key: cord-333403-imx3990a
authors: Christianson, K. K.; Ingersoll, J. D.; Landon, R. M.; Pfeiffer, N. E.; Gerber, J. D.
title: Characterization of a temperature sensitive feline infectious peritonitis coronavirus
date: 1989
journal: Arch Virol
DOI: 10.1007/bf01311080
sha: 
doc_id: 333403
cord_uid: imx3990a

The characteristics of a temperature sensitive feline infectious peritonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 °C (permissive temperature) but not at 39 °C (nonpermissive temperature). This temperature preference of TS-FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38–39 °C) prevail. Viral structural proteins and RNA were synthesized at 39 °C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS-FIPV was more thermolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protective immune responses in cats.

Feline infectious peritonitis (FIP) is a complex and fatal disease of cats caused by infection with feline infectious peritonitis virus (FIPV). Previous attempts to consistently protect cats by immunization with other antigenically related coronaviruses such as porcine transmissible gastroenteritis virus (TGEV) [26] , canine coronavirus (CeV) [2] , and human coronavirus [1] have been unsuccessful. Inconsistent protection was found when cats were given a sublethal dose of virulent FIPV and cats vaccinated with an avirulent FIPV were more easily infected than were nonvaccinated cats [14] . Recently, it has been demonstrated that an intranasally (IN) administered temperature sensitive FIP¥ (TS-FIPV) vaccine is efficacious and safe upon FIPV challenge [5] . The pathogenesis of F I P is complicated and not fully understood. Evidence indicates that FIP is an immune-mediated disease [10] . The virus has been shown to replicate initially in the upper respiratory tract and small intestine [20] . Macrophages, the primary F I P V target cell, may then cross the mucosal barrier and spread virus throughout the cat [8, 24, 25] . Furthermore, a correlation between FIPV virulence in vivo and ability to infect macrophages in vitro has been observed [18] . It has been suggested that a strong cell-mediated immune response to F I P V may be more important than humoral immunity in protecting cats from this disease [15, 19] . However, the role of local i m m u n e responses in the upper respiratory tract and intestinal tract has not been carefully evaluated and may represent an important i m m u n e defense mechanism against FIP.

TS-FIPV was developed by serial passages at a reduced temperature, followed by ultraviolet irradiation. The selected virus will propagate at its permissive temperature (31 °C) but not at its nonpermissive temperature (39 °C). Temperature sensitivity was accompanied by the appearance of various characteristics that distinguish TS-FIPV from its virulent parent strain. The purpose of this report is to present these distinguishing characteristics which include plaque size, temperature stability, ability to synthesize R N A , expression of structural proteins and temperature dependent replication in vitro and in vivo.

Norden Laboratories feline kidney (NLFK) cells were used from passage 80 to 92. Cells were propagated in Basal Medium Eagle (BME) supplemented with 5% fetal bovine serum (FBS) and 10raM Hepes buffer.

The DF2 wild type FIP virus (WT-FIPV) was originally isolated from a cat liver explant. After several passages of tissue homogenates in specific pathogen free (SPF) cats, the virus was adapted to NLFK cells by cocultivation with infected primary spleen cells. The DF2 WT-FIPV strain was grown on NLFK cells at 39 °C for passages I through 60 and then passed at 31 °C up to passage 99. The virus collected at passage 99 was ultraviolet irradiated (5 cm distance with a Westinghouse 782-30 lamp, 118 V, 60 cycles, 0.5 amps) for 5 min. The virus was plaque purified and designated TS-FIPV. Passage 13 of the plaque purified TS-FIPV was used in the characterization studies.

The plaque assay was done with confluent cell monolayers inoculated with 0.2 ml of a tenfold virus dilution. After adsorption for 1 h at 31 °C or 39 °C, the cells were overlaid with 4ml of 0.75% carboxymethylcellulose in 199 medium. Monolayers were fixed and stained with a solution of 10% formalin, 1% crystal violet and 20% ethanol 72h postinfection.

Viral growth curves were conducted to compare the growth of TS-FIPV and WT-FIPV at both 31 °C and 39 °C. TS-FIPV and WT-FIPV were inoculated onto confluent cell monolayers at a multiplicity of infection (m.o.i.) of approximately 0.1. At predetermined times over 65 h, viral fluids were aseptically removed, stored at -8 0 °C, and titrated for infectivity at the completion of the growth curve. The TCIDs0 assay for virus infectivity was done in 96-well microtiter plates by infection of 5 wells each with 100 gl of a tenfold virus dilution. TS-FIPV was titered at 31 °C and WT-FIPV was titered at 39 °C. Wells were examined for cytopathic effects (CPE) on day 5 and the method of Reed and Muench [16] was used to calculate the TCIDs0.

Thermolability studies were performed at 54 °C with WT-FIPV and TS-FIPV. Each virus was diluted 1:5 in serum-free BME. At intervals between 5 and 75m in, samples were removed, immediately cooled in an ice bath and titrated for residual infectivity.

NLFK cells were used to determine TS-FIPV virus-specific RNA synthesis. Cell monolayers were infected at an m.o.i, of approximately 10 for 1 h at 31 °C. After removal of the inoculum, 2 ml of BME containing 2% dialyzed FBS, 1 ~g/ml actinomycin D and 25 txCi [5-3H]uridine was added to duplicate cultures. Following incubation for 12 h, the radiolabeled cellular RNA was extracted according to Miller et al. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . The RNA pellets were solubilized in Readi-Protein (Beckman, Fullerton, CA) and counted in a liquid scintillation counter (Model LS3801, Beckman Instruments, Fullerton, CA).

A parallel set of cultures without radiolabel was used to monitor infectivity. The unlabeled cells and medium collected at 12 h were titrated after one freeze-thaw cycle.

Spleen cells from BALB/c mice immunized with either CCV or WT-FIPV were fused with NS 1/Ag4 mouse plasmacytoma cells (American Type Cell Culture, Rockville, MD) using standard procedures [13] . Hybridoma culture fluids were screened for specific antibody production by ELISA and Western blot. Selected hybridomas were cloned twice by limiting dilution and ascites was produced by intraperitoneal injection of 5 × 10 6 hybridoma cells into pristane-treated BALB/c mice.

The monoclonal antibodies chosen for use in this study were broadly cross-reactive against CCV, FIPV, and feline enteric coronavirus (FECV). Three antibodies designated Mab-P, Mab-N, and Mab-E were selected based on their specificities for the peplomer protein (Mab-P), nucleocapsid protein (Mab-N) and envelope protein (Mab-E). Mab-P and Mab-N were obtained from a fusion performed with a CCV-immunized mouse and Mab-E originated from a mouse immunized with WT-FIPV. All three antibodies were used as the primary antibody in the Western blot and indirect immunofluorescence assays (IFA) (see below).

The structural proteins of WT-FIPV grown at 39 °C and TS-FIPV grown at 31 °C were examined by SDS-PAGE and Western blot. SDS-PAGE was performed by a modified Laemmli system [9] . After electrophoresis, the proteins were blotted to Immobilon PVDF Transfer Membrane (Millipore, Bedford, MA) by the procedure of Towbin et al. [22] . Following transfer, the blot was blocked with 2% nonfat dried milk in PBS, incubated with Mab-P, Mab-N, or Mab-E and then reacted with alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) (Kirkegaard and Perry, Gaithersburg, MD).

TS-FIPV proteins in supernatants from cells infected at 31 °C and 39 °C were examined by Western blot. NLFK monolayers were infected at an m.o.i, of approximately 1 or 10 for K . K . Christianson et al.

1 h at 31 °C or 39 °C. The inocula were removed, the cultures were washed twice with BME and finally 2 ml of BME was added. At intervals between 0 and 96 h post-adsorption, the culture medium was removed and centrifuged (t3,000 x g) for 10 min. The supernatants were frozen at -8 0 °C until Western blot analysis.

WT-FIPV and TS-FIPV intracellular and surface antigens expressed at 31 °C and 39 °C were examined by an indirect IFA [7] . Cell monolayers were infected with WT-FIPV and TS-FIPV at an m.o.i, of approximately 0.1. At the appropriate time point the cells were rinsed with PBS and then fixed in acetone for 20min at -2 0 °C. The slides were then exposed to Mab-P, Mab-N, or Mab-E for 30 rain at 37 °C. Following incubation, the slides were rinsed in PBS and then exposed to fluorescein-conjugated goat anti-mouse IgG (H + L) (Kirkegaard and Perry, Gaithersburg, MD) for 30 rain at 37 °C. Detection of viral surface antigens by indirect IFA was done as described for detection of intracetlular antigens except the monolayers were not fixed. The cells were observed and photographed with a Zeiss Axioskop photomicroscope equipped with a Zeiss x 40 objective.

In vivo fate of the virus SPF cats (Liberty Labs, Liberty Corner, NJ), 10 to 20 months old, were used in this study. Three cats were vaccinated IN with 1 ml of TS-FIPV (106.75 TCIDs0/ml ). At 1, 2, and 4 days after inoculation one TS-FIPV vaccinated cat was sacrificed. One cat was infected orally with WT-FIPV (106.23 TCIDs0/ml ) and sacrificed on day 4.

After sacrifice, tissues were aseptically removed from the cats and frozen at -8 0 °C. Tissues were then thawed and pulverized with a manually operated tissue grinder to make a 5% to 20% (w/v) suspension using BME with antibiotics as a diluent. The suspensions were clarified by centrifugation (10,000 x g) for 3min and 0.2ml was then added to the cell monolayers for 1 h at 31 °C for TS-FIPV and 39 °C for WT-FIPV. After incubation the tissue suspensions were removed and the monolayers were washed once with BME followed by the addition of 1 ml BME. The plates were incubated at the optimal temperature for each virus and were observed for viral CPE daily for 7 days following inoculation. A standard virus neutralization test [7] was done with Mab-P on the virus positive tissue homogenates to confirm the presence of coronavirus. Cat tissues were examined by direct IFA for viral antigen.

The effect o f incubation temperature on the in vitro growth o f T S -F I P V is shown in Table 1 . T S -F I P V h a d optimal infectivity when p r o p a g a t e d and titrated at 31 °C. The CPE produced at 31 °C was characterized by formation of syncytia. When grown at 31 °C, TS-FIPV showed infectivity initially when titrated at 39 °C. However, after 4 days at 39 °C CPE was observed on less than ten percent of the monolayer and all plaques that formed were healed within 5 days. When grown at 39 °C, TS-FIPV had no detectable infectivity when assayed at 39 °C, but showed minimal infectivity when assayed at 31 °C.

The growth curves (Fig. 1) 

As can be seen in Fig. 2 , TS-FIPV was rapidly inactivated at 54 °C. TS-FIPV infectivity decreased by five logs in 15 min. WT-FIPV was more stable at 54 °C than TS-FIPV since only two logs of infectious virus were inactivated within 15 min.

The synthesis of TS-FIP virus-specific RNA, as measured by 3H-uridine incorporation, was compared at permissive and nonpermissive temperatures. Viral RNA synthesis at 31 °C and 39 °C was similar 12 h following infection, indicating that normal viral entry and initial synthesis occurred at the nonpermissive temperature. The 3H-uridine incorporation at 31 °C was 98,920 cpm and at 39 °C was 95,913cpm. However, a difference in the number of mature, infectious virions was apparent by 12 h post-infection. Virus grown for 12 h at the permissive temperature had a titer of 1.0 × 105 TCIDs0/ml, while virus grown at the nonpermissive temperature had a titer of only 2.1 × 103 TCIDs0/ml. Thus, at the nonpermissive temperature, it appears that early viral RNA synthesis occurred without a concomitant virus maturation process.

The structural proteins of WT-FIPV and TS-FIPV grown at 39 °C and 31 °C, respectively, were compared by Western blot using coronavirus-specific monoclonal antibodies. The Western blots showed that WT-FIPV and TS-FIPV had structural protein profiles characteristic of that reported for other coronaviruses [17] . The peplomer protein band had a molecular weight of 200 kDa and the apparent molecular weight of nucleocapsid was 58 kDa for both viruses (Fig. 3) . Conspicuous differences appeared in the pattern of blotted envelope proteins for the wild type and temperature sensitive viruses. The WT-FIPV envelope (Fig. 3, E, a) , whereas the envelope protein of TS-FIPV produced several protein bands from 30 kDa to 46 kDa (Fig. 3, E, b) . The 28 kDa component of TS-FIPV was present but not at the same intensity as it was for WT-FIPV. The differences in molecular weight of the envelope protein of the two viruses were confirmed using immune cat sera. Immune sera from both TS-FIPV vaccinated cats and WT-FIPV challenged cats showed the same differences in the envelope protein of the two viruses as did the monoclonal antibody (data not shown). Thus, the differences in the molecular weights of the envelope polypeptides were not due to differences in reactivity to the monoclonal antibody. There was not a difference in the molecular weights of the envelope polypeptides from TS-FIPV grown at 31 °C or 39 °C (data not shown). The specificity of the monoclonal antibodies used for antigen detection is demonstrated by the lack of reactivity with NLFK cell extracts (Fig. 3, c) . The culture supernatant from TS-FIPV infected cells was monitored for the appearance of structural proteins at both the permissive and nonpermissive temperatures. TS-FIPV grown at 31 °C for 24, 48, 72, or 96h had all three structural proteins detected in the culture supernatant (Fig. 4A) . When TS-FIPV was grown at 39 °C for similar lengths of time, only nucleocapsid was found (Fig. 4B) . Nucleocapsid may be the only protein released at 39 °C or the less abundant peplomer and envelope proteins were not detected. Figure 4 also shows that the nucleocapsid protein released at 39 °C may be associated with infectious RNA or a few viral particles were released at 39 °C because these culture supernatants were infective at 31 °C. However, at 24 h the infectivity of Intracellular synthesis of TS-FIPV and WT-FIPV proteins was compared at 31 °C and 39 °C. Synthesis was monitored by IFA using coronavirus-specific monoclonal antibodies on acetone-fixed, infected cells. All three structural proteins of TS-FIPV were detected in the cells by IFA at both the permissive and nonpermissive temperatures ( Table 2 ). The TS-FIPV proteins appeared at both temperatures within 6 h post-infection. Similar results were observed with WT-FIPV infected cells at 39°C. When incubated at 31 °C, WT-FIPV proteins appeared somewhat later. Nucleocapsid was visible by 8 h while peplomer and envelope proteins were apparent by 9 h.

Examination of the surface of infected cells by immunofluorescence using a peplomer monoclonat antibody indicated that TS-FIPV was present on the surface of living cells by 12h post-infection at 31 °C but not 39°C (Table2). Small areas of envelope surface fluorescence were observed for TS-FIPV by 18h at its permissive temperature only. After 12h at 39°C peplomer and envelope antigens were detected on the surface of WT-FIPV infected cells, but at 31 °C only slight peplomer fluorescence was found after 15 h. Nucleocapsid was not detected on the surface of cells infected with either virus.

In order to determine if the temperature sensitive characteristics of TS-FIPV observed in vitro were reflected by growth of the virus in vivo, cats were inoculated with either TS-FIPV or WT-FIPV. At predetermined times postinoculation, cats were sacrificed and tissues were examined for the presence of virus. Evidence of TS-FIPV replication in the upper respiratory tract was found by virus isolation and immunofluorescence (Table 3) . Virus was isolated from the cervical lymph node, tonsil, trachea and turbinate at 1, 2, or 4 days postvaccination. TS-FIPV antigen was identified by direct IFA in the mandibular lymph node and the tonsil. In contrast, by 4 days after oral infection with WT-FIPV, the virus had disseminated throughout the cat. WT-FIPV was isolated from four different lymph nodes (cervical, mandibular, mediastinal and ruesenteric), the oral/nasal/pharyngeal area, as well as from the thymus and spleen. All of these tissues except the thymus were positive for viral antigen by direct IFA. 

The differences in the characteristics of TS-FIPV and WT-FIPV may account for the protective immunity afforded by TS-FIPV [5] and the onset of disease by WT-FIPV. TS-FIPV and WT-FIPV differ in temperature specificity and stability, plaque size, and structural protein expression. In addition, TS-FIPV and WT-FIPV disseminate differently in the cat. Although the lesions responsible for the temperature sensitive defect have not been located, the characteristics of TS-FIPV in relation to its parent strain suggest a maturation defect. TS-FIPV growth is impaired at the optimal temperature (39 °C) for WT-FIPV but TS-FIPV replicates normally at its permissive temperature (31 °C). This temperature preference allows TS-FIPV to grow in the upper respiratory tract of cats but retards systemic growth in cats where the temperature is 39 °C. Replication of TS-FIPV in the upper respiratory tract may stimulate mucosal responses that may be required to protect cats against FIPV challenge. Mucosal immunity provided by TS-FIPV may be important in stopping the primary infection of FIPV since Stoddart et al. [20] has shown that FIPV administered orally replicated initially in the tonsil and small intestine. Also, the temperature preference of TS-FIPV may prevent a vaccine-induced sensitization of the cat. This contrasts with a previous study by Pedersen and Black [14] in which vaccination with a modified-live FIPV not only failed to protect cats against disease, but appeared to sensitize cats so they were more susceptible to FIPV challenge than were nonvaccinated cats.

WT-FIPV produced larger plaques in NLFK cells than did TS-FIPV. Tupper et al. demonstrated that two virulent FIPV strains (79-1146 and NOR 15, a plaque purified DF2 WT-FIPV) produced larger plaques than the nonvirulent FECV strain 79-1683 [23] . McKeirnan and coworkers also documented this difference in plaquing profiles between these same feline coronavirus strains [11] . It appears that avirulent feline coronaviruses produce small plaques, whereas virulent FIPV produces large plaques.

TS-FIPV RNA synthesis occurred normally at 39 °C for approximately 12 h, even though viral growth did not proceed normally. The absence of intact virion production in the presence of RNA synthesis at 39 °C suggests a defect in the maturation and assembly of the virion which has been shown in other temperature sensitive viruses [3, 4, 21] .

The detection of TS-FIPV structural proteins by indirect IFA in NLFK cells at 39 °C without concomitant virus production also indicates a maturation defect at 39 °C. Surface expression of TS-FIPV and WT-FIPV peplomer and envelope proteins but not nucleocapsid at the optimal temperature for each virus resembles the situation in FIPV-infected macrophage-like cells [8] . The expression of viral antigen on the cell surface may be important in the pathogenesis of FIPV. The absence of TS-FIPV surface antigen at 39 °C may account for the lack of sensitization in TS-FIPV vaccinated cats.

A difference was evident in the molecular weights of the envelope polypep-tides of TS-FIPV and WT-FIPV as shown by Western blotting. The low molecular weight (28 kDa) component was detected to a lesser degree for TS-FIPV than WT-FIPV. This same kind of difference was observed with two virulent strains of FIPV; UCD 1 did not have the low molecular weight component that was present in the Dahlberg strain of FIPV I-6]. These observed differences in the envelope protein may be due to differences in glycosylation. Further investigation by two-dimensional gels is needed to clearly differentiate the envelope proteins of TS-FIPV and WT-FIPV. Different characteristics are apparent between TS-FIPV and its parent strain WT-FIPV. The importance of these TS-FIPV characteristics in relation to the protective response of the virus in cats is under investigation.

Experimental inoculation of cats with human coronavirus 229 E and subsequent challenge with feline infectious peritonitis virus

Experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus

Isolation and characterization of conditionaMethal mutants of sindbis virus

Physiological characterization of heatdefective (temperature-sensitive) poliovirus mutants: preliminary classification

Protection against feline infectious peritonitis (FIP) by a temperature sensitive-FIP virus vaccine inoculated intranasally

Virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus

Comparison of serologic assays for measurement of antibody response to coronavirus in cats

Expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrpohage-like cells

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Feline infectious peritonitis (FIP)--the present state of knowledge

Comparative properties of feline coronaviruses in vitro

Temperature sensitive coronavirus characterization

Kinetics of accumulation and processing of simian virus 40 RNA in Xenopus laevis oocytes injected with simian virus 40 DNA

Immunoglobulin-producing hybrid cell lines

Attempted immunization ofcats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus

Experimental studies with three new strains of feline infectious peritonitis virus: FIPV-UCD2, FIPV-UCD 3, and FIPV-UCD4

A simple method for estimating fifty percent endpoints

The biology of coronaviruses

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence

Virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus

The sites of early viral replication in feline infectious peritonitis

Semliki forest temperature-sensitive mutants: isolation and characterization

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications

Antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus

Pathogenesis of feline infectious peritonitis: nature and development of viremia

Pathogenesis of feline infectious peritonitis: pathogenic changes and immunofluorescence

Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses

The authors thank Dr. B. Huseman for performing the necropsies and B. Suiter and Dr. M. Mellencamp for monoclonal antibody production. The critical review of this manuscript by Dr. W. Beckenhauer, S. Christianson, Dr. K. Haffer and Dr. A. Torres was greatly appreciated.

Received July 24, 1989