【病毒外文文獻】2014 Comparative In Vivo Analysis of Recombinant Type II Feline Coronaviruses with Truncated and Completed ORF3 Region

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Comparative In Vivo Analysis of Recombinant Type II Feline Coronaviruses with Truncated and Completed ORF3 Region A da mBa lint 1 Attila Farsang 2 Zolta nZa dori 3 Sa ndor Bela k 4 1National Food Chain Safety Office Veterinary Diagnostic Directorate Budapest Hungary 2National Food Chain Safety Office Directorate of Veterinary Medicinal Products Budapest Hungary 3Institute for Veterinary Medical Research Centre for Agricultural Research Hungarian Academy of Sciences Budapest Hungary 4Department of Virology Immunobiology and Parasitology National Veterinary Institute SVA Uppsala Sweden Abstract Our previous in vitro comparative study on a feline coronavirus FCoV pair differing only in the intactness of their ORF3abc regions showed that the truncated ORF3abc plays an important role in the efficient macrophage monocyte tropism of type II feline infectious peritonitis virus FIPV In the present study we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses While parent virus FIPV DF 2 developed feline infectious peritonitis in all the infected cats its recombinant virus PBFIPV DF 2 differing only in seven nucleotides proved to be surprisingly low virulent although caused an acute febrile episode similarly to the original FIPV DF 2 PBFIPV DF 2 infection induced significantly lower virus neutralization titers than its parent virus and lacked the second phase of viremia and development of fatal course of the disease The recombinant PBFIPV DF 2 R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus FECV and FIPV biotypes such as intensive replication in the gut absence of viremia and weak or no serological response Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo Citation Ba lint A Farsang A Za dori Z Bela k S 2014 Comparative In Vivo Analysis of Recombinant Type II Feline Coronaviruses with Truncated and Completed ORF3 Region PLoS ONE 9 2 e88758 doi 10 1371 journal pone 0088758 Editor Frederick C C Leung University of Hong Kong China Received August 21 2013 Accepted December 21 2013 Published February 20 2014 Copyright C223 2014 Ba lint et al This is an open access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited Funding This study was supported by Award of Excellence from the Swedish University of Agricultural Sciences Agria Animal Insurance Company Agria Djurfo rsa kring Orsza gos Tudoma nyos Kutata si Alapprogramok OTKA and Nemzeti Kutata si e s Technolo giai Hivatal NKTH Mobilita s 08 C OTKA 81187 and Ja nos Bolyai Fellowship from the Hungarian Academy of Sciences BO 00414 10 The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript Competing Interests The authors received funding of this work from a commercial source Agria Animal Insurance Company This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials E mail balintad nebih gov hu Introduction Feline coronaviruses FCoVs members of the Alphacoronavirus genus within the Coronaviridae family are major pathogens of Felidae with worldwide distribution 1 FCoV occurs in two pathotypes feline enteric coronavirus FECV primarily replicates in the lower portion of intestinal tract spreads by fecal oral route and its clinical appearance is characterized by mild or unapparent enteritis 2 3 In contrast feline infectious peritonitis virus FIPV efficiently replicates in macrophages monocytes and can lead to feline infectious peritonitis FIP a highly lethal systemic granu lomatous disease 4 8 FIPVs arise most likely from FECV in the infected cat via genetic changes 9 Characteristic changes can be detected in the spike S gene 10 11 in the ORF7ab 9 12 13 and the ORF3abc 9 14 16 regions FECVs have three open reading frames ORFs in the ORF3abc region 6 that code proteins conserved both in length and sequence in different isolates On the contrary the majority of FIPVs contain genetic alterations non synonymous mutations deletions and termination codons mostly in ORF3c but not rarely in ORF3a and ORF3b 9 14 The first in vitro comparison of a recombinant FCoV pair differing only in the intactness of their ORF3abc revealed that completion of the truncated ORF3abc reduces virus replication rate by 2log 10 titer in feline peripheral blood monocytes 17 supporting the long time suspected but never experimentally proved theory that completion of this region alters the in vivo characteristics and pathogenesis of FCoV 8 In the present study using the parent FIPV DF 2 strain and its recombinant derivates we aimed to collect in vivo data how the completed ORF3abc alters virulence virus shedding viremia viral load of organs and humoral immune response against type II FCoV The data of our experiments show that completion of ORF3abc vested the highly virulent FIPV DF 2 with properties that are characteristic to FECV Materials and Methods Cells and Viruses Felis catus whole fetus 4 FCWF 4 cells originally purchased from the American Type Culture Collection were used for virus propagation titration and virus neutralization tests The cell line was maintained as monolayer culture in Dulbecco s Modified PLOS ONE www plosone org 1 February 2014 Volume 9 Issue 2 e88758 Eagle Medium Sigma Aldrich Saint Louis MO USA supple mented with 10 fetal bovine serum FBS 0 3 mg ml glutamine 100 U ml penicillin 0 1 mg ml streptomycin 0 25 mg ml am photericin B 1 mM sodium pyruvate and 1 non essential amino acids Sigma Aldrich The FIPV DF 2 strain was kindly provided by Berndt Klingeborn SVA Uppsala Sweden FIPV DF 2 is a regular tissue culture adapted strain that has been well described in the literature and also used by many other investigators under this name or as FIPV 79 1146 or FIPV Nor15 16 Generation of the recombinant PBFIPV DF 2 and PBFIPV DF 2 R3i was described elsewhere 17 Briefly PBFIPV DF 2 is a virus that originated as a molecular clone of FIPV DF 2 and then was successfully transfected into cat cells where it was replicated for several generations before use in this study PBFIPV DF 2 R3i is a derivate of PBFIPV DF 2 that was re engineered to contain the intact ORF3abc region of canine coronavirus and was also transfected into cat cells and cultivated for several generations before being used in this study Sequence Analysis The complete genome of PBFIPV DF 2 was reverse transcribed using the high fidelity SuperScript III First Strand Synthesis System Invitrogen Carlsbad CA USA and gene specific primers Long PCR fragments overlapping the whole genome were amplified with Phusion Hot Start High Fidelity DNA Polymerase Finnzymes Espoo Finland and sequenced using the Ion Proton System Life Technologies Carlsbad CA USA Sequences were aligned and analyzed with the SeqMan Ngen software Lasergene Madison WI USA Animal Experiments Specific pathogen free IQHsdCpb kittens Isoquimen SL Barcelona Spain were used in the challenge experiments Kittens arrived at the facility at the age of 8 12 weeks They were acclimated and used in the studies at the age of 14 18 weeks The animals were kept in separate groups in a closed facility Their FCoV negative status was checked with PCR ELISA and virus neutralization tests Kittens were inoculated oronasally with 10 3 50 tissue culture infective doses TCID 50 of the parent virus FIPV DF 2 n 4 and the recombinant viruses PFIPV DF 2 n 4 and PFIPV FD 2 R3i n 4 respectively Kittens were clinically examined on a daily basis for 42 days Cats were scored for several clinical signs as described earlier 18 Briefly scoring was based on depression inactivity for three consecutive days 1 point anorexia not eating for three consecutive days 1 point and neurological disorders swaggering 1 point on a daily basis while fever 40 1uC 1 point jaundice yellow plasma 1 point weight loss loss of 2 5 of body weight per week 1 point and lymphopenia lymphocyte count of 0 5610 9 liter was scored on weekly basis Kittens showing signs of terminal FIP were euthanized in order to avoid unnecessary suffering while healthy animals were exterminated at day 42 postinfection p i followed by full postmortem examination All animal experiments were approved and supervised by the Ethical and Animal Welfare Committee of National Food Chain Safety Office Permission No 2866 2011 The total number of animals was carefully deter mined by considering two main principles i the number of animals should be ensured proper amount of samples for statistical analysis and ii the 3R rules Replacement Reduction and Refinement must be implemented Detection of Virus Shedding To determine virus shedding in feces fecal swabs were collected at days 0 7 14 21 28 35 and 42 p i and placed in 500 mlof phosphate buffered saline PBS After vortexing and 30 min incubation the swabs were removed and the extract was centrifuged at 1000 x g for 10 min to remove cell debris The supernatant was used for subsequent PCR Viral RNA was purified using the QIAamp Viral RNA Mini Kit Qiagen Hilden Germany All RNA was stored at 80uC until used To measure the copy numbers of the genome and replicative forms of CoVs two TaqMan based quantitative real time PCR qRT PCR assays targeting the 59 end of the FIPV DF 2 genome and the N gene subgenomic sg mRNA were applied respectively 17 Each RNA sample was analyzed in duplicates in two different runs Differences in original template RNA levels were normalized by using housekeeping gene b actin PCR 19 Means of the four normalized data per sample were used for further analysis Detection of Viremia RNA was extracted from whole EDTA anticoagulated blood taken at days 0 7 14 21 28 35 and 42 p i using the QIAamp RNA Blood Mini Kit Qiagen according to the manufacturer s instructions and was subjected to genomic and subgenomic qRT PCRs Viral Load of Organs To detect virus load in different organs liver spleen kidney lung tonsil mesenteric lymph nodes brain and ileum approx imately 0 5 g pieces of organs diluted in sterile phosphate buffered saline PBS were homogenized with Tissue Lyser Qiagen Hilden Germany to obtain 50 w v suspension and then were centrifuged at 1000 x g for 10 min to remove cell debris RNA was extracted from the supernatant using the QIAamp Viral RNA Mini Kit Qiagen and was subjected to subsequent genomic and subgenomic qRT PCRs Serological Assays Serum samples were taken using VacuetteH tube Greiner Bio One Germany at days 0 7 14 21 28 35 and 42 p i For antibody ELISA tests the FCoV EIA Kit BV European Veterinary Laboratory The Netherlands was used according to the recommendations of the manufacturer For virus neutralization VN assay two fold dilutions of heat inactivated serum from kittens 50 ml were incubated for 1 hour at 37uC with equal aliquots of FIPV DF 2 50 mlof10 3 5 TCID 50 ml The viruses were then added to FCWF 4 cells showing 70 confluency in a 96 well plate and incubated for 48 h until the development of cytopathic effect Neutralizing activity was determined by end point dilution 20 Statistical Analysis To determine the statistically significant difference between the VN titers generated after inoculation with the three FCoVs the unpaired two tailed Student T test with equal variances was applied The p value under 0 05 was considered as a statistically significant difference Results Virulence of Recombinant Viruses Cats inoculated with the parent virus FIPV DF 2 showed rapid development of FIP at day 10 16 p i The animals exhibited depression and anorexia in most cases with fever jaundice weight loss and lymphopenia and they had to be euthanized between days 21 25 Table 1 Pathological examinations proved the characteristic lesions of effusive FIP with multiple dispersed In Vivo Analysis of FIPV DF 2 Recombinants PLOS ONE www plosone org 2 February 2014 Volume 9 Issue 2 e88758 pyogranulomas in the abdominal organs such as livers spleens and kidneys Surprisingly cats challenged with PBFIPV DF 2 a recombi nant FCoV containing truncated ORF3abc like the parent virus showed only clinical signs of the acute phase of the disease including transient fever from day 3 to 8 anorexia and slight lymphopenia Table 1 All cats fully recovered and survived until termination of the experiment day 42 p i Macroscopically no lesions were observed in these animals Cats inoculated with PBFIPV DF 2 R3i the recombinant FCoV containing complemented ORF3abc showed neither any clinical signs typical of FIP nor diarrhea Table 1 All cats survived and showed no macroscopic lesions except for slight enlargement of mesenteric lymph nodes in two animals In order to elucidate the unexpected low virulent phenotype of PBFIPV DF 2 the full length genomic sequence of the virion was determined using next generation sequencing and data revealed that besides the 1 nucleotid nt change at position 24429 G A that resulted in an amino acid aa change in the fusion domain of S protein at position 1332 V I and the 1 nt silent mutation at position 26064 T C in the M gene found also in the infectious clone 17 additional mutations are present in the viral genome In the ORF1ab gene three nucleotide substitutions were found at positions 3098 A G 5241 G A and 7632 C T resulting in aa changes at positions 930 T A 1644 G D and 2441 S L affecting non structural proteins nsps 3 and 4 Furthermore a single nt change was present at position 27817 C G affecting the last nucleotide of ORF 7 transcription regulatory sequence TRS and a 1 nt substitution also occurred at position 28492 G C causing an amino acid change at position 121 K N in the 7b protein The possible role of mutation of the TRS of ORF7 in decreased ORF7 mRNA transcription was examined by an ORF7 specific subgenomic qRT PCR assay and similar sub genomic ORF7 mRNA levels were detected after inoculating FCWF cells with the wild type and recombinant FCoVs indicating no effect of this mutation to mRNA transcription data not shown The genome of PBFIPV DF 2 R3i contained only the point mutations observed in the infectious clone Virus Shedding Shedding of FIPV DF 2 and PBFIPV DF 2 was detected from day 3 p i to euthanasia of the PIP diseased animals at very low and variable amounts of an average value close to the detection limit of the genomic qRT PCR 1 9610 1 FCoV RNA copies per ml fecal extract Fig 1 with undetectable virus replication using the subgenomic qRT PCR assay data not shown Virus shedding decreased to undetectable levels from day 21 p i in the PBFIPV inoculated animals The PBFIPV DF 2 R3i infected cats began to shed the virus from day 3 p i virus shedding peaked at day 7 p i with 8 3610 5 FCoV RNA copies per ml fecal extract remained high until day 14 p i then began to decrease until reaching 1 2610 2 FCoV RNA copies per ml fecal extract at day 35 p i and remained at this level until the end of the experiment Fig 1 FCoV Viremia A classic biphasic viremia was observed in FIPV DF 2 infected cats FCoV RNA was detected from day 3 p i reached a first peak of 4 8610 3 FCoV RNA copies per ml blood by day 7 p i then decreased quickly after the emergence of neutralizing antibodies A second wave of viremia was detected from day 14 p i until death peaking at 5 8610 4 FCoV RNA copies per ml blood Fig 2 The PBFIPV DF 2 infected cats developed only the first phase of viremia FCoV RNA was detected in blood from day 3 p i reached a peak of 1610 3 FCoV RNA copies per ml blood by dy 7 p i and decreased to undetectable level at day 21 Fig 2 In cats inoculated with PBFIPV DF 2 R3i complete absence of viremia was observed the presence of FCoV genomic RNA in Table 1 Total clinical scores of cats challenged oronasally with the parent virus FIPV DF 2 n 4 and recombinant FCoVs PBFIPV DF 2 n 4 and PBFIPV DF 2 R3i n 4 Virus and animal no Clinical score Total clinical score Day of death postinfection Fever Depression Anorexia Jaundice Neurological disorder Weight loss Lymphopenia FIPV DF 2 1 22231 321521 2 0 22135 3 23331 321721 4 22 2214 PBFIPV DF 2 5 11100 115 6 7 11000 002 8 1 014 PBFIPV DF 2 R3i 9 00000 000 10 00000 000 12 doi 10 1371 journal pone 0088758 t001 In Vivo Analysis of FIPV DF 2 Recombinants PLOS ONE www plosone org 3 February 2014 Volume 9 Issue 2 e88758 blood was not detected until the termination of the experiment Fig 2 FCoV Viral Load in Tissues Cats infected with FIPV DF 2 showed high viral load 4 6610 4 1 2610 7 genomic RNA copies per g tissue in the examined organs with intensive virus replication but very limited RNA copy numbers 1 1610 2 genomic RNA copies per g tissue were obtained from the gut Fig 3 In cats infected with PBFIPV DF 2 no detectable FCoV RNA copies were found in the examined organs The PBFIPV DF 2 R3i challenged animals tested highly positive 3 6610 4 genomic RNA copies per g tissue for FCoV RNA in the ileum In addition significantly lower level of positivity was observed in the mesenteric lymph node of two cats 3610 2 genomic RNA copies per g tissue Fig 3 No other organs contained genomic RNA The subgenomic qRT PCR showed replication only in the gut High copy number 10 3 genomic qRT PCR results were confirmed with subgenomic qRT PCR assay not only from organs but all fecal and blood samples data not shown Humoral Immune Response According to the pre experimental data no FCoV antibodies were detected at day 0 p i in any cat sera using ELISA and VN In the FIPV DF 2 inoculated animals neutralizing antibodies appeared by day 10 p i and reached high titers 2 4610 3 at the time of euthanasia Fig 4 The PBFIPV DF 2 challenged cats developed neutralizing antibodies from day 10 p i that elevated to lower levels 6 4610 2 by day 35 p i than in the FIPV DF 2 inoculated animals Fig 4 The difference between VN titers generated after FIPV DF 2 and PBFIPV DF 2 was statistically significant p 0 037 The PBFIPV DF 2 R3i infected cats showed variable results As ELISA and VN assays showed two animals did not seroconvert data not shown Two animals seroconverted by day 14 p i and their VN titers remained at low levels 9 6610 1 compared with those of the PBFIPV DF 2 infected cats Fig 4 The difference between VN titers generated after PBFIPV DF 2 and PBFIPV DF 2 R3i was statistically significant p 0 013 Discussion The distinctive factor of the different pathogenesis of the two FCoV biotypes is the increased macrophage tropism of FIPV 10 21 while FECV is tropic for the mature intestinal epithelium 22 23 Although alterations of several different genes of FCoV are suspected in the background of phenotypic characteristics the most widely accepted theory suggests the possible role of the truncated ORF3abc in the altered tropism and consequent pathogenesis of the two biotypes 9 14 16 18 24 However an identical FCoV pair differing only in the intactness of ORF3abc has not been tested yet in vivo due to the lack of a cell culture for propagation of type I FECV and a true type II FECV isolate 8 Our previous in vitro experiments added further evidence to the involvement of truncated ORF3abc to the increased macro phage tropism of type II FCoV 17 In the present study characterizing the parent FIPV DF 2 and the recombinant FCoV pair in in vivo experiments we were able to distinguish significant differences in their biological properties Development of typical clinical signs and post mortem lesions of classical FIP were observed in cats infected with the parent virus FIPV DF 2 similarly as it was reported earlier 5 18 25 Unexpectedly kittens inoculated with PBFIPV DF 2 showed only the acute phase of the disease with similar tropism as its wild type parent FIPV DF 2 Sequencing of the pBFIPV DF 2 infectious clone 17 and the recovered virus PBFIPV DF 2 that was passaged in FCWF three times revealed poi