2016 Feline Coronavirus 3c Protein_ A Candidate for a Virulence Marker_

Research ArticleFeline Coronavirus 3c Protein A Candidate fora Virulence Marker

A S Hora12 P O Tonietti3 S A Taniwaki2 K M Asano12 P Maiorka3

L J Richtzenhain12 and P E Brandatildeo12

1Department of Preventive Veterinary Medicine and Animal Health School of Veterinary Medicine University of Sao PauloAvenida Professor Dr Orlando Marques de Paiva 87 Cidade Universitaria 05508-270 Sao Paulo SP Brazil2Coronavirus Research Group School of Veterinary Medicine University of Sao PauloAvenida Professor Dr Orlando Marques de Paiva 87 Cidade Universitaria 05508-270 Sao Paulo SP Brazil3Department of Pathology School of Veterinary Medicine University of Sao PauloAvenida Professor Dr Orlando Marques de Paiva 87 Cidade Universitaria 05508-270 Sao Paulo SP Brazil

Correspondence should be addressed to A S Hora alineshoragmailcom

Received 11 February 2016 Revised 1 April 2016 Accepted 10 April 2016

Academic Editor Nikolai V Ravin

Copyright copy 2016 A S Hora et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis(FIP) whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomaticinfections Some candidates for geneticmarkers capable of differentiating these twopathotypes of a unique virus (feline coronavirus)have been proposed by several studies In the present survey in order to search for markers that can differentiate FECV and FIPVseveral clones of the 3andashc E and M genes were sequenced from samples obtained from cats with or without FIP All genes showedgenetic diversity and suggested the presence of FCoVmutant spectrum capable of producing a virulent pathotype in an individual-specific way In addition all the feline coronavirus FIPV strains demonstrated a truncated 3c protein and the 3c gene was the onlyobserved pathotypic marker for FCoVs showing that 3c gene is a candidate marker for the distinction between the two pathotypeswhen the mutant spectrum is taken into account

1 Introduction

Feline coronavirus (FCoV) (Nidovirales Coronaviridae Cor-onavirinae Alphacoronavirus 1) is a widespread pathogenamong the domestic cat population FCoV occurs as twopathotypes feline enteric coronavirus (FECV) and felineinfectious peritonitis virus (FIPV) both of which with strainsof serotypes I and II [1]

Phenotypically the main difference between the path-otypes is that FIPV is able to infect monocytes and macr-ophages causing systemic infection and fatal disease whileFECV is limited to replication in the mature intestinalepithelium which mainly results in asymptomatic infectionHowever FECVmay undergo a short systemic viremia phasethat also involves monocytes [2 3]

The FCoV genome (single-stranded positive-sense linearRNA 29 kb) encodes proteins directly involved in viral

replication (ORF1) the structural spike protein (S) envelopeprotein (E) membrane protein (M) and nucleocapsid pro-tein (N) as well as the accessory proteins 3andashc and 7a-b [4]

There is a major controversy regarding the existence ofgenetic markers to differentiate between FECV and FIPVpathotypes [5 6] Numerous markers that correlate with themanifestation of FIP have been proposed for S [7ndash12] 3andashc[8 13ndash16] 7a-b [16ndash18] and M [5] genes

The most studied gene is 3c [7 9 13ndash15 19 20] whichis present in the truncated form in the majority of all strainsobtained from animals with FIP However previous studieson 3c genes focused only on the dominant sequence andnot on the mutant spectrum or the clonal diversity and theinformation obtained from a dominant sequence may notbe suitable to detect mutations [10] with the exception of astudy [7] in which clones were used to assess the presence ofalterations in the gene 3c in ascites andor faeces fromFIP and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 8560691 9 pageshttpdxdoiorg10115520168560691

2 BioMed Research International

healthy cats and again 3c truncated protein was not observedin 100 of FIPV samples

Considering (i) the current discussion on molecularmarkers capable of distinguishing the pathotypes of FCoVand (ii) the lack of knowledge of the diversity of suchmarkersin the mutant spectrum of RNA the present study wasdesigned to investigate the clonal diversity of the 3andashc Eand M genes in FCoV strains present in tissue andor faecalsamples from cats with and without FIP

2 Materials and Methods

21 Animals and Samples From 2010 to 2013 tissue (eyecerebrum cerebellum lung heart muscle thoracic lymphnode thymus liver spleen stomach mesenteric lymphnode peripancreatic lymph node kidneys large and smallintestines and urinary bladder) abdominal thoracic andpericardial effusions aqueous humor and faecal sampleswere collected during the necropsies of 6 cats with clinicalmanifestations and histopathological changes consistent withFIP such as localized inflammation with macrophages neu-trophils lymphocytes and plasma cells Vascular lesionswerefound surrounded by a proliferation of inflammatory cells inheart lung omentum kidneys cerebrum and cerebellumFocal accumulations of inflammatory cells and necrotic-proliferative lesions were observed in granulomatous lesionsIntestinal samples were collected including mucosa submu-cosa muscular layer and serosa Only 2 animals (USP6 andUSP7) showed signs of dry-form FIP (neurological changes)while the remaining 4 (USP8 USP16 USP17 and USP18)showed an accumulation of effusion in body cavities (effusiveFIP)

All FIP-positive cats were sampled from different geo-graphic locations with the exception of 2 cats (USP17 andUSP18) that lived in the same house USP17 was euthanized 1month prior to USP18 due to FIP related reasons

In parallel faecal samples were also obtained from 2 cats(USP2 andUSP14) without clinical signs of FIP and from twodifferent locations not related to the locations from the FIP+cats

This study has been approved by Commission on Ethicson Animal Use from School of Veterinary Medicine Univer-sity of Sao Paulo (CEUA N 2055211113)

22 Messenger RNA Detection for the M Gene of FCoVsAll tissues were complete homogenized after tissue macera-tion and before RNA extraction Total RNA extraction andRT-PCR to detect mRNA of the M gene of FCoVs wereperformed in all samples as previously described [21] as ascreening test for FCoV replication

23 Amplification of the 3andashc E and M Genes of FCoVsAll samples from 6FIP+ and 2FIPminus cats were submitted toamplification of the 3andashc E and M genes of FCoVs evenif the mRNA detection was negative To amplify all thesefive genes of FCoVs in only one amplicon a single pairof previously described primers [14] was used (nucleotides

Table 1 Primers used to obtain full coverage of genes 3andashc E andM

Primers Sequence (51015840-31015840) Positiona

2B YATYACWGTKTAYRABTT 487ndash5053B ACRACWYTNAKKTTTGTA 1058ndash10754B AATAGCATTGCTAAATRT 1479ndash14965B CGGCTTTAGTRTYGCAGG 1999ndash2008aPosition defined according to genome FCoV-WSU 79-1146 (DQ010921)

24380 to 26996 corresponding to the FCoV-WSU 79-1146genome GenBank DQ010921)

cDNA synthesis was performed with 6 120583L total RNAextracted with TRIzoltrade Reagent (Life Technologies Corpo-ration Carlsbad CA USA) and ThermoScripttrade (Life Tech-nologies Corporation Carlsbad CA USA) using RandomPrimerstrade (Life Technologies Corporation Carlsbad CAUSA) according to the manufacturerrsquos instructions

For PCR 5 120583L of cDNA was added to 1x PCR buffer(Life Technologies Corporation Carlsbad CA USA) 2mMMgSO

4 02mM of each dNTP 025120583M of each primer 15 U

Platinumtrade Taq DNA Polymerase High Fidelity (Life Tech-nologies Corporation Carlsbad CA USA) and DEPC waterfor a final reaction volume of 50 120583L The cycling conditionswere as follows 94∘C2min 10 cycles of 94∘C15 s 50∘C15 swith an increase of 05∘C per cycle and 68∘C4min 10 cyclesof 94∘C15 s 55∘C15 s and 68∘C4min 25 additional cycles of94∘C15 s 55∘C15 s and 68∘C4min with an increase of 10 sper cycle and a final extension of 72∘C10min

Specific bands were excised from the gels and purifiedusing the Gene Jettrade Gel Extraction kit (Thermo Fisher Scien-tific Inc Vilnius Lithuania) according to the manufacturerrsquosinstructions

24 Molecular Cloning The purified amplicons (2617 bpcontaining 3abc-E-M) were cloned using pGEMtrade-T EasyVector Systems (Promega Madison WI USA) and E coliJM109 competent cells according to the manufacturerrsquos rec-ommendations After the transformation protocol trans-formation culture was plated onto duplicate LBampicillinIPTGX-Gal plates and the plates were incubated overnight(16 hours) at 37∘CThen the transformed colonieswere trans-ferred to LB liquidmedium andwere incubated overnight (16hours) at 37∘C and 180 rpm Recombinant plasmid extractionwas performed with the Illustratrade plasmidPrep Mini SpinKit (GE Healthcare Buckinghamshire UK) as per manufac-turerrsquos recommendations

For an easier identification of the clones the followingnomenclature was used cat identificationsample sourcepresence (+) or absence (minus) of FIPcat originclone number

25 DNA Sequencing Sense primers were designed at appro-ximately every 500 nucleotides to obtain full coverage ofgenes 3andashc E and M (Table 1) Primers for the plasmid(pUCM13F and R) were also used to reach the full lengthof the amplicon sequence

BioMed Research International 3

Table 2 Identification sorting according to the presence of FIP detection of mRNA FCoVs and the number of clones obtained from cats

ID Location FIP Sample mRNANumber of clones obtained from each gene

TotalE M 3a 3b 3c249nt 792nt 213nt 222nt 714nt

USP2 1 Absent Faeces Pos 4 6 6 6 4 26

USP6 7 Noneffusive Stomach Pos 10 11 10 9 6 46Faeces Neg 0 3 8 7 5 23

USP7 3 Noneffusive Cerebrum Neg 5 5 5 5 2 22Faeces Pos 3 7 10 10 5 35

USP8 6 Effusive Stomach Pos 1 1 1 1 1 5Urinary bladder Pos 0 0 3 3 2 8

USP17 2 Effusive Cerebrum Neg 3 5 8 8 8 32Large intestine Pos 3 1 4 4 2 14

USP14 4 Absent Faeces Pos 1 1 12 12 9 35USP16 5 Effusive Cerebrum Pos 6 0 9 9 9 33USP18 2 Effusive Cerebrum Pos 2 3 3 3 2 13Total of clones (119899 = 292) 38 43 79 77 55 292ID identification Pos positive Neg negative

DNA sequencing with each primer in duplicate was per-formed using the BigDyetrade Terminator v31 Cycle SequencingKit (Life Technologies Corporation Carlsbad CA USA)with a Genetic Analyzer 3500trade capillary sequencer (AppliedBiosystems Foster City CA USA) according to themanufac-turersrsquo instructions

26 Sequence Editing Chromatograms were generated induplicate for each segment and evaluated for their basequality using the online application Phred (httpasparagincenargenembrapabrphph) Only those chromatogramswith scores greater than 20 (probability lower than 1 errorper 100 nucleotides) were used The chromatograms wereanalyzed andmanually editedwith the software FinchTV ver-sion 14 (httpwwwgeospizacomProductsfinchtvshtml)The final sequences of each sample were obtained using theCAP contig application with the software BioEdit version7090 [22]

27 Phylogenetic Analyses The nucleotides and amino acidscorresponding to the 3andashc E and M genes were alignedusing the applicationClustalW inBioEdit 7090 [22] Cloneswere classified in serotypes based on comparative analysis ofthe amino acid sequences encoded by the 3a gene with thesequences available in GenBank (serotype I DQ160294 andDQ848678 and serotype II DQ010921) Nucleotide phyloge-netic trees (neighbor-joining MCL model 1000 bootstrapreplicates) were constructed using the computer softwareMega version 505 [23]

3 Results

31 Messenger RNA Detection for the M Gene and Ampli-fication Sequencing and Cloning of the 3andashc E and MGenes of FCoVs mRNA was not obtained from all samples

nevertheless sequences for the 3andashc E andMamplicons wereobtained from a total of 292 clones originating from8 animals[6FIP+ (USP6 USP7 USP8 USP17 USP16 and USP18) and2FIPminus (USP2 and USP14)] Sequences from extraintestinaltissue samples and faecalintestines samples were obtainedsimultaneously for 3 animals (USP6 USP7 and USP17) withFIP (Table 2) Due to poor sequence quality for some clonesandor regions in a clone not all full length amplicons couldbe sequenced or used in the analysesThe sequences obtainedin the present study were deposited in GenBank under accessnumbers KJ879334 to KJ879438

32 Phylogenetic Analysis of the 3andashc E and M Genes ofFCoVs All sequences of the 3a genes obtained from a total of79 clones belong to serotype I Phylogenetic trees generatedfor the 3a 3c E and M genes showed similar topologiesand thus only the 3c and 3b trees are shown (Figures 1and 2 resp) Sample from cat USP14 (non-FIP) showed aremarkable clonal diversity as their clones are present in fivedifferent clusters of the 3b tree High clonal diversity wasobserved in samples from FIP+ (USP8 USP16 USP17 andUSP18) and FIPminus cat (USP14) however FIP+ cats USP6 andUSP7 and USP2 FIPminus cat showed a lower clonal diversityIn the 3c gene tree for cats USP6 and USP7 differencescan be noticed between the FCoV strains obtained fromtissue and faecal clones that is for each animal there wasa distinction between systemic (FIPV) and enteric (FECV)strains However no generic grouping per pathotype (FECVor FIPV) was observed for all samples and trees generatedfrom this study

Conversely in the 3b gene tree samples obtained from catUSP7 (FIP+) could not be separated into systemic (cerebrum)and enteric (faeces) strains

The overall nucleotide identity of all clones (FIP+ andFIPminus) obtained from the 5 genes was ge9209 for faecal


USP17CerebrumFIP+local2C12USP17CerebrumFIP+local2C24

USP17CerebrumFIP+local2C2USP17Large intestineFIP+local2C3

USP17Large intestineFIP+local2C4USP17CerebrumFIP+local2C6USP17CerebrumFIP+local2C23



USP18CerebrumFIP+local2C57USP14FaecesFIPminuslocal4C2USP14FaecesFIPminuslocal4C7USP14FaecesFIPminuslocal4C9

USP14FaecesFIPminuslocal4C3USP14FaecesFIPminuslocal4C11


USP16CerebrumFIP+local5C3USP16CerebrumFIP+local5C4USP16CerebrumFIP+local5C13







USP7FaecesFIP+local3C13USP7FaecesFIP+local3C24

USP7FaecesFIP+local3C17USP7FaecesFIP+local3C14USP7FaecesFIP+local3C20


USP8Urinary bladderFIP+local6C19USP8Urinary bladderFIP+local6C32

USP6FaecesFIP+local7C53USP6FaecesFIP+local7C56USP6FaecesFIP+local7C54USP6FaecesFIP+local7C51USP6FaecesFIP+local7C49

USP8StomachFIP+local6C22USP6StomachFIP+local7C8USP6StomachFIP+local7C15USP6StomachFIP+local7C24USP6StomachFIP+local7C28

USP6StomachFIP+local7C16USP6StomachFIP+local7C2987

100

100

98

100

65

100

100

100

6098

98

7465

5068

62

61100

99

100

63

6399

0005

Figure 1 Phylogenetic tree constructed using the neighbor-joining method and the maximum composite likelihood substitution model forthe FCoV 3c gene The numbers next to each node represent the values of 1000 bootstrap repetitions and only those above 50 are shownThe scale represents the number of substitutions per site For easy identification of the samples the following nomenclature was used catidentificationsample sourcepresence (+) or absence (minus) of FIPcat originclone number



USP7FaecesFIP+local3C27USP7FaecesFIP+local3C24USP7FaecesFIP+local3C23USP7FaecesFIP+local3C20USP7FaecesFIP+local3C17USP7FaecesFIP+local3C14USP7FaecesFIP+local3C13USP7FaecesFIP+local3C12USP7FaecesFIP+local3C10USP7FaecesFIP+local3C8 USP7CerebrumFIP+local3C15USP7CerebrumFIP+local3C13

USP14FaecesFIPminuslocal4C10USP16CerebrumFIP+local5C6

USP16CerebrumFIP+local5C9USP16CerebrumFIP+local5C5USP16CerebrumFIP+local5C3USP16CerebrumFIP+local5C4USP16CerebrumFIP+local5C13


USP16CerebrumFIP+local5C8USP14FaecesFIPminuslocal4C1

USP14FaecesFIPminuslocal4C6USP14FaecesFIPminuslocal4C20USP8Urinary bladderFIP+local6C32USP8Urinary bladderFIP+local6C20

USP8Urinary bladderFIP+local6C19USP14FaecesFIPminuslocal4C4



USP17Large intestineFIP+local2C4USP17Large intestineFIP+local2C1



USP17CerebrumFIP+local2C22USP17CerebrumFIP+local2C23USP17CerebrumFIP+local2c2



USP14FaecesFIPminuslocal4C8USP14FaecesFIPminuslocal4C7USP14FaecesFIPminuslocal4C2

USP14FaecesFIPminuslocal4C3USP2FaecesFIPminuslocal1C10USP2FaecesFIPminuslocal1C12USP2FaecesFIPminuslocal1C5USP2FaecesFIPminuslocal1C3USP2FaecesFIPminuslocal1C2USP2FaecesFIPminuslocal1C1

USP8StomachFIP+local6C22USP6FaecesFIP+local7C56USP6FaecesFIP+local7C61USP6FaecesFIP+local7C54USP6FaecesFIP+local7C53USP6FaecesFIP+local7C51USP6FaecesFIP+local7C50USP6FaecesFIP+local7C49

USP6StomachFIP+local7C3USP6StomachFIP+local7C4USP6StomachFIP+local7C8USP6StomachFIP+local7C12USP6StomachFIP+local7C15USP6StomachFIP+local7C16USP6StomachFIP+local7C24USP6StomachFIP+local7C28

USP6StomachFIP+local7C29

7099

5163

80

99

97

68

6199

83

64

99

95

73

51

84

51

50

5598

6675

001

Figure 2 Phylogenetic tree constructed using the neighbor-joining method and the maximum composite likelihood substitution model forthe FCoV 3b gene The numbers next to each node represent the values of 1000 bootstrap repetitions and only those above 50 are shownThe scale represents the number of substitutions per site For easy identification of the samples the following nomenclature was used catidentificationsample sourcepresence (+) or absence (minus) of FIPcat originclone number


samples and ge9009 for tissue samples (Table 3) Thegreatest nucleotide diversity when all available sequences foreach gene were compared together was observed for the3b gene for which a 9009 identity was found with theexception of comparisons between cats living in the samehouse for which the identity between the 3b gene sequenceswas 9709

For tissue and faecal samples 3a was the most conservedgene (nucleotide identities of 9938 and 9770 resp)considering each individual cat The mean identity betweenclones from the two cohabitating FIP+ cats (USP17 andUSP18) ranged from 9617 (3c gene) to 9983 (E gene)

Premature stop codons were observed in the 3a (USP1833 cerebrumclones) 3b (USP17 18 cerebrumUSP7 110 fae-ces) 3c (USP17 88 cerebrum and 22 large intestine USP1822 cerebrum USP7 22 cerebrum USP16 19 cerebrumUSP8 11 stomach USP6 66 stomach) and M (USP7 15cerebrum) genes fromFIP+ cats No premature stop codon inthe 3andashc or E genes was observed in faecal samples from catswithout FIP with exception of M gene (USP2 16 faeces)

Stop codons in 3c were evident in at least one of the clonesfrom tissue samples from cats with FIP The faecal samplesfrom cats with or without FIP did not demonstrate thistruncated gene in any of the clones and no other molecularmarker capable of differentiating the biotypes (FECV andFIPV) was observed in the sequenced genes

4 Discussion and Conclusions

The existence of genetic markers to differentiate these twobiotypes is currently a topic of the highest interest on the FIPfield not only from the clinical and epidemiological pointsof view but also as a basis for an in-deep comprehension ofthe pathogenesis of this highly complex disease Consideringthat in the present study clones of structural (E and M)and accessory (3andashc) genes were obtained from cats withand without FIP and the results demonstrate a highly likelycandidate FCoV pathotypic marker in the 3c gene

Amplicons obtained from the cerebrum and large intes-tine of the cat USP17 (FIP+) were also cloned and sequencedWhen assessing the 46 clones (32 clones from cerebrum and14 from large intestine) obtained from this cat the nucleotideidentity among the five genes was greater than 99 andaccording to the phylogenetic trees generated clones fromthis cat resulted in a single cluster In the 3c gene a singlenucleotide transversion (G rarr T) was observed at position10 in all clones from samples from this cat resulting in pre-mature stop codons and truncated proteins The observationof truncated proteins in large intestine observed herein is incontrast to what has been observed for FECV in other studies[13 14 20]

Possibly because it serves as a preferential organ for viralreplication and therefore for pathological changes in FIPcases the intestine of the cat USP17 (FIP+) was also affectedby the systemic strain Chang et al [20] suggested that viruseswith an inactivated 3c gene rarely replicate in the intestinewhich helps to explain the rare incidence of FIP outbreaksdue to a less frequent faecal-oral transmission A prematurestop codon in the 3c gene was previously reported in a strain

of FCoV from the jejunum of a cat with FIP and the sameintestinal strain showed 100 identity with a strain obtainedfrom a liver sample of the same cat [6]

In recent studies [13 15] 3c genes with premature ter-mination were detected in faecal and extraintestinal samplesfrom cats with FIP although it was not determined whetherthe viruses were replicating Also after an experimentalinoculation of cats with truncated and complete 3abc therole of the truncation in this region on FIP pathogenesiscould not be clearly determined despite it led to lower viralreplication in the gut [24] However in the present study thesample from the large intestine of the cat USP17 (FIP+) fromwhich the studied clones originated and which demonstrateda truncated 3c gene was positive for FCoVmRNAThis resultindicated the presence of viral replication in that sample

When comparing the sequences obtained from theextraintestinal organs and faeces of the FIP+ cats USP7 andUSP6 the presence of a premature stop codon in the 3cgene was not observed in faecal clones from both cats incontrast to what was observed for tissue clones Moreoverthe nucleotide identity of the 3c gene for tissue and faecalsamples was 9909 and 9558 for the cats USP7 and USP6respectively In a study comparing FCoV from extraintestinalFIP lesions and the faeces of the same cat the identitybetween the sequences of the structural and accessory genesevaluated (S M N E 3andashc and 7a-b) was always higher than99 corroborating the hypothesis of in vivomutation [14]

As FIPV lineages may be derived from FECV mutationsit was expected that nucleotide diversity between faecaland tissue samples from the cat USP6 (FIP+) would belower However the difference was 884 for the 3b genesconsisting exclusively in point mutations indicating that thiscat could be coinfected with an FCoV strain that is distinctfrom the systemic strain Furthermore the high nucleotideidentity between samples from the cerebrum and faeces ofthe cat USP7 (FIP+) along with the topology of the generatedtrees showed that the intestinal viral replication of the entericpathotypemay bemaintained even during FIP in some casesas demonstrated by the presence of mRNA

As previously described and according with results fromthe present study FIPV can both emerge endogenously andbe transmitted to different cats as a ldquoreadyrdquo virulent patho-type which has major implications for the understanding ofthe dynamics of viral transmission [5 15 21]

Among the structural proteins genes (E and M) studiedherein premature stop codons were detected only in the MgeneTheMprotein of FCoVs is themost abundant structuralprotein [25] The presence of a premature stop codon in aconstitutive gene such as the M gene is incompatible withthe functionality of the virion However this defective virusis no less important than the others as it provides geneticbackground for recombination with other FCoVs resultingin greater genetic diversity and possibly increased virulenceof some viral quasispecies Recent studies suggest that the3c gene which encodes a small protein (238aa) of unknownfunction is associated with most FIPV isolates when presentin truncated form although not all FCoV strains in FIP+samples present this 3c form [13 14 20] In the present studythe 3c truncated protein was observed in all clones obtained


Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall


from tissue samples from FIP+ cats with the exception ofthose obtained from the cat USP16 where only 11 (19) ofthe clones demonstrated the truncated protein

It is essential to note that if the predominant viralpopulation in the present study had been assessed by directamplicon sequencing only as in studies conducted by otherauthors [13ndash15 19 20] without using the previous cloningstep the truncated 3c gene could have been undetected as itwas only present in a minority of the sequences observed inthe cerebrum of the cat USP16 (FIP+)

The transversions observed at position 10 of the 3c genewhich resulted in premature stop codons were the sameas those observed in the clone sequences obtained fromthe cerebrum and stomach of the FIP+ cats USP17 andUSP6 respectively With the exception of this finding andin agreement with the results of Pedersen et al [14 15] the3c gene mutations were unique to each cat including thesequences obtained from the cohabitating FIP+ cats (USP17and USP18)

It can be concluded that the presence of a truncated3c protein is a suggestive factor for differentiation betweenthe FECV and FIPV pathotypes and therefore serves as agenetic marker of virulence It still remains to be answeredwhether 3c is expendable for FCoV replication in phagocyticcells and a nontruncated 3c is thus selected-against duringthe FECVFIPV transition Moreover the 3andashc E and Mgenes showed a level of genetic diversity indicative of theconstitution of FCoV quasispecies or mutant spectrum andthe likelihood of emergence of a highly virulent individual-specific biotype

Competing Interests

None of the authors has any financial or personal relation-ships that could inappropriately influence or bias the contentof the paper

Acknowledgments

Funding for this study was provided by Fundacao de Amparoa Pesquisa do Estado de Sao Paulo Brazil (Grants nos0917535-9 and 1007492-8) Conselho Nacional de Desen-volvimento Cientıfico e Tecnologico (Grant no 3005132010-0) andCoordenacao deAperfeicoamento de Pessoal deNıvelSuperior (CAPESPROEX 23272015) The authors are alsograteful to the veterinarians and cat ownerswho provided FIPcases and cat faecal samples for this study

References

[1] N C Pedersen ldquoA review of feline infectious peritonitis virusinfection 1963ndash2008rdquo Journal of Feline Medicine and Surgeryvol 11 no 4 pp 225ndash258 2009

[2] A Kipar M L Meli K E Baptiste L J Bowker and H LutzldquoSites of feline coronavirus persistence in healthy catsrdquo Journalof General Virology vol 91 no 7 pp 1698ndash1707 2010

[3] L Vogel M van der Lubben E G te Lintelo et al ldquoPathogeniccharacteristics of persistent feline enteric coronavirus infectionin catsrdquo Veterinary Research vol 41 no 5 article 71 2010

[4] P S Masters ldquoThemolecular biology of coronavirusesrdquo Advan-ces in Virus Research vol 65 pp 193ndash292 2006

[5] M A Brown J L Troyer J Pecon-Slattery M E Roelke andS J OrsquoBrien ldquoGenetics and pathogenesis of feline infectiousperitonitis virusrdquo Emerging Infectious Diseases vol 15 no 9 pp1445ndash1452 2009

[6] C Dye and S G Siddell ldquoGenomic RNA sequence of felinecoronavirus strain FCoV C1Jerdquo Journal of Feline Medicine andSurgery vol 9 no 3 pp 202ndash213 2007

[7] B R Bank-Wolf I Stallkamp SWiese AMoritz G Tekes andH-J Thiel ldquoMutations of 3c and spike protein genes correlatewith the occurrence of feline infectious peritonitisrdquo VeterinaryMicrobiology vol 173 no 3-4 pp 177ndash188 2014

[8] H-W Chang H F Egberink R Halpin D J Spiro and P JM Rottie ldquoSpike protein fusion peptide and feline coronavirusvirulencerdquo Emerging Infectious Diseases vol 18 no 7 pp 1089ndash1095 2012

[9] C S Lewis E Porter D Matthews et al ldquoGenotyping coron-aviruses associated with feline infectious peritonitisrdquo Journal ofGeneral Virology vol 96 pp 1358ndash1368 2015

[10] B N Licitra J K Millet A D Regan et al ldquoMutation in spikeprotein cleavage site and pathogenesis of feline coronavirusrdquoEmerging Infectious Diseases vol 19 no 7 pp 1066ndash1073 2013

[11] A D Regan R Shraybman R D Cohen and G R WhittakerldquoDifferential role for low pH and cathepsin-mediated cleavageof the viral spike protein during entry of serotype II felinecoronavirusesrdquo Veterinary Microbiology vol 132 no 3-4 pp235ndash248 2008

[12] P J M Rottier K Nakamura P Schellen H Volders andB J Haijema ldquoAcquisition of macrophage tropism during thepathogenesis of feline infectious peritonitis is determined bymutations in the feline coronavirus spike proteinrdquo Journal ofVirology vol 79 no 22 pp 14122ndash14130 2005

[13] L-E Hsieh W-P Huang D-J Tang Y-T Wang C-T Chenand L-L Chueh ldquo3C protein of feline coronavirus inhibits viralreplication independently of the autophagy pathwayrdquo Researchin Veterinary Science vol 95 no 3 pp 1241ndash1247 2013

[14] N C Pedersen H Liu K A Dodd and P A Pesavento ldquoSigni-ficance of coronavirus mutants in feces and diseased tissues ofcats suffering from feline infectious peritonitisrdquo Viruses vol 1no 2 pp 166ndash184 2009

[15] N C Pedersen H Liu J Scarlett et al ldquoFeline infectiousperitonitis role of the feline coronavirus 3c gene in intestinaltropism and pathogenicity based upon isolates from residentand adopted shelter catsrdquo Virus Research vol 165 no 1 pp 17ndash28 2012

[16] H Vennema A Poland J Foley and N C Pedersen ldquoFelineinfectious peritonitis viruses arise by mutation from endemicfeline enteric coronavirusesrdquo Virology vol 243 no 1 pp 150ndash157 1998

[17] A Dedeurwaerder D A J Olyslaegers LM B Desmarets I DM Roukaerts S Theuns and H J Nauwynck ldquoORF7-encodedaccessory protein 7a of feline infectious peritonitis virus as acounteragent against IFN-120572-induced antiviral responserdquo Jour-nal of General Virology vol 95 no 2 pp 393ndash402 2014

[18] M Kennedy N Boedeker P Gibbs and S Kania ldquoDeletions inthe 7a ORF of feline coronavirus associated with an epidemicof feline infectious peritonitisrdquo Veterinary Microbiology vol 81no 3 pp 227ndash234 2001

[19] C M Borschensky and M Reinacher ldquoMutations in the 3c and7b genes of feline coronavirus in spontaneously affected FIP


catsrdquo Research in Veterinary Science vol 97 no 2 pp 333ndash3402014

[20] H-W Chang R J de Groot H F Egberink and P J M RottierldquoFeline infectious peritonitis Insights into feline coronaviruspathobiogenesis and epidemiology based on genetic analysis ofthe viral 3c generdquo Journal of General Virology vol 91 no 2 pp415ndash420 2010

[21] A S Hora K M Asano J M Guerra et al ldquoIntrahost diversityof feline coronavirus a consensus between the circulating vir-ulentavirulent strains and the internal mutation hypothesesrdquoThe Scientific World Journal vol 2013 Article ID 572325 8pages 2013

[22] T A Hall ldquoBioedit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquo inNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[24] A Balint A Farsang Z Zadori and S Belak ldquoComparative invivo analysis of recombinant type II feline coronaviruses withtruncated and completed ORF3 regionrdquo PLoS ONE vol 9 no2 Article ID e88758 2014

[25] P J M Rottier ldquoThe coronavirus membrane glycoproteinrdquo inTheCoronaviridae S G Siddell Ed pp 115ndash140 Plenum PressNew York NY USA 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


healthy cats and again 3c truncated protein was not observedin 100 of FIPV samples

Considering (i) the current discussion on molecularmarkers capable of distinguishing the pathotypes of FCoVand (ii) the lack of knowledge of the diversity of suchmarkersin the mutant spectrum of RNA the present study wasdesigned to investigate the clonal diversity of the 3andashc Eand M genes in FCoV strains present in tissue andor faecalsamples from cats with and without FIP

2 Materials and Methods

21 Animals and Samples From 2010 to 2013 tissue (eyecerebrum cerebellum lung heart muscle thoracic lymphnode thymus liver spleen stomach mesenteric lymphnode peripancreatic lymph node kidneys large and smallintestines and urinary bladder) abdominal thoracic andpericardial effusions aqueous humor and faecal sampleswere collected during the necropsies of 6 cats with clinicalmanifestations and histopathological changes consistent withFIP such as localized inflammation with macrophages neu-trophils lymphocytes and plasma cells Vascular lesionswerefound surrounded by a proliferation of inflammatory cells inheart lung omentum kidneys cerebrum and cerebellumFocal accumulations of inflammatory cells and necrotic-proliferative lesions were observed in granulomatous lesionsIntestinal samples were collected including mucosa submu-cosa muscular layer and serosa Only 2 animals (USP6 andUSP7) showed signs of dry-form FIP (neurological changes)while the remaining 4 (USP8 USP16 USP17 and USP18)showed an accumulation of effusion in body cavities (effusiveFIP)

All FIP-positive cats were sampled from different geo-graphic locations with the exception of 2 cats (USP17 andUSP18) that lived in the same house USP17 was euthanized 1month prior to USP18 due to FIP related reasons

In parallel faecal samples were also obtained from 2 cats(USP2 andUSP14) without clinical signs of FIP and from twodifferent locations not related to the locations from the FIP+cats

This study has been approved by Commission on Ethicson Animal Use from School of Veterinary Medicine Univer-sity of Sao Paulo (CEUA N 2055211113)

22 Messenger RNA Detection for the M Gene of FCoVsAll tissues were complete homogenized after tissue macera-tion and before RNA extraction Total RNA extraction andRT-PCR to detect mRNA of the M gene of FCoVs wereperformed in all samples as previously described [21] as ascreening test for FCoV replication

23 Amplification of the 3andashc E and M Genes of FCoVsAll samples from 6FIP+ and 2FIPminus cats were submitted toamplification of the 3andashc E and M genes of FCoVs evenif the mRNA detection was negative To amplify all thesefive genes of FCoVs in only one amplicon a single pairof previously described primers [14] was used (nucleotides

Table 1 Primers used to obtain full coverage of genes 3andashc E andM

Primers Sequence (51015840-31015840) Positiona

2B YATYACWGTKTAYRABTT 487ndash5053B ACRACWYTNAKKTTTGTA 1058ndash10754B AATAGCATTGCTAAATRT 1479ndash14965B CGGCTTTAGTRTYGCAGG 1999ndash2008aPosition defined according to genome FCoV-WSU 79-1146 (DQ010921)

24380 to 26996 corresponding to the FCoV-WSU 79-1146genome GenBank DQ010921)

cDNA synthesis was performed with 6 120583L total RNAextracted with TRIzoltrade Reagent (Life Technologies Corpo-ration Carlsbad CA USA) and ThermoScripttrade (Life Tech-nologies Corporation Carlsbad CA USA) using RandomPrimerstrade (Life Technologies Corporation Carlsbad CAUSA) according to the manufacturerrsquos instructions

For PCR 5 120583L of cDNA was added to 1x PCR buffer(Life Technologies Corporation Carlsbad CA USA) 2mMMgSO

4 02mM of each dNTP 025120583M of each primer 15 U

Platinumtrade Taq DNA Polymerase High Fidelity (Life Tech-nologies Corporation Carlsbad CA USA) and DEPC waterfor a final reaction volume of 50 120583L The cycling conditionswere as follows 94∘C2min 10 cycles of 94∘C15 s 50∘C15 swith an increase of 05∘C per cycle and 68∘C4min 10 cyclesof 94∘C15 s 55∘C15 s and 68∘C4min 25 additional cycles of94∘C15 s 55∘C15 s and 68∘C4min with an increase of 10 sper cycle and a final extension of 72∘C10min

Specific bands were excised from the gels and purifiedusing the Gene Jettrade Gel Extraction kit (Thermo Fisher Scien-tific Inc Vilnius Lithuania) according to the manufacturerrsquosinstructions

24 Molecular Cloning The purified amplicons (2617 bpcontaining 3abc-E-M) were cloned using pGEMtrade-T EasyVector Systems (Promega Madison WI USA) and E coliJM109 competent cells according to the manufacturerrsquos rec-ommendations After the transformation protocol trans-formation culture was plated onto duplicate LBampicillinIPTGX-Gal plates and the plates were incubated overnight(16 hours) at 37∘CThen the transformed colonieswere trans-ferred to LB liquidmedium andwere incubated overnight (16hours) at 37∘C and 180 rpm Recombinant plasmid extractionwas performed with the Illustratrade plasmidPrep Mini SpinKit (GE Healthcare Buckinghamshire UK) as per manufac-turerrsquos recommendations

For an easier identification of the clones the followingnomenclature was used cat identificationsample sourcepresence (+) or absence (minus) of FIPcat originclone number

25 DNA Sequencing Sense primers were designed at appro-ximately every 500 nucleotides to obtain full coverage ofgenes 3andashc E and M (Table 1) Primers for the plasmid(pUCM13F and R) were also used to reach the full lengthof the amplicon sequence














3 Results





























100

100

98

100

65

100

100

100

6098

98

7465

5068

62

61100

99

100

63

6399

0005
























7099

5163

80

99

97

68

6199

83

64

99

95

73

51

84

51

50

5598

6675

001


















Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results





























100

100

98

100

65

100

100

100

6098

98

7465

5068

62

61100

99

100

63

6399

0005
























7099

5163

80

99

97

68

6199

83

64

99

95

73

51

84

51

50

5598

6675

001


















Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























100

100

98

100

65

100

100

100

6098

98

7465

5068

62

61100

99

100

63

6399

0005
























7099

5163

80

99

97

68

6199

83

64

99

95

73

51

84

51

50

5598

6675

001


















Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























7099

5163

80

99

97

68

6199

83

64

99

95

73

51

84

51

50

5598

6675

001


















Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Table3Mean

maxim

umand

minim

umnu

cleotideidentities

of3andashcE

and

MFC

oVgenesfrom

different

clones

Identifi

catio

n3a

gene

3bgene

3cgene

Egene

Mgene

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

Mean(

)Max

()

Min

()

USP

2FaecesPIFminuslo

cal1

9983

10000

9950

10000

10000

10000

9945

9950

9940

9975

10000

9950

9952

9980

9930

USP

6Stom

achPIF+

local7

9967

10000

9950

10000

10000

10000

9992

10000

9980

9990

10000

9950

9977

9980

9970

USP

6FaecesPIF+local7

10000

10000

10000

9903

9950

9860

9920

9920

9920

lowastlowast

lowast9977

9980

9970

USP

7CerebrumPIF+local3

9902

10000

9810

9639

10000

8810

9979

10000

9970

9953

10000

9910

9948

10000

9940

USP

7FaecesPIF+local3

9942

10000

9710

9216

10000

8330

9729

10000

9550

10000

10000

10000

9954

10000

9910

USP

8UBPIF+

local6

9870

9950

9810

9989

10000

9950

9973

10000

9920

lowastlowast

lowastlowast

lowastlowast

USP

14FaecesPIFminus

local4

9586

10000

9150

9896

10000

9770

9948

10000

9880

lowastlowast

lowastlowast

lowastlowast

USP

16C

erebrumPIF+local5

9942

10000

9900

9933

9950

9900

10000

10000

10000

9983

10000

9950

9970

10000

9940

USP

17C

erebrumPIF+local2

9925

10000

9850

9792

10000

9450

9933

10000

9850

9967

10000

9950

9918

9960

9870

USP

17LIP

IF+local2

9950

10000

9900

9903

9950

9860

9950

9950

9950

9967

10000

9950

lowastlowast

lowast

USP

18C

erebrumPIF+local2

9870

9950

9810

9868

10000

9810

9980

9980

9980

10000

10000

10000

9953

9960

9940

Intestinalsam

ples

9770

10000

9150

9209

10000

8150

9564

10000

9320

9387

10000

9110

9959

10000

9910

Extraintestin

alsamples

9938

10000

9810

9009

10000

7560

9537

10000

9270

9519

10000

8990

9953

10000

9870

Maxm

axim

umM

inm

inim

umLIlargeintestin

eUB

urinarybladder

lowastSamples

thaton

lyon

eclone

was

obtained

orno

clone

was

obtained

atall






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Competing Interests


Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

2016 Feline Coronavirus 3c Protein_ A Candidate for a Virulence Marker_

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