Feline Interferon Research Articles

Correlation Between MicroRNA by Extracellular Vesicle Mediated and Antiviral Effects of Interferon Omega in Feline Peripheral Blood.

Feline interferon omega (IFN-ω) has been proven to have high antiviral activity; however, its in-depth antiviral effects remain unknown. Extracellular vesicles (EVs) have been demonstrated to participate in the regulation of the immune response pathway for the body through various active substances, especially through the microRNA (miRNA) carried by them. In this study, we isolated EVs from feline peripheral blood by differential centrifugation, and further found that the content of IFN-ω in EVs increased continuously within 24 h after IFN-ω treatment, and a large number of miRNAs were significantly downregulated in EVs within 12 h after IFN-ω treatment. These significantly differentially expressed miRNAs were important for regulating changes in antiviral cytokines. This study reveals for the first time the correlation between EVs-mediated miRNA in feline peripheral blood and IFN-ω on antiviral immune response, which may provide strong data support for the development of novel antiviral nanomedicine and the research of the antiviral effects of IFN-ω.

Cloning and codon optimization of a novel feline interferon omega gene for production by Pichia pastoris and its antiviral efficacy in polyethylene glycol-modified form

ABSTRACT Feline viral diseases, such as feline panleukopenia, feline infectious peritonitis, and feline coronaviral enteritis, seriously endanger the health of cats, and restrict the development of pet industry. Meanwhile, there is a current lack of effective vaccines to protect against feline viral diseases. Thus, effective therapeutic agents are highly desirable. Interferons (IFNs) are important mediators of the antiviral host defense in animals, particularly type I IFNs. In this study, a novel feline IFN omega (feIFN-ω) gene was extracted from the cat stimulated with feline parvovirus (FPV) combined with poly(I:C), and following codon optimization encoding the feIFN-ω, the desired gene (feIFN-ω’) fragment was inserted into plasmid pPICZαA, and transformed into Pichia pastoris GS115, generating a recombinant P. pastoris GS115 strain expressing the feIFN-ω’. After induction, we found that the expression level of the feIFN-ω’ was two times more than that of feIFN-ω (p < 0.01). Subsequently, the feIFN-ω’ was purified and modified with polyethylene glycol, and its antiviral efficacy was evaluated in vitro and in vivo, using vesicular stomatitis virus (VSV) and FPV as model virus. Our results clearly demonstrated that the feIFN-ω’ had significant antiviral activities on both homologous and heterologous animal cells in vitro. Importantly, the feIFN-ω’ can effectively promote the expression of antiviral proteins IFIT3, ISG15, Mx1, and ISG56, and further enhance host defense to eliminate FPV infection in vivo, suggesting a potential candidate for the development of therapeutic agent against feline viral diseases.

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Оцінка ефективності препарату «Феліферон»® в комплексній терапії інфекційного ринотрахеїту котів

Relevance of the topic. Infectious feline rhinotracheitis, like herpesvirus infection is an acute disease characterized by damage to the eyes and respiratory system. Cats of all breeds are ill regardless of age. Kittens can become infected from mothers carrying the virus. In small populations of healthy animals the prevalence of the virus is less than 1 %, while in large populations it can reach 20 %. In shelters, the risk of contamination and infection is higher - up to 50 %. Almost all animals with primary infection become latent carriers throughout their lives. The incidence can reach 55%, the mortality rate is 5 – 25 %. The causative agent - a DNA-containing virus has a tropism for the epithelial cells of the mucous membranes of the nasal cavity, tonsils, trachea, conjunctiva. Diagnostic methods for detecting latent carriage have not yet been developed, since the virus remains in the form of genomic DNA in the nucleus of latently infected neurons without replication. Laboratory diagnosis of feline rhinotracheitis is currently most often performed by isolating the pathogen or by PCR using swabs from the oral cavity and conjunctiva. The PCR method allows detecting the virus and is the laboratory “gold standard” of diagnostics. Antibodies to the pathogen can be detected using a neutralization test or ELISA in serum, conjunctival mucus and cerebrospinal fluid. Direct methods for diagnosing herpesvirus infection in chronic or latent course have not been developed. For the treatment of viral diseases of small pets, immunomodulators are widely used, which stimulate the immune system. Some drugs tend to induce the production of its own interferon in the body. However, their use often does not give a therapeutic effect due to the peculiarities of the pathogenesis of the disease and the replication of the pathogen. To date, preparations based on the species-specificity of feline interferon have not been available on the domestic market, and attempts to use human interferon for the treatment of viral infections in cats have not yielded the expected result with prolonged and repeated courses due to the formation of neutralizing antibodies in the animal's body.

In vitro Effect of Recombinant Feline Interferon-Ω (rFeIFN-Ω) on the Primary CanineTransmissible Venereal Tumor Culture.

Background: Interferons (IFNs), signaling proteins produced by host cells, are secreted in response to pathogen activity as well as to tumor cells, and display antiviral, antiproliferative, and immunomodulatory effects. Recombinant feline interferon omega (rFeIFN-ω) has in vitro growth inhibition activities on various canine and feline tumor cell lines. Canine transmissible venereal tumor (CTVT) is used as an animal model for immunotherapy due to its specific growth phase. Previous studies have usually focused on the interaction between tumor infiltrating lymphocytes (TILs) and CTVT cells. However, the specific effects of rFeIFN-ω on CTVT cells remains poorly defined.Aims: The aims of this study, therefore, were to evaluate the in vitro effect of rFeIFN-ω on primary CTVT cells and to study the mRNA expression of apoptotic genes and drug resistance genes.Materials and Methods: Purified CTVT cells were treated with various concentrations of rFeIFN-ω and the viability of the cultured cells was ascertained at 24, 48, and 72 h post treatment (hpt) and a dose-response curve plotted. The mRNA expression of apoptotic (BAX and BCL-2) and drug resistance (ABCB1 and ABCG2) genes was performed by reverse transcription quantitative real-time PCR at 72 hpt.Results: rFeIFN-ω displayed an effect against CTVT cell viability, which decreasing viability in a dose-dependent manner within 72 hpt. The relative mRNA expression of BCL-2 was upregulated only at a rFeIFN-ω concentration of 104 IU/100 μl. However, higher concentrations of rFeIFN-ω gave a higher level of relative mRNA expression of ABCB1 transporter gene.Conclusion: This study provided the information of in vitro effect of rFeIFN-ω on CTVT cell viability in a dose dependent manner, as well as, the alteration of BCL-2 and ABCB1 gene expression after treatment. These results encourage future in vivo studies to evaluate the potential efficacy of this treatment in CTVT cases.

Novel recombinant feline interferon carrying N-glycans with reduced allergy risk produced by a transgenic silkworm system

BackgroundThe generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity.ResultsA cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN.ConclusionsThe lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ was recovered after purification with a specific activity of 1 × 106IUmg-1. By this method, we obtained 8.9 × 104 IU of purified rFeIFNβ per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 104 IUmL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNβ was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNβ production in laboratory research or veterinary medicine applications.

Identification of Feline Interferon Regulatory Factor 1 as an Efficient Antiviral Factor against the Replication of Feline Calicivirus and Other Feline Viruses.

Interferons (IFNs) can inhibit most, if not all, viral infections by eliciting the transcription of hundreds of interferon-stimulated genes (ISGs). Feline calicivirus (FCV) is a highly contagious pathogen of cats and a surrogate for Norwalk virus. Interferon efficiently inhibits the replication of FCV, but the mechanism of the antiviral activity is poorly understood. Here, we evaluated the anti-FCV activity of ten ISGs, whose antiviral activities were previously reported. The results showed that interferon regulatory factor 1 (IRF1) can significantly inhibit the replication of FCV, whereas the other ISGs tested in this study failed. Further, we found that IRF1 was localized in the nucleus and efficiently activated IFN-β and the ISRE promoter. IRF1 can trigger the production of endogenous interferon and the expression of ISGs, suggesting that IRF1 can positively regulate IFN signalling. Importantly, the mRNA and protein levels of IRF1 were reduced upon FCV infection, which may be a new strategy for FCV to evade the innate immune system. Finally, the antiviral activity of IRF1 against feline panleukopenia virus, feline herpesvirus, and feline infectious peritonitis virus was demonstrated. These data indicate that feline IRF1 plays an important role in regulating the host type I IFN response and inhibiting feline viral infections.

The Use of Recombinant Feline Interferon Omega Therapy as an Immune-Modulator in Cats Naturally Infected with Feline Immunodeficiency Virus: New Perspectives.

Type I interferons (IFNs) are well-known cytokines that, among their main functions, are key components of the host immune response against viral infections. Due to its immune modulation properties, they are commonly used in the therapeutic approach of various retroviral infections, namely human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV). In HIV infection, it has been shown that IFN therapy limits early viral replication, particularly useful on post-exposure prophylaxis. In veterinary medicine, recombinant feline interferon omega (rFeIFN-ω) was the first interferon licensed for use in cats. Several studies have recently shown that this compound seems to stimulate the innate immunity, decreasing clinical signs and co-infections in naturally FIV-infected cats. More than summarizing the main conclusions about rFeIFN-ω in cats, this review emphasizes the immune-modulation properties of IFN therapy, opening new perspectives for its use in retroviral infections. Either in FIV-infected cats or in HIV individuals, type I IFNs seem to induce an innate immune-modulation and should not be overlooked as a therapeutic option in retroviral infections.

Evaluation of viremia, proviral load and cytokine profile in naturally feline immunodeficiency virus infected cats treated with two different protocols of recombinant feline interferon omega

This study assesses viremia, provirus and blood cytokine profile in naturally FIV-infected cats treated with two distinct protocols of interferon omega (rFeIFN-ω).Samples from FIV-cats previously submitted to two single-arm studies were used: 7/18 received the licensed/subcutaneous protocol (SC) while 11/18 were treated orally (PO). Viremia, provirus and blood mRNA expression of interleukin (IL)-1, IL-4, IL-6, IL-10, IL-12p40, Interferon-γ and Tumor Necrosis Factor-α were monitored by Real-Time qPCR. Concurrent plasma levels of IL-6, IL-12p40 and IL-4 were assessed by ELISA.IL-6 plasma levels decreased in the SC group (p = 0.031). IL-6 mRNA expression (p = 0.037) decreased in the PO group, albeit not sufficiently to change concurrent plasma levels. Neither viremia nor other measured cytokines changed with therapy. Proviral load increased in the SC group (p = 0.031), which can be justified by a clinically irrelevant increase of lymphocyte count.Independently of the protocol, rFeIFN-ω seems to act on innate immunity by reducing pro-inflammatory stimulus.

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28±0.15)×106Uml−1 and (1.3±0.2)×106Uml−1 respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7±0.2)×106Uml−1 and (3.5±0.4)×106Uml−1 at 2dpi in Rachiplusia nu larvae and (1.1±0.2)×106Uml−1 and (1.0±0.15)×106Uml−1 at 5dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.

Oral and subcutaneous therapy of canine atopic dermatitis with recombinant feline interferon omega

Canine atopic dermatitis (CAD) is a common allergic skin disease that has been treated with subcutaneously administered interferons (IFN). Recombinant feline IFN-ω (rFeIFN-ω) was reported to be efficacious for CAD. Whether dogs develop neutralizing antibodies against rFeIFN-ω during long-term treatment and whether orally administered IFNs are efficacious in CAD is unknown. The aim of this study was to evaluate the potential development of antibodies against rFeIFN-ω in atopic dogs and to compare subcutaneous and oral IFN therapy.Twenty-six atopic dogs were randomly assigned to two groups. The first group (n=15) received eight subcutaneous injections of rFeIFN-ω (Virbagen® omega, Virbac, Carros, France) over four months, the second group (n=11) received rFeIFN-ω daily orally. Concurrent medication was permitted, except systemically acting glucocorticoids and cyclosporin, which had to be withdrawn at least two weeks prior to the study. Serum samples for antibody detection were collected before and after the study. On days 0, 60 and 120 skin lesions and pruritus were evaluated using a validated lesion score (Canine Atopic Dermatitis Extent and Severity Index=CADESI) and a validated pruritus score. Concurrent medications were recorded. For every visit a total score, consisting of CADESI, pruritus score and medication score was created. For antibody detection an indirect ELISA, using Virbagen® omega as antigen, was performed.Comparison of pruritus scores, CADESI and total scores between days 0 and 120 showed improvement in both groups, however, significant improvement could only be detected in the oral group with CADESI and total scores (61%, P=0.04 and 36%, P=0.02 respectively). Serum antibodies against rFeIFN-ω could not be detected in any of the dogs.In this study antibody production could not be demonstrated. It suggests better efficacy with oral IFN administration, which should be further verified in larger, randomized, controlled studies.

Effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats

To determine whether 14-day topical ocular administration of high doses of feline recombinant interferon omega (FelFN) or human recombinant interferon alpha-2b (HulFN) solution improves clinical disease and decreases virus shedding in cats with naturally acquired viral keratoconjunctivitis. 36 cats with upper respiratory tract disease and ocular involvement. Cats received 1 drop of FelFN solution (1 × 10(6) U/mL), HulFN solution (1 × 10(6) U/mL), or saline (0.9% NaCl) solution (12 cats/group) in each eye twice daily for 14 days (beginning day 1). Oropharyngeal and conjunctival swab samples were collected from each cat before (day 0) and on day 14 of treatment for virus isolation (VI) and real-time quantitative PCR (RT-qPCR) testing to detect feline herpesvirus-1 and feline calicivirus. Subjective clinical scores were recorded on days 0, 3, 7, 10, and 14. The number of cats for which feline herpesvirus-1 was detected via VI or RT-qPCR assay was generally (albeit not always significantly) lower on day 14, compared with day 0 findings; however, findings on days 0 or 14 did not differ among groups. The number of cats for which feline calicivirus was detected via VI or RT-qPCR assay did not differ significantly between days 0 and 14 for any group. Clinical scores significantly decreased over the 14-day period but did not differ among groups. In cats with naturally occurring viral keratoconjunctivitis, bilateral ocular administration of high doses of FelFN or HulFN twice daily for 14 days did not improve clinical disease or virus shedding, compared with treatment with saline solution.

Relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter

Feline Immnunodeficiency (FIV) and Feline Leukemia (FeLV) viruses are common infectious agents in stray cats and shelter environments. Recombinant feline interferon-ω (rFeIFNω) has shown an antiviral action not only against FIV and FeLV but also against herpesvirus (FHV-1) and calicivirus (FCV).Sixteen naturally infected FIV/FeLV cats were followed during rFeIFNω therapy in order to monitor clinical signs and to correlate with excretion of concomitant viruses (FCV, FHV-1, feline coronavirus (FCoV) and parvovirus (FPV)). Cats were submitted to clinical evaluations and concomitant virus excretion assessement.Comparing D0–D65, 10/16 cats improved clinical scores. Of the 10 cats positive for FHV-1 on D0, 4 were negative and 6 reduced viral loads. Of the 11 FCoV positive cats, 9 reduced viral loads. The 13 FCV positive cats and the FPV positive cat were negative on D65.In conclusion, rFeIFNω improves clinical signs and reduces concurrent viral excretion in naturally infected retroviral cats.

Manufacturing Pharmaceutical-Grade Interferons Using Silkworm- Baculovirus System

To develop feline and canine interferons as medicine for companion animal, silkworm-baculovirus expression system was established as a manufacturing technology. Thus produced interferons were successfully cleared for a sale in commercial market. For expression of feline interferon in the silkworm, recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) was constructed carrying feline interferon cDNA. When the recombinant BmNPV was injected to silkworm larvae, as much as 1.2 × 10 8 unit/mL of feline interferon was produced in their hemolymph. This productivity is 100–fold higher than those of E. coli, yeast and mammalian cell expression system. Similarly, canine interferon-γ was produced in the largest amount in silkworm. Two or more forms of interferons were produced due to various processing in silkworm. For feline interferon, two forms of protein molecule resulting from two different cleavage sites in signal peptide sequence was solved by reconstitution of a single amino acid in signal sequence. In the case of canine interferon- γ, several variations were produced due to glycosylation pattern and C-terminal degradation, but removal of glycosylation sites and elimination of C-terminal 16 amino acids sequence from native sequence lead to uniformity of the expressed protein. Drug formulated two recombinant animal interferons were approved as a treatment of viral diseases and atopic dermatitis by regulatory authorities. Especially, feline interferon is now sold in 20 countries including Japan and Europe.

Comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, double-blind study in 39 cats

Chronic caudal stomatitis with alveolar/buccal mucositis in calicivirus-positive cats is the most severe presentation of feline chronic gingivostomatitis. Refractory cases are helped by antibiotic and anti-inflammatory treatments often including glucocorticoids. In order to evaluate the comparative efficacy of oromucosal administration of recombinant feline interferon omega (rFeIFN-ω) versus oral administration of glucocorticoids, a randomised, multi-centre, controlled, double-blind study was performed in 39 cats. The progression of behavioural, clinical and lesional scores was assessed over 90 days. Daily oromucosal treatment with 0.1 MU of rFeIFN-ω was associated with a significant improvement of clinical lesions (caudal stomatitis and alveolar/buccal mucositis) and a decrease of pain scores from D0 to D90. Although no such statistical improvement was noticed in the prednisolone group, there was, however, no significant difference between the two groups for most of the parameters, except pain at D60 and D90.

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)- γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8+fIFN-γ+ cells, but not CD4+fIFN-γ+ cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.

Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in the spike 2 domain and the nucleocapsid protein of feline infectious peritonitis virus

The antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection has been recognized in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis (FIP). In the present study, we synthesized eighty-one kinds of peptides derived from the spike (S)2 domain of type I FIPV KU-2 strain, the S2 domain of type II FIPV 79-1146 strain, and the nucleocapcid (N) protein of FIPV KU-2 strain. To detect the T helper (Th)1 epitope, peripheral blood mononuclear cells (PBMCs) obtained from FIPV-infected cats were cultured with each peptide, and Th1-type immune responses were measured using feline interferon (fIFN)-γ production as an index. To detect the linear immunodominant antibody-binding epitope, we investigated the reactivity of plasma collected from FIPV-infected cats against each peptide by ELISA. Four and 2 peptides containing Th1 epitopes were identified in the heptad repeat (HR)1 and inter-helical (IH) regions of the S2 domain of type I FIPV, respectively, and these were located on the N-terminal side of the regions. In the S2 domain of type II FIPV, 2, 3, and 2 peptides containing Th1 epitopes were identified in the HR1, IH, and HR2 regions, respectively, and these were mainly located on the C-terminal side of the regions. In the S2 domain of type I FIPV, 3 and 7 peptides containing linear immunodominant antibody-binding epitopes were identified in the IH and HR2 regions, respectively. In the S2 domain of type II FIPV, 4 peptides containing linear immunodominant antibody-binding epitopes were identified in the HR2 region. The Th1 epitopes in the S2 domain of type I and II FIPV were located in different regions, but the linear immunodominant antibody-binding epitopes were mostly located in the HR2 region. Eight peptides containing Th1 epitopes were identified in N protein, and 3 peptides derived from residues 81 to 100 and 137 to 164 showed strong inductivity of fIFN-γ production in PBMCs isolated from type I FIPV- and type II FIPV-infected non-FIP cats. In N protein, 4 peptides containing linear immunodominant antibody-binding epitopes were identified, and 2 peptides derived from residues 345 to 372 showed strong reactivity with plasma of type I FIPV- and type II FIPV-infected cats. The Th1 and linear immunodominant antibody-binding epitopes were located at different positions in both the S2 domain and N protein. Our results may provide important information for the development of peptide-based vaccine against FIPV infection.

Molecular Characterization of Feline Type I Interferon Receptor 2

The cDNA sequence of feline interferon receptor 2 (feIFNAR2) was generated using RT-PCR method in present study. This gene included 1,572 bp and encoded a 523 aminoacid (aa) protein with a 35 aa signal peptide. The deduced protein shared 61% amino acid identity to the human IFNAR2. There were two fibronectin type III (FBN-III) domains of about 110 residues in the extracellular domain. Homology modeling of feIFNAR2 presented a similar structure with other IFN receptors. The ELISA and FACS experiments demonstrated that the protein could bind to feIFN-alpha or feIFN-omega. However, antiviral activity assay found that feIFN-omega had broader species spectrum compared with feIFN-alpha. To define the functional differences, several point mutations of feIFNAR2 were constructed and the relative affinities of feIFN-alpha or feIFN-omega for feIFNAR2 and mutants were evaluated. The results suggested that feIFN-alpha and feIFN-omega had different binding sites on feIFNAR2. T75 and M77 on feIFNAR2 were hotspots for binding to feIFN-alpha, but not to feIFN-omega. These findings suggested that the cloned feline IFNAR2 interacted with both feIFN-alpha and feIFN-omega, however, not sharing the same binding sites.

Sensitivity of FCV to recombinant feline interferon (rFeIFN)

Feline calicivirus cause feline respiratory diseases, and inactivated and attenuated vaccines are available for its prevention. Moreover, the presence of vaccine breakdown strains (VBS) is problematic. In Japan, feline recombinant interferon (rFeIFN) has been used for its treatment. However the method of compare with each strains has not established. To examine the relationship between the breakdown vaccine strain and rFeIFN sensitivity, the sensitivity of 47 field isolates to rFeIFN was determined. The Log PDD(50) values were normally distributed within the range 1.1-3.7, with a mean value of 2.3 +/- 0.64. Since 68.3% of the PDD values fell in the range of the mean +/- standard deviation, the values in the range 1.7-2.9, the lower values, and the higher values were defined as representing moderate, low, and high sensitivity, respectively. Among the 15 vaccine breakdown strains, strain Fukuoka9 showed a low sensitivity, but strains ML89, T58, and N74 were highly sensitive, showing no association with vaccine breakdown. The amino acid sequence changes specific to the low rFeIFN-sensitive Fukuoka-9 strain were found, suggesting that these sites are involved in rFeIFN sensitivity.