2015 Molecular epidemiological study of feline coronavirus strains in Japan using RT-PCR targeting nsp14 gene

2015·1.09 MB·English

Tanakaetal.BMCVeterinaryResearch (2015) 11:57
DOI10.1186/s12917-015-0372-2
METHODOLOGY ARTICLE Open Access
Molecular epidemiological study of feline
coronavirus strains in Japan using RT-PCR
targeting nsp14 gene
Yoshikazu Tanaka1*†, Takashi Sasaki1†, Ryo Matsuda1, Yosuke Uematsu2 and Tomohiro Yamaguchi2
Abstract
Background: Feline infectious peritonitis is a fatal disease ofcats causedby infection with feline coronavirus
(FCoV). For detectingor genotyping ofFCoV,some RT-PCR plus nested PCR techniques have been reported
previously. However, referring to thewholegenome sequences (WGSs) registered atNCBI, there are nodetection
methods that can tolerate the genetic diversity among FCoV population. Inaddition, the quasispecies nature
ofFCoV,which consistsof heterogeneous variants, has been also demonstrated; thus, a universal method for
heteropopulations of FCoV variants inclinical specimens is desirable.
Results: Todevelop an RT-PCR method for detectionand genotyping of FCoV, we performed comparative genome
analysis using WGSs of32 FCoV,7 CCoVand 5TGEV strains obtained from NCBI. As the PCR target, wefocused
onthe nsp14 coding region, which is highlyconserved and phylogenetically informative, and developed a PCR
method targeting nsp14 partial sequences. Among 103 ascites, 45 pleural effusion and 214blood specimens from
clinically ill cats, wecould detectFCoV in 55 (53.4%), 14 (31.1%)and 19 (8.9%) specimens using the present method.
Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I- and II-FCoV
serotypes. Our nsp14aminoacid sequence typing (nsp14 aa ST) showed that the FCoV clone with sequence type
(ST)42, which was themost predominantgenotype of WGSstrains,was prevalent indomestic cats in Japan.
Conclusions: Our nsp14 PCR scheme will contribute to virus detection, epidemiology and ecology of FCoV strains.
Keywords: Feline coronavirus,Epidemiology, Phylogenetic study
Background FECVs/FIPVs predominate throughout the world, but
Felinecoronavirus(FCoV)canbeclassifiedintotwobio- type II strains appear to be more adaptable to tissue cul-
types, namely low virulent feline enteric coronavirus ture. However, type I strains are more likely to cause
(FECV) and highly virulent feline infectious peritonitis clinical FIP signs [3]. FCoV is also common in healthy
virus (FIPV) [1]. Clinical appearance of FECV, if any, is cats worldwide; less than 10% of FCoVseropositive cats
characterized by mild enteritis. In contrast, FIPV effi- develop FIP [5-9]. Therefore, measuring antibody levels
ciently replicates in macrophages/monocytes, and can against FCoV is rarely of diagnostic value in FIP. Thus,
lead to FIP, which is a highly lethal systemic granuloma- Histopathological examination of infected tissues is
tous disease [2]. FIPV exists in two serotypes based on needed for theaetiologicaldiagnosisofFIP[10,11].
virus neutralizing antibodies, type I and type II [1,3]. RT-PCR plus nested PCR and real-time PCR tech-
Serotype I virus has a distinctive spike protein, while the niques have allowed the detection or genotyping of
spike protein of serotype II is a recombinant protein be- FCoV [3,12-15]. However, they have not been fully ree-
tween feline and canine enteric coronaviruses [4]. Type I valuated whether they can tolerate the genetic diversity
of FCoV. In recent years, whole genome sequences of
*Correspondence:[email protected] clinical strains of FCoV have been registered in the
†Equalcontributors NCBI database by Rottier et al. at the J. Craig Venter
1DepartmentofVeterinaryHygiene,VeterinarySchool,NipponVeterinary&
Institute, and we have been able to readily obtain nu-
LifeScienceUniversity,1-7-1Kyounan,Musashino,Tokyo180-8602,Japan
Fulllistofauthorinformationisavailableattheendofthearticle cleotide sequences of the FCoV genome. With regard
©2015Tanakaetal.;licenseeBioMedCentral.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
unlessotherwisestated.
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page2of9
to genome information, there have been no reports on pleural fluid, ascites, pericardial effusion and cerebral
PCR-based methods that can tolerate the genetic diver- fluid), and fcwf-4 cells infected with FCoV (79–1146
sity in whole genome sequenced FCoV strains. Thus, a strain) according to the manufacturer’s protocol. Total
detection method for all variants of FCoV is highly RNA was reverse-transcribed using the PrimeScript RT-
desirable. PCR kit (Perfect Real Time; Takara Bio, Shiga, Japan), as
In the present study, in order to construct a universal reportedpreviously[22].
method for FCoV variants, we performed a genome-
wide analysis of FCoV, and developed an RT-PCR
ConstructionofPCRmethodfordetectionand
method for detecting FCoV in clinical specimens.
genotypingofFCoVstrains
Consequently, direct sequencing of PCR products and
In order to construct a PCR method that detects vari-
phylogenetic analysis allowed discrimination between
ants in FCoV strains, primers were designed by mul-
type I and II serotypes of FCoV. Using this method,
tiple alignments of nucleotide sequences of the nsp14
we investigated the population genetics of FCoV
genes in all whole genome-sequenced FCoV, closely
strains from diseased cats in Japan.
related subspecies, CCoV and TGEV strains. The pri-
mer set nsp14-F (5′-GTGATGCTATCATGACTAG-
Methods
3′) and nsp14-R (5′-CACCATTACAACCTTCTAA-
BioinformaticsforFCoV,CCoVandTGEV
3′) was used. The expected size of PCR products was
As shown in Additional file 1, thirty-two FCoV strains,
417 bp. The reaction mixture for PCR consisted of 4
seven canine coronavirus (CCoV) strains, four transmis-
μl of cDNA in a total volume of 25 μl composed of 1
sible gastroenteritis coronavirus (TGEV) strains, a por-
U of Ex Taq (Takara-Bio), 10 pmol of each primer, 0.2
cine respiratory coronavirus (PRCV) strain and a Mink
mM deoxynucleoside triphosphate mixture and 1× re-
coronavirus (MiCoV) strain were used for comparative
action buffer (Takara-Bio). Reaction mixtures were
genome analysis. All sequences were obtained from the
thermally cycled once at 95°C for 2 min; 40 times at
NCBI database. Gene searches and annotation were car-
95°C for 30 s, 48°C for 35 s, and 72°C for 45 s; and
ried out by GeneMarkS (http://opal.biology.gatech.edu/
then once at 72°C for 5 min. Using 6 μl of PCR sam-
genemarks.cgi) [16], RASTannotation server(http://rast.
ple, DNA fragments were analyzed by electrophoresis
nmpdr.org) [17] and the blast-based method. MAFFT
in1×Tris-acetate-EDTAona1%agarosegelstainedwith
v7.037 was used for multiple sequence alignment [18],
ethidium bromide. In addition, these PCR products were
and Aminosan v1.0.2011 was performed for amino-acid
directly sequenced using a Big Dye terminator (version
substitution model selection [19]. Phylogenetic inference
3.1) cycle sequencing kit (Applied Biosystems, Tokyo,
using maximum likelihood (ML) and bootstrapping was
Japan) with an ABI Prism 3100 genetic analyzer (Applied
performedusingMEGAv5.05[20].
Biosystems).
In order to perform a genome-wide comparison
Total RNA was extracted from the fcwf-4 cells in-
among type I- and II-FCoV, CCoVand TGEVstrains, we
fected with FCoV strain 79–1146, and was reverse-
used the Genotyping tool at NCBI (http://www.ncbi.nlm.
transcribed into cDNA. Viral cDNA was quantified
nih.gov/projects/genotyping/formpage.cgi),whichhelps
using a real-time PCR method, as reported previously
identify genotype and recombinant sequences using the
[22]. Using cDNA samples of known copy numbers, we
blast-basedmethod[21].Defaultvalueswereused;300for
evaluated the detection limit of our PCR method target-
“window”and100for“increment”.
ingnsp14.
Cellcultureandvirus
Felis catus whole fetus-4 (fcwf-4; American Type Studypopulationinmolecularepidemiologicalstudyof
Culture Collection,VA, USA) cells were maintained in FCoVstrainsfromclinicallyillcatsinJapan
Dulbecco’s modified Eagle’s medium (D-MEM, Sigma- Intheperiod between2007and2014inJapan,372spec-
Aldrich, Tokyo, Japan) supplemented with 10% fetal imens (103 ascites, 45 pleural effusion, 214 blood, 9
bovine serum (JRH, Nissui,Tokyo, Japan). We purified cerebral fluid and 1 pericardial effusion), which were ob-
FCoV using linear sucrose gradient ultracentrifugation tained in the examination of clinically ill cats for the
from FCoV 79–1146 strains (a gift from Tsutomu presence of FCoV, were used in the present study. To
Hodatsu, Kitasato University, Japan) propagated in detect FCoV in clinical specimens, we performed RT-
fcwf-4 cells. PCR using a random primer plus a single PCR targeted
nsp14 constructed in the present study. To differentiate
RNAextractionandreversetranscription-PCR type I-FCoVfrom type II-FCoV or CCoVgenotypes, dir-
Isogen-LS (Nippon Gene, Toyama, Japan) was used for ect sequencing of their PCR products and phylogenetic
RNA preparation from clinical specimens (whole blood, analysis were carriedout.
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page3of9
In order to determine the significance of differences in belonged, the coding regions from nsp14 to nsp16
detection rate between ascites, pleural effusion and were the most highly conserved (Figure 1).
blood specimens, relative risk and odds ratio were calcu-
lated using MedCalc software (http://www.medcalc.org/ PCRmethodtargetingnsp14fordetectionofFCoV
index.php). strainsfromclinicalspecimens
Based on a genome-wide comparison using the Geno-
typing tool at NCBI, we focused on protein-coding se-
nsp14aminoacidsequencetyping(nsp14aaST)and
quences within nsp14 to nsp16 as a candidate PCR
comparisonofgeneticdiversityinFCoVstrainsbetween
target. The sequence region was suited to universal de-
differentspecimens
tection of variants because of its highly conserved se-
In order to devise a genotyping method for FCoV, we
quence. Among the primer sets designed in nucleotide
assigned an ST number to all distinct nsp14 partial
sequences conserved in all FCoV, CCoV, TGEV and
amino acid sequences (135 aa) found in all FCoVstrains,
PRCV strains, we determined the best primer set,
which were sequenced in the present study and previ-
nsp14-F and nsp14-R, which allowed amplification of
ously registered in the NCBI database. We compared
417 bp of the nsp14 partial sequences. Consequently,
the population genetic structures of FCoV strains in
thisPCRisuniversallyapplicabletoallalpha-coronavirus1
Japan with whole genome-sequenced strains from 6 dif-
subspecies.
ferent countries.
We were able to detect FCoV strains from ascites,
pleural effusion, pericardial effusion and blood samples
using the present method. Few nonspecific bands were
ComparisonofdiversityandevennessindexesofFCoV
found in agarose gel electrophoresis (Figure 2). Specific
strainsbyspecimen
amplification of target sequences against all PCR prod-
The diversity and evenness of ST distribution by spe-
cimen type were calculated using Simpson’s diversity ucts was confirmed by sequencing analysis. To evaluate
index (1-λ) and Pielou’s evenness index (J’) [23,24]. the detection limit, the present method was applied to
cDNA samples of known copy number using PCR. The
These parameters have generally been used for compar-
detectionlimitwas4.59×102copies/mL.
isons of biodiversity between geographically or eco-
logically separated environments. Both values range
Sequence-baseddifferentiationoftypeI-FCoVfromtype
from 0 (no diversity or evenness) to 1 (extreme diver-
II-FCoVorCCoV
sity or evenness).
The nsp14 coding region was located on the recombin-
ation hotspot in type II-FCoV, and sequencing analysis
Results of the region permitted discrimination between type I
Genome-widecomparisonbetweenFCoVstrainsand and II serotypes. The nucleotide identity of the nsp14
closelyrelatedsubspecies,CCoVandTGEV partial sequence among FCoV, CCoV, TGEV and PRCV
In order to compare the genome structures between strains ranged from 89.9 to 100%. By phylogenetic ana-
type I-FCoV and type II-FCoV, and the closely related lysis based on nsp14 partial nucleotide sequences (406
subspecies CCoV, TGEV and PRCV, we carried out a bp), we were able to successfully distinguish the type I-
blast-based genome-wide comparison. After performing FCoV genotype from type II-FCoV, CCoV or TGEV
genotyping, genomic regions of 300 bp in each viral (Figure 3). Unfortunately, our method had no discrim-
strain were color coded according to scores (0 to 300) inating power for differentiating between type II-FCoV
based on nucleotide similarities against FCoV strain andCCoVgenotypes.
UU9 (Figure 1). In the genome structures of all strains
withtype II-FCoVgenotype,eventsoflarge-scalerecom- ComparisonofFCoVdetectionratefromascites,pleural
bination were found in a locus that stretches for about effusionandbloodincats
12,000 bp from the first half of nsp12 to upstream of the Using the PCR and direct-sequencing method targeting
nucleocapsid gene, as reported previously [25,26]. The nsp14 in the present study, we surveyed FCoV strains
recombination site varied slightly from one strain to an- from clinically ill cats on which FIP was suspected but
other. In the phylogenetic tree based on concatenated not confirmed by a complete clinico-pathological or
sequences of nsp13, nsp14, nsp15, nsp16 and spike, pathological workup in veterinary hospitals in Japan. In
which are protein-coding regions located within recom- peritoneal and pleural fluid samples, FCoV was detected
binant sequences, all type II-FCoVstrains were clustered in 55 of 103 (53.4%) and 14 of 45 (31.1%), respectively.
into a clade belonging to canine coronavirus strains AlloftheseexhibitedthetypeI-FCoVgenotype.Inacom-
(data not shown). Among the alpha-coronavirus 1 parison of positivity rate of FCoV between peritoneal and
subspecies, to which FCoV, CCoV, TGEV and PRCV pleural effusion samples, relative risk and odds ratio were
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page4of9
Figure1Genome-widecomparisonofFCoV,CCoV,TGEVandPRCVstrainsregisteredatNCBIdatabase.Genomicregionsofevery300
bpofstrainUU9werecomparedwith43wholegenomesequencedstrainsusingBLASTGenotypingtool[21].Theblastscorewerevisualizedin
ared-yellow-greengradientwithgreenbeingthetopscore(300)andredbeingthebottomscore(0),indicatingnucleotidesimilarityagainstthe
correspondingregioninFCoVstrainUU9.
1.7164 and 2.5372 (95% confidence interval=1.21-5.32, z assigned ST numbers of nsp14 aa ST in whole genome-
statistic=2.465,P=0.0137),respectively,andwefoundsta- sequencedstrainsareshowninAdditionalfile1.
tisticallysignificantdifferencesbetweenthetwo. As shown in Figure 4, our nsp14 aa ST indicated that
On the other hand, positive results on nsp14 PCR the most predominant genotype among domestic cats in
from 214 blood samples, 9 cerebral fluid samples and 1 Japan was ST42 (24 of 89, 27.0%), which was also found
pericardial effusion sample from 224 clinically ill cats, most frequently in whole genome-sequenced strains,
wereseenin19(8.9%),0(0%)and1(100%),respectively. followed by ST8 (n=10), and ST25 (n=7), and the top
Only one of the blood samples exhibited type II-FCoV three STs accounted for 46.1% of total FCoV-positive
orCCoVgenotype. samples. Among ascites specimens in the present study,
ST42 was the most frequent genotype (n=18), followed
PopulationgeneticsofFCoVstrainsinJapanbynsp14 by ST8 (n=5) and ST25 (n=5). Of pleural effusion and
aminoacidsequencetyping(nsp14aaST) blood samples, multiple strains of ST42 (n=4), ST8
Using nsp 14 amino acid sequence typing (nsp14 aa ST), (n=2) and ST8 (n=3), ST25 (n=2), ST42 (n=2) were
which was an allelic analysis based on 135 amino acid identified, respectively. We were unable to find any cor-
residue of nsp14, we identified fifty-three unique STs relationsbetween typesofspecimen andSTs.
among the 89 FCoV-positive samples in this study, and AsshowninTable1,populations ofFCoVstrainsfrom
39wholegenome-sequencedFCoVandCCoVstrainsregis- blood specimens showed high values for both diversity
teredintheNCBIdatabase(Additional file2) [27-29]. The andevennessindexes,whichindicatesanalmostrandom
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page5of9
reportedthatquasispeciescompositioniscorrelatedwith
the seriousness of clinical form and lesions in the organs
[37]. Therefore, detection of a population that consists
of a complex of heterogeneous variants is necessary in
the diagnosis of FCoV infection. Consequently, this may
have resulted in improved sensitivity for FCoV detection
by the present PCR targeting nsp14, which is a highly
conserved region among alpha-coronavirus1 subspecies.
The present method is the most reliable PCR method
thatcan tolerate thegeneticdiversity ofFCoV.
Using the scheme of nsp14 aa ST, we found that a
clone of FCoV, ST42, is prevalent in domestic cats in
Japan. It is likely that the ST42 FCoV clone is also en-
demic worldwide, as it accounts for most of the whole
genome-sequenced strains registered at NCBI. The glo-
bal distribution of ST8 and ST25 FCoV clones, which
Figure2Agarosegelelectrophoresisofnsp14PCR are second and third most common STs after ST42 in
amplificationproductsfromclinicalspecimens.Lane1; Japan, remains to be clarified. Further epidemiological
pleuralfluid,lane2;ascites,lane3;pericardialeffusion,lane4; study isthusneeded.
FCoVpositivewholebloodbyaqPCRmethod,lane5;FCoV
Aggregated distribution of specific STs in FCoVstrains
negativewholebloodbyaqPCRmethod,lane6;fcwf-4cells
infectedwithFCoVstrain79–1146.M.W.M.(molecularweight from ascites was in contrast to that of blood specimens
marker;100bpladder)isloadedontheagarosegel. that formed an almost random pattern, suggesting that a
tropic ascites clone is present among FCoV populations.
A previous study also reported differences of quasispe-
pattern. The lowest values for both diversity and evenness cies compositions of FCoV in ORF7b and N region be-
indexes in ascites suggested the presence of ascites-tropic tween organs [38]. Interestingly, diversity and evenness
clones. In addition to FCoV detection rate, population of FCoV strains from pleural effusion were similar to
structuresofFCoVstrainsbetweenascitesandpleuraleffu- those of blood but not ascites. With regard to both viral
sionspecimensalsoexhibiteddifferenttrends. detection rate and population structures, differenttrends
between ascites and pleural effusion specimens suggest
Discussion that pathology of FCoV relevant-pleural effusion or
In previous epidemiological studies, nested PCR tech- pleural FIP have markedly different characteristics from
niques targeting 3′-UTR and spike sequences have been those of FCoV relevant-ascites or peritoneal FIP. Previ-
widely used for viral detection and discrimination be- ous studies reported that genomic alteration or variabil-
tween type I and II serotypes, respectively [3,12,30-36]. ity of viral population during infection could affect the
However, previous methods are time-consuming, and organ-specificity, severity and immunological escape
nonspecific amplification often occurs in the course of [37,39,41]. Although, an onset of FCoV infection or FIP
nested PCR. Our nsp14 PCR method allowed detection highly depends on host factors, FCoValso might exhibit
ofFCoVusingasinglePCR,andlittlenonspecificampli- thepathogenicdiversitybyviralstrains.
fication occurred during PCR in various clinical speci- Theso-calledinternalmutationhypothesis,whichpos-
mens, suggesting that the primer set exhibited high tulates that viruses transition from avirulent to virulent
specificity to only viral genome. Consequently, our viacertainmutationsleadingtoFIPpathogenesis,isgen-
method allows to ascertain accurately the presence of erally believed in veterinary medicine [10]. However,
FCoV in any type of specimen without confirmation by there have been no reports on the identification of con-
sequencing of PCR products. Recently, Soma et al. re- sensus mutations in any FCoV strains; no relationship
ported that positivity of FCoV was 44.1% (377 of 854) in between viral phylogeny and virulence has previously
ascites from cats suspected of wet FIP in Japan using the been found [42-44].Our diversity analysis did not search
3′-UTR nested PCR method [36]. Although a simple for mutations in the FCoV genome responsible for FIP
comparison was not clear because of different study pathogenesis, suggesting that genetically diverse FCoV
populations, detection sensitivity of FCoV in ascites clones are present in domestic cats and that dynamic-
specimens of our nsp14 single PCR was comparable or ally selected clones can cause FCoV-related ascites or
better (P=0.0746), compared with the study using the wet FIP. Other reports also suggested that sequential
nested PCR method. There have been some reports on emergence of variants and replacing the pre-existing
the quasispecies in FCoV [31,37-41]; Battilani et al. population occurred in FCoV under the host immune
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page6of9
Figure3Phylogenetictree(MLmethod)basedonnsp14partialnucleotidesequences(406bp).Clinicalstrainsdetectedinthepresent
studyandthoseobtainedfromNCBIdatabasewereindicatedbyblueandblackletters,respectively.
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page7of9
Figure4Phylogenetictree(MLmethod)basedonnsp14partialaminoacidsequences(135aa).STdistributionandnumberofstrains
fromascites,pleuraleffusion,bloodandpericardialeffusioninpopulationgeneticstructureofFCoVareindicated.
pressure [39,41]. Thus, transition to FIP may occur been reports on detection of strains with the FCoV-
through changes in viral populations in a feline host genotype in host species of Carnivora other than domestic
rather than internal mutations. cats [35,47-49], our ecological understanding of this virus
Recently, severe acute respiratory syndrome (SARS) in remains insufficient. The present method, which was
2002/2003 and Middle East respiratory syndrome (MERS) applicable to all alpha-coronavirus1 subspecies, will also
in 2012 have emerged as human infectious disease from contributetoourecologicalunderstandingofthisvirus.
zoonotic coronaviruses [45,46]. To trace the original infec-
tioussourceofcoronaviruszoonotictransmission,weneed Conclusions
to understand the ecology and population structures of In conclusion, we developed a detection and genotyping
viral strains invarious animal species.Although therehave toolfor allvariantsofFCoV,andconfirmed thepresence
Table1DiversityandevennessindexesofFCoVstrainsbyspecimens
Kindofspecimen No.ofspecimens No.ofstrains No.ofSTs Simpson’sindex(1-λ) Pielou’sindex PredominantST(s)a
Ascites 103 55 26 0.881 0.661 ST42
Pleuraleffusion 45 14 10 0.923 0.812 ST8,ST42
Blood 214 19 15 0.971 0.895 ST8,ST25,ST42
aST(s)whichaccountedfornotlessthan10%ofclonesinthepopulation.
Tanakaetal.BMCVeterinaryResearch (2015) 11:57 Page8of9
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