC PoSaV at passage level 30 in LLC-PK cells
was sequenced in this study and compared with those of WT
PoSaV at pig passage levels 5 and 13, TC PoSaV at cell culture
passage level 20 (25), and the infectious clone pCV4A carrying the
cDNA of TC PoSaV-2005 (cell culture passage level 27) (20). Two
and six conserved amino acid mutations were observed in the
RdRp (residues 1252 and 1379) and VP1 of (residues 178, 289,
291, 295, 324, and 328) regions TC PoSaV (Fig. 1 and Table 3),
respectively.
The VP1 region is critical for cell culture adaptation of
PoSaV. To examine whether the RdRp or the VP1 region was
critical for PoSaV adaptation to cells, we engineered two chimeric
genomes (Fig. 1) based on the previously established PoSaV re-
verse-genetics system, pCV4A: (i) TC-WTRdRp, whose amino acid
residues 1252 and 1379 of the polyprotein (RdRp region) were
mutated from the TC to the WT phenotype (H1252Y and
K1379R), and (ii) TC-WTVP1, whose partial VP1 region (nt 5227
to 6060 and amino acids 30 to 308, excluding amino acid residues
324 and 328) was replaced with the corresponding WT fragment.
The capped genomic RNA transcripts were transfected into
BHK-T7 cells. After infection of LLC-PK cells with BHK-T7 cell
lysates, the PoSaV VP1 proteins were detected exclusively by an
immunofluorescence assay (IFA) in LLC-PK cells inoculated with
TC-WTRdRp- but not TC-WTVP1-transfected products. We fur-
ther compared the growth kinetics and plaque sizes of the TC-
WTRdRp virus to those of the TC-pCV4A virus in LLC-PK cells (Fig.
2). TC-WTRdRp had plaque sizes (0.168 ⫾ 0.052 mm2) similar to
those of TC-pCV4A (0.146 ⫾ 0.066 mm2) and had growth kinetics
similar to those of TC-pCV4A in LLC-PK cells, showing increasing
titers between 0 and 72 hpi with similar peak titers (7.1 ⫾ 0.2 log10
TCID50/ml for TC-pCV4A and 7.0 ⫾ 0.0 log10 TCID50/ml for TC-
WTRdRp). These results suggested that the VP1 region, but not the
RdRp region, was critical for cell culture adaptation of PoSaV.
Four (residues 178, 289, 324, and 328) of the six amino acid
residues in the VP1 region were essential for PoSaV adaptation to
LLC-PK cells. To address which of the individual mutations in the
VP1 region was essential for PoSaV adaptation, each of the sites
was mutated to the WT sequence individually (Fig. 1): TCVP1-
S178C, TCVP1-H289Y, TCVP1-D291N, TCVP1-R295K, TCVP1-
I324M, and TCVP1-G328E. The TCVP1-D291N and TCVP1-R295K
mutants replicated in LLC-PK cells. No infectious virus was res-
cued from LLC-PK cells infected with the transfection lysates of
TCVP1-S178C, TCVP1-H289Y, TCVP1-I324M, or TCVP1-G328E in-
fectious clones. Furthermore, when the point mutations of TC-
WTVP1 were mutated back to TC sequences, the double mutant
(TC-WTVP1-C178S&Y289H, carrying TC amino acids at residues
324 and 328), instead of the individual point mutants (TC-
WTVP1-C178S and TC-WTVP1-Y289H), was rescued in LLC-PK
cells. Infectious virus was rescued from infectious clones of the
TCVP1-I324M¡M324I and TCVP1-G328E¡E328G back muta-
tions. These results indicate that the 4 amino acids at residues 178,
289, 324, and 328 in the VP1 region are essential for PoSaV adap-
tation to LLC-PK cells.
Single amino acid substitutions in the VP1 region alter PoSaV
growth kinetics in vitro. To examine whether the amino acid sub-
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FIG 4 Changes in ⌬G for the C178S, Y289H, N291D, K295R, M324I, and
E328G point mutations. Mutations C178S, Y289H, N291D, and E328G de-
creased thermodynamic stability, whereas K295R and M324I compensated for
the changes.
stitutions D291N, R295K, and TC-WTVP1-C178S&Y289H can al-
ter viral replication in LLC-PK cell cultures, the growth kinetics
and plaque sizes of TCVP1-D291N, TCVP1-R295K, and TC-
WTVP1-C178S&Y289H were compared to those of TC-pCV4A in
LLC-PK cells (Fig. 2). The TCVP1-D291N (0.041 ⫾ 0.011 mm2) and
TCVP1-R295K (0.047 ⫾ 0.016 mm2) viruses and especially the TC-
WTVP1-C178S&Y289H (0.018 ⫾ 0.003 mm2) virus formed signif-
icantly smaller (P ⬍ 0.05) plaques than those formed by TC-
pCV4A (0.146 ⫾ 0.066 mm2) and TC-WTRdRp (0.168 ⫾ 0.052
mm2) (Fig. 2A). The infectious titers of TCVP1-D291N, TCVP1-
R295K, and TC-WTVP1-C178S&Y289H increased postinocula-
tion. However, the peak titers of TC-pCV4A (7.1 ⫾ 0.2 log10
TCID50/ml) and TC-WTRdRp (7.0 ⫾ 0.0 log10 TCID50/ml) were
the highest, followed by TCVP1-R295K (6.6 ⫾ 0.0 log10 TCID50/
ml), TCVP1-D291N (6.4 ⫾ 0.0 log10 TCID50/ml), and TC-WTVP1-
C178S&Y289H (4.9 ⫾ 0.1 log10 TCID50/ml) (Fig. 2B). These data
led us to conclude that the D291N and R295K amino acid substi-
tutions in the VP1 region reduced PoSaV replication in LLC-PK
cells.
Comparative structural analyses of the VP1 proteins of TC
and WT PoSaVs predicted the location and potential function of
amino acid residues. The 3D structural models of both the TC
and WT PoSaV Cowden strains matched the template structure
(FCV VP1 [PDB accession no. 3M8L]) (Fig. 3A). Subsequently,
3D structural analysis was performed to examine whether the
residue changes in VP1 between the WT and TC PoSaVs re-
sulted in structural changes. Amino acid residue 178 was lo-
cated in the S domain near the dimer-dimer interface (Fig. 3B).
C178 of the WT was exposed, but S178 of the TC strain was
hidden. Amino acid residues 289, 291, 324, and 328 in PoSaV
were located at the P2 region, while amino acid 295 was located
at the interface of two monomeric VP1 proteins forming a
dimer at P2 (Fig. 3B).
To investigate whether the mutations at residues 178, 289, 291,
295, 324, and 328 influenced the stability of WT PoSaV, we ana-
lyzed the changes in thermodynamic stability by the mutations
using the Protein Design application in the MOE. A positive num-
ber indicated decreased stability, while a negative number indi-
cated increased stability. The changes in ⌬G caused by the C178S,
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FIG 5 Geometric mean titers (GMT) of viral RNA shedding of TCVP1-D291N and TCVP1-R295K compared with those of TC-pCV4A and the WT PoSaV
Cowden strain in Gn pigs on various PIDs. Gn pigs were inoculated with the corresponding virus inoculum. Rectal swabs were collected daily for titration of viral
RNA shedding. The WT PoSaV Cowden strain had the highest peak titer and longest duration of viral RNA shedding among the experimental groups. The TC
PoSaV Cowden strain had the lowest peak titer and shortest duration of shedding among the experimental groups. Peak viral RNA shedding titers and durations
of shedding in TCVP1-D291N- and TCVP1-R295K-inoculated Gn pigs were intermediate between the WT and TC groups.

Y289H, N291D, K295R, M324I, and E328G point mutations were
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 (Fig. 4). The C178S,
Y289H, N291D, and E328G mutations decreased the stability,
whereas K295R and M324I, especially K295R, compensated for
the changes.
Single amino acid substitutions in the VP1 region alter
PoSaV replication in pigs. To investigate whether the amino acid
substitutions from the TC to the WT sequence in VP1 could re-
store the virulence of WT PoSaV in vivo, we performed pathogen-
esis studies in Gn pigs. Among the experimental groups, the WT
PoSaV-inoculated Gn pigs had a significantly longer duration of
viral RNA shedding (30.3 ⫾ 3.8 days) and higher peak viral RNA
titers (10.8 ⫾ 0.4 log10 GE/g) than those of the other three inocu-
lated Gn pig groups. Relative to the mutants, the TCVP1-D291N-
inoculated Gn pigs had a longer duration of viral RNA shedding
(23.2 ⫾ 3.4 days) and a higher peak RNA titer (8.6 ⫾ 0.8 log10
GE/g) than those of the TCVP1-R295K- or TC PoSaV Cowden
strain-inoculated Gn pigs (Fig. 5 and Table 4).
Clinical signs and histopathological lesions are observed ex-
clusively in WT PoSaV-infected Gn pigs. Moderate diarrhea (fe-
cal score of 2) was observed in three of seven (43%) WT PoSaV-
inoculated Gn pigs. Diarrhea developed by PIDs 3 to 12 and
persisted for 2 to 16 days. No diarrhea or other clinical signs were
observed in TC PoSaV Cowden-, TCVP1-D291N-, TCVP1-R295K-,
or mock-inoculated Gn pigs.
Microscopically, histopathological lesions were not observed
in organs from the TC PoSaV-, TCVP1-D291N-, or TCVP1-R295K-
inoculated or control Gn pigs. WT PoSaV-inoculated Gn pigs
euthanized at the acute phase of infection exhibited mild-to-mod-
erate, diffuse, and atrophic enteritis, demonstrating shortened
and blunt villi from the duodenum to the midjejunum of the small
intestine (Fig. 6A to C). Duodenal, proximal, and midjejunal tis-
sues showed moderate, diffuse villous atrophy (Fig. 6A to C). The
VH/CD ratios for duodenal and midjejunal tissues were 3.54 ⫾
1.06 and 3.98 ⫾ 1.84, respectively, significantly lower than those
for control pigs (5.42 ⫾ 2.26 and 5.95 ⫾ 2.25, respectively) (Table
5). Interestingly, the VH/CD ratio for jejunal tissues from the

TABLE 4 Comparative viral RNA shedding parameters for TC, WT, and mutant PoSaV Cowden strains
Groupa No. of Gn pigs Time of onset (PIDs)b Duration (days) (SD)c Peak titer (log10 GE/ml) (SD)c Time of peak titer (PIDs)d
a a
TC 6 1–4 19.8 (2.6) 7.7 (0.4) 3–14
295 5 1–4 20.8 (3.4)a 7.9 (1.0)a 5–15
291 6 1–6 23.2 (3.4)b 8.6 (0.8)b 3–19
WT 4 1–3 30.3 (3.8)c 10.8 (0.4)c 6–10
NC 2 NAe NA NA NA
a
TC, 295, 291, WT, and NC refer to the TC PoSaV Cowden strain, TCVP1-R295K, TCVP1-D291N, the WT PoSaV Cowden strain, and the negative control, respectively.
b
The onset of RNA shedding refers to the PIDs when fecal viral RNA was first detected by RT-qPCR.
c
Superscript letters denote significant differences among the groups (determined by one-way ANOVA followed by Duncan’s multiple-range test).
d
Time of peak titer refers to PIDs that have the highest viral RNA titers by RT-qPCR.
e
NA, not applicable.

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FIG 6 Histopathological examination of small intestinal samples from WT PoSaV Cowden- or mock (negative-control [NC])-infected Gn pigs. Shown are H&E
staining (left) and IF staining (right) of samples collected from different regions of the small intestine. (A to C) Samples from WT PoSaV Cowden-inoculated Gn
pigs at PID 5, showing fusion, shortening (arrows), or blunting (arrowhead) of villi in the duodenum (A), proximal jejunum (B), and midjejunum (C). (D to F)
Samples from mock-inoculated Gn pigs at PID 5, showing normal villi in the duodenum (D), proximal jejunum (E), and midjejunum (F). Intestinal samples were
collected in duplicate for IF staining and for H&E staining. Bar, 50 ␮m.

TCVP1-D291N-inoculated Gn pigs was statistically lower than titrated (Fig. 8A to C). From PIDs 6 to 27, Gn pigs inoculated with
those for the TCVP1-R295K- and TC PoSaV Cowden-inoculated the WT had the highest IgG, IgA, and VN serum antibody titers.
and control Gn pigs (Table 5), indicating more severe villous at- Gn pigs inoculated with TCVP1-D291N had higher serum IgG,
rophy in the jejuna of TCVP1-D291N-inoculated Gn pigs than in IgA, and VN antibody titers than did those inoculated with TC or
TCVP1-R295K- and TC PoSaV-inoculated and control Gn pigs. TCVP1-R295K mutant PoSaV at PIDs 6 to 27. Gn pigs inoculated
Virus VP1 antigens were detected in the frozen tissues of WT with the WT also had the highest IgA mucosal antibody titers at
PoSaV-inoculated Gn pigs by IF staining. Most antigen-positive PID 27 (Fig. 8D). Gn pigs inoculated with TCVP1-D291N and TC
cells were distributed in the mature enterocytes lining the intesti- PoSaV had significantly higher IgA mucosal antibody titers than
nal villi of the midjejunum and, to a lesser extent, in the duode- did those inoculated with TCVP1-R295K (P ⬍ 0.05 by one-way
num and proximal jejunum (Fig. 6). Antigen-positive cells were ANOVA followed by Duncan’s multiple-range test on log4-trans-
rarely detected in the distal jejunum and ileum. The mean num- formed titers).
bers of antigen-positive cells per villus differed significantly
among different regions of the small intestine, with most of the DISCUSSION
positive epithelial cells being observed in the duodenum to midje- The genomes of the WT (Gn pig passage level 5) and TC PoSaV
junum, with an increasing trend from duodenum (1.18 ⫾ 0.55 Cowden strains (cell culture passage level 20) were reported in
cells) to proximal jejunum (2.45 ⫾ 0.42 cells) and to midjejunum 1999 (25). It was also reported that the WT PoSaV Cowden strain
(3.78 ⫾ 1.30 cells) (Fig. 7). Antigen-positive cells were not ob- grew in porcine primary kidney cell culture after 13 passages in Gn
served in any tissue sections from TC PoSaV-, TCVP1-D291N-, or pigs but only with mock Gn pig intestinal contents in medium
TCVP1-R295K-inoculated Gn pigs. (14). Therefore, we selected one Gn pig intestinal sample from
IgG, IgA, and VN antibody titers in pig serum samples were both the 5th and the 13th passages of WT PoSaV for genomic
sequence analysis. Besides the reported 6 amino acid substitutions
between WT and TC PoSaVs (25), we identified two additional
TABLE 5 Ratios of villus length to crypt depth in different regions of substitutions at amino acid residues 324 and 328 of VP1. We com-
small intestines of Gn pigs pared seven genomes from different passages of the WT and TC
Avg VL/CD ratio (SD)b PoSaV Cowden strains (Table 3). Compared to the WT PoSaV
Cowden strain, 8 of 19 amino acid mutations were conserved
Groupa Duodenum Jejunum Ileum
among all TC PoSaV genomes. The differences between the re-
TC 5.21 (0.86)a 5.93 (1.60)a 4.68 (0.52) ported WT PoSaV Cowden genome and our results are likely due
295 5.97 (1.15)a 6.44 (0.78)a 5.26 (0.66) to the intestinal content samples from different pigs and the dif-
291 5.32 (0.94)a 5.09 (1.47)b 5.20 (0.76)
ferent passage levels tested. Because the two newly identified sites
WT 3.54 (1.06)b 3.98 (1.84)c 5.23 (1.20)
NC 5.42 (2.26)a 5.95 (2.25)a 6.57 (0.33)
(residues 324 and 328 in the VP1 region) were conserved in the
a
newly sequenced genomes from the 5th and 13th Gn pig passages
TC, 295, 291, WT, and NC refer to the TC PoSaV Cowden strain, TCVP1-R295K,
TCVP1-D291N, the WT PoSaV Cowden strain, and the negative control, respectively.
of WT PoSaV, both sites were also included in this study. The
b
Superscript letters denote significant differences among the groups not sharing the establishment of the reverse-genetics system pCV4A for the
same letter (determined by one-way ANOVA). PoSaV Cowden strain provided an important tool to study

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FIG 7 Distribution of antigen-positive cells per villus in different regions of the small intestine of WT PoSaV-inoculated pigs. Mean numbers of antigen-positive
cells per villus were significantly different among the duodenum, proximal jejunum, and midjejunum of the small intestine (P ⬍ 0.05 by one-way ANOVA).

the molecular mechanisms of PoSaV cell culture adaptation (20).
In this study, we generated a series of PoSaV mutants to test which
of the 8 amino acids were critical for cell culture adaptation. In
previous reports, a one-step in vitro transcription-and-capping
procedure was used to generate infectious PoSaV genomic RNA
for transfection (20). Infectious PoSaV Cowden virions were suc-
cessfully rescued from LLC-PK cells by using the one-step method
(20, 35). However, for unknown reasons, we failed to rescue in-
fectious virus. Because infectious murine norovirus (MNV) was
rescued from RAW 264.7 cells by using two-step in vitro transcrip-
tion followed by capping (36), we tried the two-step method and
rescued infectious PoSaV from LLC-PK cells. The genome-linked

FIG 8 Serum and mucosal antibody responses in Gn pigs. (A) Geometric mean titers (GMT) of IgG antibodies to the corresponding PoSaV strains or mock (NC)
in serum samples from Gn pigs. (B) Geometric mean titers of IgA antibodies to the corresponding PoSaV strains or mock infection in serum samples of Gn pigs.
(C) Geometric mean titers of virus-neutralizing antibodies to the corresponding PoSaV strains or mock in serum samples of Gn pigs. (D) Geometric mean titers
of IgA antibodies to the corresponding PoSaV strains in ileal mucosal samples from Gn pigs. Serum samples were collected from each Gn pig before inoculation
and at PIDs 1, 3, 6, 9, 16, 23, and 27. Ileal mucosal samples were collected at PID 5 and PID 27. Data points marked with different letters at each day differed
significantly (P ⬍ 0.05 by one-way ANOVA followed by Duncan’s multiple-range test on log10-transformed titers).

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Lu et al.
virus protein (VPg) is encoded by the NS5 gene and is covalently
linked to the 5= end of the SaV genomic RNA (20, 37, 38). In our
system, the cap structure analog m7G(5=)ppp(5=)G was added to
the 5= end of the SaV genomic RNA transcripts to simulate SaV
VPg (36, 39).
Among the mutants, TCVP1-S178C, TCVP1-H289Y, TCVP1-
I324M, and TCVP1-G328E could not be recovered in 4 to 5 repeats
of the experiment. Whether virus particles were formed was un-
known. However, HEK 293T/17 cells transfected with in vitro-
transcribed and -capped RNAs of TCVP1-S178C, TCVP1-H289Y,
TCVP1-I324M, and TCVP1-G328E showed several VP1-positive
cells by IHC staining using hyperimmune serum against the VLPs
of the PoSaV Cowden strain (data not shown). This suggests that
the defect may occur at any step post-VP1 expression, such as
virion assembly and binding to the receptors on LLC-PK cells or
entry into LLC-PK cells. TCVP1-D291N, TCVP1-R295K, and TC-
WTVP1-C178S&Y289H were culturable in the LLC-PK cell line,
although the titer of the TC-WTVP1-C178S&Y289H strain was 2
log10 units lower than those of the other culturable strains. In the
in vitro study, both the TCVP1-D291N and TCVP1-R295K mutants
showed reduced replication compared with that of TC PoSaV.
However, in our Gn pig study, both the TCVP1-D291N and TCVP1-
R295K mutants had relatively higher peak viral RNA titers, a lon-
ger duration of fecal viral RNA shedding, and higher-level serum
and mucosal antibody responses than those of TC PoSaV but
lower than those of WT PoSaV. This indicates that the replication
efficiencies of PoSaV mutants were discordant in vitro and in vivo.
In vitro, peak virus infectivity titers (highest to lowest) were TC
PoSaV ⬎ TCVP1-R295K ⬎ TCVP1-D291N ⬎ TC-WTVP1-
C178S&Y289H; in vivo, peak viral RNA titers (highest to lowest)
were WT PoSaV ⬎ TCVP1-D291N ⬎ TCVP1-R295K ⬎ TC PoSaV.
Because the critical mutation sites are all located in VP1, different
receptors were probably used by the PoSaV Cowden strain in vivo
to infect small intestinal epithelial cells in pigs and in vitro to infect
porcine kidney LLC-PK cells.
Three passages of MNV1 in the macrophage cell line RAW
264.7 resulted in a total of 3 amino acid substitutions, which in-
cluded V716I and H845R in the 3A-like protease (NS4) region and
E296K in the P2 region of VP1. Only V716I and E296K were sus-
pected to be related to decreased virulence in mice and increased
titers in RAW 264.7 cells (40, 41). Using reverse genetics, the
K296E but not the I716V back mutation restored the virulence of
the MNV1 revertant in mice (41). Therefore, a single amino acid
substitution in the P2 region of VP1 of a norovirus may affect its
virulence in the host and replication efficacy in cell culture. A
previous study concluded that the TC PoSaV Cowden strain was
attenuated in vivo compared with the WT PoSaV Cowden strain
after oral inoculation of Gn pigs with a TC PoSaV Cowden strain
cell culture supernatant (cell culture passage 20) or a filtrate of the
WT PoSaV Cowden strain (18). In our study, we concentrated the
TC PoSaV Cowden strain, TCVP1-D291N, and TCVP1-R295K,
which were 1-log10-higher doses than those used in the previous
study. However, clinical signs were observed exclusively in the WT
PoSaV-infected pigs but not in the TC PoSaV Cowden strain-,
TCVP1-D291N-, or TCVP1-R295K-infected Gn pigs. These results
indicated that even at a 1-log10-higher inoculation dose, the TC
PoSaV Cowden strain did not cause diarrhea in Gn pigs. Although
the TCVP1-D291N and TCVP1-R295K revertants did not cause di-
arrhea in Gn pigs, they replicated more efficiently and induced
relatively higher-level immune responses than TC PoSaV, sug-
1356 jvi.asm.org Journal of Virology
gesting that the two amino acid substitutions in the P2 subdomain
of VP1 affected PoSaV virulence in the host and could be better
candidates than the TC PoSaV Cowden strain for PoSaV vaccine
development.
The first calicivirus structure was reported in 1994 for a pri-
mate calicivirus (42). Currently, structures of VLPs or virion par-
ticles of caliciviruses have been determined for GI.1 and GII.10
human NoVs (43, 44), GV MNV (45, 46), San Miguel sea lion
virus (47), Tulane virus (48), rabbit hemorrhagic disease virus
(49), and FCV (50). Therefore, structures are available for all clas-
sified genera of the family Caliciviridae, except for the genera Sa-
povirus and Nebovirus. Chen et al. reported that sapovirus showed
more structural similarity to vesivirus (22). The FCV VP1 protein
has the closest phylogenetic relatedness (37% amino acid identity)
Downloaded from http://jvi.asm.org/ on February 18, 2021 by guest
to that of the PoSaV Cowden strain among the existing calicivirus
VP1 structures. To understand the location of the mutation sites
based on structural predictions, we performed 3D structure mod-
eling and analysis of the VP1 protein of the PoSaV Cowden strain
using FCV VP1 as the template. In a previous study, the complete
removal of the P domain of recombinant Norwalk virus-like par-
ticles resulted in the formation of smooth particles, which dem-
onstrated that the S domain is sufficient for assembly of the capsid
(51). Since the P2 region of caliciviruses is the most protruding
part of VP1 and is highly variable, it has been considered respon-
sible for binding to host receptors (52). A recent study indicated
that the ␣2,3- and ␣2,6-linked sialic acids on O-linked glycopro-
teins are receptors on LLC-PK cells for the PoSaV Cowden strain
(53). In our study, based on their locations in the structural
model, amino acid position 178 was located in the S domain,
which may influence VP1 oligomerization, virion assembly, and
stability. Amino acid positions 289, 291, 324, and 328 were located
in the P2 region, which affects binding to the receptors on LLC-PK
cells and may impair virus replication. Amino acid position 295
was located in the P2 region at the interface of two monomeric
VP1 proteins, which may influence VP1 dimerization.
The changes in thermodynamic stability caused by the muta-
tions indicate whether or not the protein structure can be main-
tained. The overall change in thermodynamic stability is the sum
of each estimated value. Decreasing the stability is a disadvantage
to maintain the structure, whereas increasing the stability is an
advantage. Therefore, mutations that decrease stability are critical
for function, e.g., cell culture adaptation, rather than structure.
The mutations that increase stability then play a role in the com-
pensation for decreasing stability. In this study, thermodynamic
stability analysis indicated that C178S, Y289H, N291D, and
E328G decrease stability, while K295R and M324I increase stabil-
ity. These results suggest that C178S, Y289H, N291D, and E328G
would provide essential functions for cell culture adaptation,
whereas K295R and M324I would compensate for the decreased
stability of C178S, Y289H, N291D, and E328G.
We compared 12 WT GIII PoSaV VP1 protein sequences avail-
able in GenBank (http://www.ncbi.nlm.nih.gov/nucleotide/). We
found that C178 in the S domain is conserved among all 12 WT
GIII PoSaVs (WT PoSaV Cowden [GenBank accession no.
KT922087], SaV1-CHN [accession no. ACP43737], HW20-
KOR [accession no. ADN84680], ID3-HUN [accession no.
ABD38714], LL14-US [accession no. AAR37376], MM280-US
[accession no. AAX32888], JJ259-US [accession no. AAX37311],
QW270-US [accession no. AAX37314], PES-VENEZ [accession
no. AAY88248], PoS6-HUN [accession no. ACS68238], PoS9-
February 2016 Volume 90 Number 3

HUN [accession no. ACS68240], and s20-JAP [accession no.
BAE94661]), suggesting that C178S may be the most critical mu-
tation during PoSaV Cowden strain tissue culture adaptation.
In this study, WT PoSaV antigen was observed in epithelial
cells of the Gn pig small intestine from the duodenum to midje-
junum but rarely in the distal jejunum or ileum. We confirmed the
region of PoSaV infection in Gn pigs, as reported previously (18).
Also, as previously reported, morphological alterations in duode-
nal and jejunal villi were observed in WT PoSaV Cowden-infected
Gn pigs (18, 54). IF staining of small and large intestinal impres-
sion smears as well as villus length measurement also confirmed
that the small intestine was the major infection site (18, 54). Our
data support results of previous studies of the pathogenesis of the
WT PoSaV Cowden strain in Gn pigs.
This study demonstrated that cell culture adaptation of the
PoSaV Cowden strain is due to amino acid substitutions in the
VP1 region. The single-revertant mutation (from the TC to
the WT phenotype) at certain positions, positions 291 and 295, in
the VP1 region reduced virus replication in vitro but partially re-
gained PoSaV replication efficiency in vivo. The genetic basis de-
lineated for cell culture adaptation of PoSaV may provide new
critical information for the rescue of other uncultivable PoSaVs
and human SaVs. In future studies, we plan to construct reverse-
genetics systems for selected PoSaV and human SaV strains by
introducing site-directed mutations at structurally corresponding
positions in the VP1 protein to rescue such unculturable SaVs.
ACKNOWLEDGMENTS
We acknowledge Juliette Hanson, Jeff Ogg, Andrew Wright, Ronna
Wood, and Megan Strother for assistance with animal care and Chun-
Ming Lin, Xiaohong Wang, and Susan Sommer-Wagner for technical
assistance.
Zhongyan Lu was supported by a scholarship provided by the China
Scholarship Council. Salaries and research support were provided by state
and federal funds provided to the Ohio Agricultural Research and Devel-
opment Center (OARDC), The Ohio State University.
The funders had no role in study design, data collection and interpre-
tation, or the decision to submit the work for publication.
FUNDING INFORMATION
HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
provided funding to Linda J. Saif under grant numbers R01 AI49742-04
and R21 AI081009-2. HHS | NIH | National Institute of Allergy and In-
fectious Diseases (NIAID) provided funding to Kyeong-Ok Chang under
grant number U01AI08001. China Scholarship Council provided funding
to Zhongyan Lu. HHS | NIH |National Institute of Allergy and Infectious
Diseases (NIAID) provided funding to Qiuhong Wang under grant num-
ber R21 AI081009-2.
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