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Short Communication |
Laboratory of Veterinary Infectious Disease, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
Correspondence
Tsutomu Hohdatsu
hohdatsu{at}vmas.kitasato-u.ac.jp
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). FCoVs are classified into types I and II according to their S protein properties (Hohdatsu et al., 1991a; Motokawa et al., 1995, 1996). FCoVs are also classified into two biotypes based on differences in pathogenicity: feline enteric coronavirus (FECV) and FIPV. FECV is asymptomatic in cats, but FIPV infection induces feline infectious peritonitis (FIP). FIP is a fatal disease in wild and domestic cat species, mainly leading to the development of immune complex vasculitis. There are types I and II FECV and FIPV in FCoV. Type II FCoVs are known to utilize feline aminopeptidase N (fAPN) as virus receptor (Hohdatsu et al., 1998).
FIPV targets the monocyte/macrophage lineage, infection of which is enhanced in the presence of antibodies (antibody-dependent enhancement or ADE). ADE activity in FIPV infection is induced when anti-FIPV-S antibody-bound viruses infect cells of the monocyte/macrophage lineage by binding to the Fc portion of Fc receptors on the cell surface (Corapi et al., 1992; Hohdatsu et al., 1991b; Olsen et al., 1992). When anti-FIPV-S antibodies are absent, FIPV infects the monocyte/macrophage lineage via fAPN (Rottier et al., 2005). However, whether the virus receptor, fAPN, is involved in ADE of FIPV infection is not clear.
It has been reported that mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) enter cells through a low pH-dependent, endosomal pathway (Qiu et al., 2006; Simmons et al., 2005). Van Hamme et al. (2007) studied the kinetics of binding and internalization, demonstrating clearly that FIPV enters the cell via endocytosis. However, it is unclear whether acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of antibodies. In this study, we investigated the involvement of the virus receptor (fAPN) in ADE of FIPV infection and the necessity of acidification of the endosome for cellular invasion by viruses.
Feline alveolar macrophages were isolated from anti-FCoV antibody-negative specific pathogen-free (SPF) cats as described previously (Hohdatsu et al., 1991b). Feline monocytes were isolated from SPF cats as described by Dewerchin et al. (2005). mAb 6-4-2 (IgG2a), used in the present study, recognizes the S protein of type II FIPV, as demonstrated by immunoblotting. It has been reported that mAb 6-4-2 exhibits a neutralizing activity in Felis catus whole fetus (Fcwf)-4 and Crandell feline kidney (CrFK) cells, but an enhancing activity in feline macrophages, depending on the reaction conditions (Hohdatsu et al., 1993). As a mAb to recognize fAPN, R-G-4 (IgG1), prepared by our laboratory (Hohdatsu et al., 1998), was used. Mouse IgG1 mAb recognizing feline gamma interferon, as control for R-G-4, was prepared by our laboratory. RNA was isolated from cells by a method reported previously (Takano et al., 2007). To synthesize cDNA from FIPV negative-strand RNA, 1 µl RNA extract and 0.02 mol sense primer for the FIPV N gene (positions 876–895, 5'-CAACTGGGGAGATGAACCTT-3') were added to Ready-to-Go RT-PCR beads (GE Healthcare Life Sciences) and the volume was adjusted to 50 µl with water. The resulting solution was incubated at 42 °C for 1 h to synthesize cDNA. cDNA was amplified by PCR using primers specific for the FCoV N gene (sense primer, positions 876–895, 5'-CAACTGGGGAGATGAACCTT-3'; antisense primer, positions 1644–1663, 5'-GGTAGCATTTGGCAGCGTTA-3'). PCR was performed as reported previously (Takano et al., (2007). Data were analysed by Student's t-test. P values of <0.01 and <0.05 are used to indicate significant differences between compared groups.
The influence of the presence of anti-fAPN antibodies on ADE of FIPV infection of the feline monocyte/macrophage lineage was investigated. When feline macrophages and monocytes were treated with a mixture of type II FIPV strain 79-1146 (kindly provided by Dr M. C. Horzinek, Utrecht University, the Netherlands) and mAb 6-4-2, which enhances FIPV infection, virus production was increased significantly (P<0.01) compared with that after inoculation with virus alone, showing that infection was enhanced (Fig. 1; Methods A and C). When feline alveolar macrophages and monocytes were incubated with mAb R-G-4 at 4 or 37 °C beforehand and then inoculated with FIPV strain 79-1146, infection was inhibited completely (Fig. 1; Method B). However, when feline alveolar macrophages and monocytes were incubated with mAb R-G-4 at 4 or 37 °C beforehand and then inoculated with a mixture of FIPV and mAb 6-4-2, infection was not inhibited, but enhanced, as in cells without mAb R-G-4 treatment (Fig. 1; Method D).
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). Virus production in the culture supernatant also decreased in a manner dependent on lysosomotropic-agent concentration (Fig. 2).
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). U937 cells (1x106) were incubated with mAb R-G-4 at 4 °C for 1 h. The cells were washed, resuspended in Hanks' balanced salt solution (HBSS) containing 0.1 % NaN3 and then incubated with fluorescein isothiocyanate-conjugated Fab of goat anti-mouse IgG antibody (MP Biomedicals) at 4 °C for 1 h. The cells were washed and the number of stained cells was determined by counting about 10 000 cells on a FACS 440 flow cytometer (Becton Dickinson). Fcwf-4 cells reacted with mAb R-G-4, but U937 cells did not. In addition to ADE of FIPV infection, we investigated the effects of lysosomotropic agents: U937 cells (2x105) were cultured in medium containing the lysosomotropic agents at 37 °C for 1 h. After washing, FIPV strain 79-1146 (1x106 TCID50) reacted with mAb 6-4-2 or HBSS at 4 °C for 1 h was added to the culture and adsorbed to the cells at 37 °C for 3 h in the presence of the lysosomotropic agents. The cells were washed and cultured at 37 °C for 48 h, and supernatants and cells were collected. When U937 cells were inoculated with a mixture of FIPV and mAb 6-4-2, the virus apparently replicated (Fig. 3). However, when cells were inoculated with the mixture in the presence of the lysosomotropic agents, intracellular expression of FIPV negative-strand RNA and virus production in the culture supernatant decreased in a manner dependent on the lysosomotropic-agent concentration (Fig. 3). When cells were inoculated with the virus alone without mAb 6-4-2 treatment, no viral replication was noted, regardless of the presence or absence of the lysosomotropic agents.
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; Sullivan, 2001; Tirado & Yoon, 2003). For human immunodeficiency virus (HIV) infection, the necessity of the HIV receptor, CD4, for ADE induction, in addition to Fc receptors, has been reported (Takeda et al., 1990). In contrast, in foot-and-mouth disease virus infection, virus receptor-independent ADE activity was detected in an experiment using a mutant virus lacking virus receptor-binding ability (Mason et al., 1994). The involvement of Fc receptors in ADE of FIPV infection has been reported, but the necessity of the virus receptor, fAPN, was unclear. First, we investigated whether anti-fAPN mAb blocks ADE of FIPV infection in feline alveolar macrophages and monocytes. When anti-fAPN mAb was incubated with cells at 37 °C, ADE of FIPV infection was not blocked. However, it was considered possible that capping could have occurred on the cell surface at 37 °C, resulting in incorporation of anti-fAPN mAb into the cytoplasm, which avoided blocking of FIPV binding to the virus receptor. Thus, we reduced the anti-fAPN mAb reaction temperature to 4 °C and repeated the experiment, but ADE of FIPV infection was not blocked, as in the experiment at 37 °C. We also clarified that FIPV alone could not establish infection in fAPN-negative, Fc receptor-positive U937 cells, but the virus apparently replicated when a mixture of FIPV and an anti-FIPV-S antibody, mAb 6-4-2, was used for inoculation, suggesting strongly that the virus receptors are not necessary for the induction of ADE of FIPV infection.
We demonstrated that (i) cellular invasion by FIPV via the virus receptors was inhibited by lysosomotropic agents in a concentration-dependent manner, and (ii) cellular invasion via Fc receptors (ADE of FIPV infection) was also inhibited by lysosomotropic agents in a concentration-dependent manner, revealing that acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of anti-FIPV-S antibodies. FIPV that entered cells of the monocyte/macrophage lineage may somehow escape digestion in late endosomes and lysosomes, and release the viral genome into the cytoplasm. Gollins & Porterfield (1986) reported that the viral core transfers to the cytosol in pre-lysosomal endosomes (late endosomes) 1–3 min after antibody-mediated entrance of West Nile virus into the cell. However, this process is limited to viruses that use virus receptors in ADE. It is not clear whether this phenomenon occurs when the virus receptors are not used, as in ADE of FIPV infection. Detailed investigation of the following by electron-microscopic and fluorescence-microscopic analyses is expected: (i) the mechanism by which FIPV that has entered cells via Fc receptors escapes digestion in late endosomes and lysosomes, and (ii) whether endosomal protease is involved in cellular invasion by FIPV, as in that by SARS-CoV and MHV-2 (Qiu et al., 2006; Simmons et al., 2005).
Addie et al. (1995) reported that FCoV reinfection of anti-FCoV antibody-positive domestic cats might not result in the development of ADE. Although the details are unclear, differences in the immunological condition of FCoV-infected cats may be the reason that the phenomenon noted in this experiment does not occur in domestic cats. It is assumed that ADE does not occur in cats that have acquired strong cellular immunity, even if they possess anti-FCoV antibodies, escaping from FIP development. It may also be possible that anti-FCoV antibodies prevent FIPV infection, avoiding ADE, when the anti-FCoV neutralizing-antibody titre is high.
We clarified that fAPN is not involved in ADE of FIPV infection and that acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of antibodies. We reported previously that ADE activity in FIPV infection of feline alveolar macrophages caused overproduction of tumour necrosis factor alpha, which may lead to serious FIP symptoms (Takano et al., 2007). ADE-associated aggravation of the pathology of FIP in cats is being elucidated, but further studies are necessary to understand the aggravation mechanism. Clarification of ADE of FIPV infection is also important for understanding ADE of other virus infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 November 2007;
accepted 5 January 2008.
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) or FIPV alone (
) at 37 °C for 1 h. After 3 days, the intracellular FIPV negative-strand RNA level or virus titre in the cell-culture supernatant was measured (n=4). (a–c) Alveolar macrophages; (d–f) monocytes. (a, d) Chloroquine treatment; (b, e) ammonium chloride treatment; (c, f) bafilomycin A1 treatment. **P<0.01; *P<0.05 versus medium without lysosomotropic agents.