key: cord-329642-5t8yuq4v
authors: Takano, Tomomi; Katoh, Yasuichiroh; Doki, Tomoyoshi; Hohdatsu, Tsutomu
title: Effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo
date: 2013-05-03
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2013.04.016
sha: 
doc_id: 329642
cord_uid: 5t8yuq4v

Feline infectious peritonitis (FIP) is a feline coronavirus-induced fatal disease in domestic and wild cats. Several studies have investigated potential treatments for FIP. However, there have been no reports on agents that have exhibited a therapeutic effect. Recently, chloroquine has been reported to antiviral effect. We investigated whether chloroquine can be used to treat FIP in vitro and in vivo. It was demonstrated that chloroquine has inhibitory effect against the replication of FIPV and anti-inflammatory effect in vitro. In vivo study using cats with experimentally induced FIP, the clinical score of chloroquine-treatment groups were better than in chloroquine-untreated group. However, alanine aminotransferase levels increased in the chloroquine-treated groups. It will be necessary to further investigate the possibility of FIP treatment with a combination of chloroquine and other agents.

Coronaviruses cause diseases of the respiratory, digestive, and nervous systems in mammals and birds. After severe acute respiratory syndrome coronavirus (SARS-CoV) was newly identified as a pathogen of fatal respiratory disease in 2003 (Ksiazek et al., 2003) , new coronavirus species have been isolated from various animals including humans. Recently, it has been suggested that coronavirus may be transmitted from animals to humans (Müller et al., 2012) .

Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus (FCoV) of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. Cats that developed FIP were affected in several organs, including the liver, lungs, spleen, and central nervous system, forming lesions accompanied by necrosis and pyogenic granulomatous inflammation (Pedersen, 2009) . In some cats, pleural effusion and ascitic fluid accumulated.

Monocytes and macrophages (monocytes/macrophages) play an important role in the pathogenesis of FIP. It has been reported that virus replication in monocytes/macrophages induced interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a production (Regan et al., 2009; Takano et al., 2007b Takano et al., , 2009 . FIPV replication and cytokine production are enhanced when monocytes/macrophages are inoculated with FIPV in the presence of anti-FIPV S antibodies (antibody-dependent enhancement: ADE) (Hohdatsu et al., 1991; Corapi et al., 1992; Olsen et al., 1992) . The phenomenon of ADE is involved in the aggravation of the pathology of FIP (Pedersen and Boyle, 1980; Takano et al., 2007b) .

Over the past forty years, several studies have investigated potential treatments for FIP (Hartmann and Ritz, 2008) . Antiviral, immunostimulating, and immunosuppressive drugs have been experimentally used for the treatment of FIP, but none of these have exhibited a sufficient therapeutic effect. Several agents that significantly inhibit FCoV replication in vitro have been identified (Balzarini et al., 2006; Barlough and Shacklett, 1994; Hsieh et al., 2010; Kim et al., 2012; Takano et al., 2008) ; however, whether or not these agents exhibit a therapeutic effect in cats with FIP has not been investigated.

The anti-malarial drug, chloroquine, has been reported to inhibit the replication of human immunodeficiency virus (HIV), influenza A/H5N1 virus, SARS-CoV, and human coronavirus 229E (Kono et al., 2008; Murray et al., 2010; Savarino et al., 2003; Vincent et al., 2005; Yan et al., 2012) . Chloroquine also has been used to treat immune-mediated inflammatory disorders (Karres et al., 1998; Landewé et al., 1994; Lesiak et al., 2010) . FIP is a viral infection that causes an immune-mediated inflammatory disease. On the basis of these findings, chloroquine may be effective as a therapeutic drug for FIP.

We investigated whether or not chloroquine inhibited FIPV replication in vitro and the FIPV-induced ADE activity of monocytes. Furthermore, we investigated the effect of chloroquine for cats with experimentally induced FIP.

Felis catus whole fetus-4 (fcwf-4) cells were grown in Eagles' minimum essential medium containing 50% L-15 medium, 5% fetal calf serum (FCS), and antibiotics. Feline monocytes were cultured in RPMI 1640 medium containing 10% FCS and antibiotics. Strain FIPV 79-1146 was grown in fcwf-4 cells. Strain FIPV 79-1146 was supplied by Dr. M.C. Horzinek (Utrecht University).

MAb 6-4-2 (IgG2a) used in the present study recognizes the Spike (S) protein of type II FIPV, as demonstrated by immunoblotting. It has been reported that MAb 6-4-2 exhibits a neutralizing activity in fcwf-4 cells, and an enhancing activity in feline monocytes/macrophages depending on the reaction conditions (Hohdatsu et al., 1993) .

Feline monocytes were isolated from specific pathogen-free (SPF) cats as previously described by Dewerchin et al. (2005) .

Confluent fcwf-4 cell monolayers were cultured in medium containing chloroquine (Wako, Japan) in 24-well multi-plates at 37°C for 1 h. After washing, FIPV strain 79-1146 (1 Â 10 4 TCID 50 ) was added to the culture and adsorbed to the cells at 37°C for 1 h in the presence of chloroquine. After washing, cells were cultured in medium containing chloroquine, and culture supernatants were collected after 1 day. The experiment using feline monocytes from SPF cats was performed as follows: feline monocytes (2 Â 10 5 cells) were cultured in this medium in 24-well multiplates at 37°C for 1 h. After washing, FIPV strain 79-1146 (1 Â 10 4 TCID 50 ) reacted with or without MAb 6-4-2 was added to the culture and adsorbed to the cells at 37°C for 1 h in the presence of chloroquine. After washing, cells were cultured in medium containing chloroquine, and culture supernatants and cells were collected after 2 days. As the virus titer in the culture supernatant, TCID 50 was calculated by the method of Reed-Muench. Cells were used to measure IL-1b, TNF-a, and IL-6 mRNA, and FCoV N genes. To evaluate the cytotoxic effects of chloroquine in feline cells, cell viability was measured by the WST-8 assay as described before (Takano et al., 2007b) . The percent viability was calculated using the following formula: Cell viability (%) = (OD of chloroquine-treated cells/OD of chloroquine-untreated cells) Â 100.

FIPV strain 79-1146 (1 Â 10 4 TCID 50 ) was added to the culture and adsorbed to the fcwf-4 cells at 37°C for 1 h. After washing, cells were cultured in medium containing chloroquine, and culture supernatants were collected after 1 day. The experiment using feline monocytes from SPF cats was performed as follows: FIPV strain 79-1146 (1 Â 10 4 TCID 50 ) reacted with or without MAb 6-4-2 was added to the culture and adsorbed to the feline monocytes at 37°C for 1 h. After washing, cells were cultured in medium containing chloroquine, and culture supernatants and cells were collected after 2 days. The experiment using feline monocytes from cats with FIP (FIP cats) was performed as follows: feline monocytes were cultured in medium containing chloroquine, and culture supernatants and cells were collected after 2 days. As the virus titer in the culture supernatant, TCID 50 was calculated. Cells were used to measure cytokine mRNA, and FCoV N genes.

RNA isolation and cDNA preparation were performed employing the method of Takano et al. (2007a) .

2.6. Determination of feline cytokine mRNA and FCoV N gene expression levels cDNA was amplified by PCR using specific primers as shown in Table 3 . PCR was performed using the method of Takano et al. (2007a) . Band density was quantified under appropriate UV exposure by video densitometry using Image J software (NIH, U.S.A.). Cytokine mRNA and FCoV N genes were quantitatively analyzed in terms of the relative density value to the mRNA of the housekeeping gene GAPDH.

Nine SPF cats were randomly assigned to three experimental groups. In accordance with the experimental schedule indicated in Fig. 5 . Group A: chloroquine (10 mg/kg) was subcutaneously administered every 3 days from 3 days before FIPV inoculation. Strain FIPV 79-1146 (10 5 TCID 50 /ml) was inoculated orally to cats. Group B: Hanks' balanced salt solution (HBSS) was subcutaneously administered every 3 days from 3 days before FIPV inoculation. Strain FIPV 79-1146 (10 5 TCID 50 /ml) was inoculated orally to cats. HBSS administration was completed 12 days after FIPV inoculation, and chloroquine (10 mg/kg) was subcutaneously administered every 3 days thereafter instead of HBSS. Group C: HBSS was subcutaneously administered every 3 days from 3 days before FIPV inoculation. Strain FIPV 79-1146 (10 5 TCID 50 /ml) was inoculated orally to cats. One animal in the group C died unexpectedly before the challenge of FIPV, and was excluded from this study. Cats were checked daily for clinical signs, and we measured their body temperature and weight. FIP diagnoses were confirmed upon postmortem examination, revealing peritoneal and pleural effusions, and pyogranuloma in the major organs. To evaluate the condition of cats, we monitored Karnofsky's score. Karnofsky's score was calculated in reference to the report by Hartmann and kuffer (1998) , Ritz et al. (2007) . The protocol for the experiments in the present Table 1 The cytotoxic effects of chloroquine in fcwf-4 cells and monocytes. Cell viability was evaluated by WST-8 assay.

Chloroquine ( 

Blood (1 ml) collected from cats using a syringe coated with EDTA were used for the collection of samples for blood biochemistry and hematological analyses. A half milliliter of the blood sam-ple was centrifuged (800g) and the supernatant was used as a plasma sample for blood biochemistry. The remainder of blood samples was used for hematological analyses.

Heparinized blood (5 ml) was 2-fold diluted with phosphate-buffered saline (PBS), and subjected to Ficoll-Hypaque , followed by inoculation with FIPV at 37°C. After 1 h, the supernatant was removed and cells were then treated with chloroquine at 37°C in the Pre/Post treatment and Post treatment groups. After 1 day, the virus titer in the fcwf-4 cell culture supernatant was measured. n = 4. (B) Monocytes were cultured with chloroquine at 37°C for 1 h in the Pre/Post treatment group, but not in the Post treatment group, followed by inoculation with FIPV at 37°C. After 1 h, the supernatant was removed and cells were then treated with chloroquine at 37°C in the Pre/Post treatment and Post treatment groups. After 1 day, the virus titer in the feline primary monocyte culture supernatant was measured. n = 8. ⁄⁄ p < 0.01 vs. without chloroquine (0 lM), ⁄ p < 0.05 vs. without chloroquine (0 lM). Monocytes were cultured with chloroquine for 1 h in the Pre/Post treatment group (black bar), but not in the None group (white bar) or Post treatment group (gray bar), followed by treatment with a reaction mixture of FIPV and anti-FIPV-S antibodies (FIPV + MAb 6-4-2) for 1 h. Then, the supernatant was removed and cells were treated with chloroquine at 37°C in the Pre/Post treatment and Post treatment groups, but not in the None group. After 2 days, the virus titer in the cell culture supernatant (C) or the intracellular FCoV N gene level (D) was measured. The FCoV N gene was quantitatively analyzed in terms of the relative density value to the mRNA of the housekeeping gene GAPDH. n = 8. with chloroquine at 37°C in the Pre/Post treatment and Post treatment groups, but not in the None group. After 2 days, intracellular IL-1b, TNF-a, and IL-6 mRNA levels were measured. Cytokine mRNAs were quantitatively analyzed in terms of the relative density value to the mRNA of the housekeeping gene GAPDH. n = 8. density gradient centrifugation at 540g for 20 min. The PBMC layer was collected, washed twice with PBS, and resuspended with growth medium at 2 Â 10 6 cells/ml. 2.8. Statistical analysis Data from two groups were analyzed by the Student's t test, and multiple groups were analyzed by a one-way ANOVA. 

The effect of chloroquine on FIPV infection in fcwf-4 cells and SPF cat-derived monocytes was investigated. The cytotoxic effect by chloroquine in feline cells was showed in Table 1 . In fcwf-4 cells, FIPV replication was inhibited in a chloroquine concentration-dependent manner in both Pre/Post and Post treatment groups (Fig. 1A) . Similarly, concentration-dependent inhibition of viral replication was noted in feline monocytes treated with chloroquine (Fig. 1B) . However, the antiviral effect of chloroquine was slightly lower in the Post treatment group than in the Pre/Post treatment group.

The effect of chloroquine on the antibody-dependent enhancement of FIPV infection in monocytes was investigated. The virus titer and intracellular expression level of the FIPV N gene were significantly higher in the culture supernatant of monocytes inoculated with a mixture of FIPV and MAb 6-4-2 than in monocytes cultured with FIPV alone ( Fig. 2A and B) . In monocytes inoculated with a mixture of FIPV and MAb 6-4-2, the virus titer was significantly lower in the Post and Pre/Post treatment groups than in the None group (Fig. 2C) . The intracellular expression level of the FIPV N gene was significantly lower in the Pre/Post treatment group than in the None group (Fig. 2D) . Intracellular IL-1b, TNFa, and IL-6 mRNA expression levels were significantly higher in monocytes inoculated with a mixture of FIPV and MAb 6-4-2 than in monocytes cultured with medium or FIPV alone (Fig. 3A) . In monocytes inoculated with a mixture of FIPV and MAb 6-4-2, IL-1b, TNF-a and IL-6 mRNA expression levels were significantly lower in the Pre/Post treatment group than in the None group (Fig. 3B) . However, no significant differences in these mRNA expression levels were noted between the Post treatment and the None groups (Fig. 3B) .

The influence of chloroquine on cytokine mRNA and FCoV N gene expressions was investigated in monocytes from cats with FIP. IL-1b, TNF-a, and IL-6 mRNA expression levels were signifi-cantly higher, with an increase in the level of FCoV N gene expression, in monocytes from cats with FIP than in monocytes from SPF cats (Fig. 4A) . These mRNA expression levels were significantly lower in monocytes from cats with FIP cultured in medium containing 10 lM of chloroquine than in monocytes cultured without chloroquine (Fig. 4B) . However, no significant difference was noted in the level of FCoV N gene expression with or without chloroquine.

Based on the in vitro results, the therapeutic effect of chloroquine was investigated using cats with experimentally induced FIP. The experiment was performed following the schedule shown in Fig. 5 . The FCoV N gene and cytokine mRNA were detected and clinical parameters were measured after FIPV inoculation until the onset of FIP.

PBMC was collected 14 and 21 days after FIPV inoculation, viral mRNA expression level was measured. The viral mRNA expression level was measured by semiquantitative RT-PCR. On day 14, viral mRNA expression level was slightly lower in group A than in group B and C. On day 21, viral mRNA expression level was slightly lower in group A and B (chloroquine-treated groups) than in group C. (Fig. 6) . Inflammatory cytokine mRNA expression levels in PBMC on day 18-21 after FIPV inoculation were measured, and the ratios of mRNA expression levels to those before inoculation were determined (Fig. 7) . Inflammatory cytokine mRNA expression ratios were slightly lower in group A and B (chloroquine-treated groups) than in group C.

Changes in body temperature after FIPV inoculation were measured (Table 2) . No major change in body temperature was noted in any group. Lymphocyte and neutrophil counts in peripheral blood decreased in all cats on day 14 after FIPV inoculation (Table  2 ), but the level of this reduction was smaller in chloroquine-treated groups than in group C. Increases in total protein (TP), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and direct bilirubin (D-Bil) levels were smaller in group A than in the other groups (Table 2) . On the other hand, alanine aminotransferase (ALT) levels increased in the chloroquine-treated groups. When the clinical condition of the cats in each group was evaluated using the Karnofsky score, the score was high in the chloroquine-treated groups ( Table 2 ). The mean number of survival days were 34.3, 31.7, and 21.0 in groups A, B, and C, respectively. Unfortunately, one animal in the group C died before the challenge of FIPV, statistical significance was not shown for any of the differences between 0.5 1 1.5

Cytokine mRNA levels (ratio to the control)

IL-1 IL-6 TNF- Fig. 7 . IL-1b, TNF-a, and IL-6 mRNA expression levels of PBMC in FIPV-infected cats treated with chloroquine. After inoculation with FIPV 79-1146 for 18-21 days, PBMCs were collected from cats. Intracellular IL-1b, TNF-a, and IL-6 mRNA levels were measured. Cytokine mRNAs were quantitatively analyzed in terms of the relative density value to the mRNA of the housekeeping gene GAPDH. Data are expressed as a ratio of the control value (at the day of inoculation with FIPV). the chloroquine-treatment groups and the group C in the days of survival.

It has been reported that chloroquine is useful for the treatment for viral infections (Kono et al., 2008; Murray et al., 2010; Savarino et al., 2003; Vincent et al., 2005; Yan et al., 2012) . However, most of studies were obtained by in vitro experiments; only a few studies have investigated the antiviral effect of chloroquine using experimental animals (Pallister et al., 2009; Vigerust and McCullers, 2007) . We investigated the effect of chloroquine as a therapeutic drug for FIP both in vitro and in vivo.

FIPV was enhanced proliferation in monocytes treated with a reaction mixture of FIPV and anti-FIPV-S antibodies. In contrast, virus proliferation was inhibited in chloroquine-treated monocytes, i.e., chloroquine inhibited the FIPV-induced ADE activity of monocytes. In addition, a significant inhibitory effect on FIPV gene expression was noted in cells with Pre/Post treatment. In contrast, no significant inhibitory effect was noted in cells with Post treatment, and the treatment of FIPV-infected monocytes collected from FIP cats with chloroquine did not change FIPV N gene expression, suggesting that the treatment of cells already infected with FIPV with chloroquine induces no antiviral effect.

FIPV-infected monocytes/macrophages are major component cells that produce the inflammatory cytokines involved in pathological deterioration (Regan et al., 2009; Takano et al., 2007b Takano et al., , 2009 . In this study, chloroquine significantly inhibited inflammatory cytokine mRNA expression levels in FIPV-infected monocytes. The mechanism of inhibiting inflammatory cytokine mRNA expression by chloroquine is still unclear. Weber and Levitz (2000) suggested the presence of lysosomotropic and non-lysosomotropic mechanisms in the chloroquine-induced inhibition of TNF-a production. The former inhibits TNF-a release from cells and the latter inhibits TNF-a mRNA expression and transcription, which suggests that the significant inhibition of inflammatory cytokine mRNA expression levels by chloroquine in FIPV-infected monocytes involves the non-lysosomotropic mechanism. However, in a more recent paper by Jang et al. (2006) , it was shown that chloroquine does inhibit TNF-a release, but does not change the TNF-a mRNA levels or the synthesis of the TNF-a precursor. The reason for this discrepancy is unclear. Furthermore, in addition to the lowering of TNF-a mRNA expression, the expression of IL-1b and IL-6 mRNA was decreased. Jang et al. (2006) reported that the stability of IL-1b and IL-6 mRNA was decreased by chloroquine. In the present study, the same mechanisms might decrease the expression of cytokine mRNA.

Chloroquine is administered orally or intravenously for the treatment of malaria. However, oral or intravenous administration of chloroquine is a burden on veterinarians and/or owners. Thus, in the present study, we chose a subcutaneous route. Unfortunately, there are no pharmacokinetic data on chloroquine after subcutaneous administration. Therefore, we must rely on pharmacokinetic data on chloroquine after intravenous administration examined under similar conditions to our study. Aderounmu et al. (1986) reported pharmacokinetic data after a single intravenous dose of 3 mg/kg chloroquine. In our present study, the subcutaneous dose of chloroquine was 2.5-3.3 mg/kg. Thus, pharmacokinetic data on chloroquine after subcutaneous administration to cats may be similar to those reported by Aderounmu et al. (1986) .

We administered chloroquine when there were some clinical symptoms in cats after inoculation with FIPV strain 79-1146. The timing of administration was established based on the report of de Groot-Mijnes et al. (2005) and our previous experiments (Takano and Hohdatsu, unpublished data). Therefore, the CQ treatment timing in group B was set to 12 days after inoculation with FIPV strain 79-1146.

The clinical score of chloroquine-treatment groups were better than in chloroquine-untreated group, which suggests that chloroquine has therapeutic effect against FIP. If chloroquine has antiviral effect against FIPV in vivo, this drug may be useful for the treatment of FIP. To obtain the antiviral effect, it is necessary to increase the chloroquine dosage. However, as mentioned above, when the chloroquine concentration is increased, a severe side effect may be induced. Combining chloroquine with other ''anti-FIPV agents'' overcomes this problem. Hsieh et al. (2010) succeeded in inhibiting FIPV replication in fcwf-4 cells using the low-cytotoxic antiviral agents. It will be necessary to further investigate the possibility of FIP treatment with a combination of chloroquine and other agents.

In conclusion, it was demonstrated that chloroquine has inhibitory effect against the replication of FIPV and anti-inflammatory effect. It was also noted experimentally that chloroquine may be an effective drug for the treatment of FIP. The present experiments only examined whether CQ is effective for treating FIP. Future studies are necessary to collect preclinical data using the pharmacokinetics data of CQ reported by Aderounmu et al. (1986) and others (Aderounmu and Fleckenstein, 1983; Gustafsson et al., 1983; Ducharme and Farinotti, 1996) . Table 3 Sequences of PCR primers for feline GAPDH, IL-1b, TNF-alpha, IL-6, and FCoV N.

Nucleotide sequence Location Length ( 

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This work was in part supported by KAKENHI (Grants-in-Aid for Young Scientists (B), no. 22780285) from the Ministry of Education, Culture, Sports, Science and Technology, and Kitasato University Research Grant for Young Researchers (2012).