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In vitro activity of cidofovir against the emerging Cantagalo virus and the smallpox vaccine strain IOC
International Journal of Antimicrobial Agents 33 (2009) 75–79
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
In vitro activity of cidofovir against the emerging Cantagalo virus and the smallpox vaccine strain IOC Desyreé Murta Jesus, Nissin Moussatché, Clarissa R. Damaso ∗ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - CCS, Ilha do Fundão, Rio de Janeiro 21941-590, Brazil
a r t i c l e
i n f o
Article history: Received 21 March 2008 Accepted 17 July 2008 Keywords: Cantagalo virus Smallpox vaccine Cidofovir Antiviral agent Vaccinia virus Poxvirus
a b s t r a c t The antiviral effect of cidofovir was evaluated against two strains of vaccinia virus: the field strain Cantagalo virus (CTGV) and the smallpox vaccine IOC. The drug severely inhibited virus replication, revealing an EC50 (drug concentration required to inhibit 50% of virus replication) of 7.68 M and 9.66 M, respectively, for CTGV and vaccine strain IOC. Similarly, other field isolates of Cantagalo-like viruses recently collected in distinct outbreaks were equally sensitive to the drug. Pre-treatment of cells prior to infection effectively established an antiviral state, inhibiting virus replication by >90% after 24 h in the absence of cidofovir. CTGV infections represent an emerging zoonosis, and outbreaks have been frequently reported in several states of Brazil. Also, the possibility of resuming the manufacture of smallpox vaccine supports the need to evaluate the effect of antiviral drugs on the Brazilian vaccine strain IOC. As there is no currently approved antipoxvirus therapy, our data are extremely encouraging. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Cantagalo virus (CTGV) is a strain of vaccinia virus (VACV) isolated during an outbreak of a pustular disease in dairy farms in Brazil [1]. Subsequent episodes of VACV-like infections have frequently been reported during the last decade [2–5]. The economical losses and occupational aspects of this emerging disorder are quite alarming, primarily affecting small rural properties [4,5]. None the less, detection of new cases is usually not managed adequately and milkers do not seek health care promptly, leading to delayed notification of the episodes. The spread of VACV-like infections in Brazil is also distressing because no antiviral therapy is currently licensed for poxvirus-associated diseases [6]. In fact, the concern is widespread because these disorders also include the adverse events following exposure of individuals to smallpox vaccine. There is a general debate worldwide regarding whether smallpox vaccination should be resumed, the choice of the population groups to receive pre-event vaccination and the safest VACV strains to be used [7]. In Brazil, the health authorities have firmly discussed whether to re-start the manufacture of smallpox vaccine [8]. The vaccine used in the Brazilian systematic smallpox vaccination campaign for several decades was produced at the Instituto Oswaldo Cruz, Rio de Janeiro, and generated primary take rates usually >90% [9]. This VACV strain has been previously charac-
∗ Corresponding author. Tel.: +55 21 2562 6510; fax: +55 21 2280 8193. E-mail address: [email protected] (C.R. Damaso).
terised and named IOC [1]. In the last few years, several studies have addressed the immunogenicity and efficacy of distinct VACV strains [7] and, most importantly, their response to antiviral drugs [6]. Nevertheless, the sensitivity of VACV-IOC and CTGV to antiviral compounds remains largely unknown, except for the reported antiviral action of the immunosuppressive drug FK506 [10]. Cidofovir (CDV) is an acyclic nucleoside phosphonate with potent activity against several DNA viruses, including the poxviruses. It is one of the most promising and effective antipoxvirus drugs studied so far [6,11]. However, CDV is licensed for clinical use only against human cytomegalovirus retinitis in acquired immune deficiency syndrome (AIDS) patients [11]. In the present work, we evaluated the antiviral efficacy of CDV on the replication of the VACV field strain Cantagalo and the smallpox vaccine strain IOC. 2. Materials and methods 2.1. Cells and viruses BSC-40 cells (African green monkey kidney) were propagated in monolayer cultures at 37 ◦ C in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 2% heat-inactivated foetal bovine serum, as described previously [10]. CTGV isolates CM-01 [1], VI03, ALE-H1 and PO-01 [5] and vaccinia virus strain IOC clone A11-1 [1] were available in the laboratory’s collection and routinely propagated and titered by plaque assay in BSC-40 cells, as described previously [1,10].
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.07.015
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D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79
2.2. Cidofovir CDV was a generous gift from Gilead Sciences, Inc. (Foster City, CA). The drug was dissolved in phosphate-buffered saline and stored at −20 ◦ C as a 5 mg/mL stock solution. 2.3. Cytotoxicity assay Cell viability was evaluated following incubation of confluent monolayers in a 96-well plate for 48 h at 37 ◦ C with CDV concentrations ranging from 3 M to 632 M. The neutral red uptake assay was performed as described previously [10]. All measurements are expressed as the average of three assays performed in eight replicates. 2.4. Plaque reduction assay For plaque reduction assays, monolayers of BSC-40 cells grown in six-well plates were infected with 100 plaque-forming units (PFU) or 200 PFU of each virus. After a 90 min adsorption period,
viral inocula were removed (zero time of infection) and the cells were incubated with fresh medium in the absence or presence of different concentrations of CDV ranging from 3.2 M to 47.4 M. After 48 h the cells were stained with 0.1% crystal violet in 10% formaldehyde and the viral plaques were counted manually [10]. In some assays, 32 M CDV was added to the cells at 1 h, 3 h, 5 h, 14 h, 17 h, 20 h or 24 h prior to infection without subsequent addition of the drug during the 48 h infection period. The percentage of inhibition of plaque formation was calculated as follows: 100 − [(mean number of plaques in test × 100)/(mean number of plaques in control)]. The EC50 values (drug concentration required to inhibit 50% of virus replication) were estimated from the plots. 2.5. Determination of the production of intracellular virus Analysis of intracellular virus yield was done as described previously [10]. BSC-40 monolayers were infected with 1000 PFU or 5000 PFU of each virus and at zero time of infection the cells were incubated in the absence or presence of CDV at concentrations ranging from 3.2 M to 47.4 M. After 24 h the monolayers
Fig. 1. Cytotoxicity and antiviral effect of cidofovir (CDV). (a) The indicated concentrations of CDV were added to BSC-40 cells and cytotoxicity was evaluated after 48 h incubation. (b) Plaque number reduction assay. Cells were infected with 100 plaque-forming units (PFU) of Cantagalo virus (CTGV) or the vaccinia virus smallpox vaccine strain IOC (VACV-IOC) in presence of the indicated concentrations of CDV. Viral plaques were counted after 48 h. (c) Dose-dependent inhibition of virus yield. Cells were infected with CTGV or VACV-IOC (1000 PFU) in the presence of the indicated concentrations of CDV. Virus yield was determined at 24 h post-infection. The values represent the mean of at least three independent assays. (d) Time-course analysis of virus yield. Infection proceeded for 48 h in the absence or presence of 47.4 M CDV and virus yield was determined at the indicated time points. (e) Inhibition of virus protein accumulation. The cells were infected and treated or not with 47.4 M CDV. Samples were collected at 24 h, 30 h and 48 h post-infection and analysed by Western blot using antibodies against VACV structural proteins. M, mock-infected cells. Molecular weight markers (kDa) are shown on the right.
D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79
were harvested for yield determination by plaque assay in BSC-40 cells, as described previously [1,10]. For time-course analysis, the infection was carried out in the absence or presence of 47.4 M CDV and the samples were harvested for yield determination at 0 h, 6 h, 18 h, 24 h, 30 h and 48 h post-infection. In some assays, CDV at 32 M (CTGV) or 48 M (VACV-IOC) was added 24 h prior to infection without addition of the drug after zero time. The percentage of yield inhibition and the EC50 values were determined essentially as described in Section 2.4. 2.6. Detection of virus protein accumulation by Western blot BSC-40 cells in 35-mm dishes were infected with 5000 PFU of each virus and treated with 47.4 M CDV at zero time of infection. After 24 h, 30 h and 48 h post-infection, the cells were collected in sodium dodecyl sulphate (SDS)-containing sample buffer and processed for SDS-polyacrylamide gel electrophoresis analysis, followed by Western blot detection of virus structural proteins (anti-total VACV proteins), essentially as described previously [1,5,10]. 2.7. Measurement of viral DNA accumulation by slot–blot hybridisation BSC-40 cells in 35 mm dishes were infected with 5000 PFU of each virus and treated with CDV at 32 M (CTGV) or 48 M (VACV-IOC) at zero time of infection. Alternatively, the cells were pre-treated with CDV 24 h prior to infection, and at zero time fresh medium without drug was added to the infected cells. At 24 h post-infection, cell extracts were obtained essentially as described previously [10] and applied in triplicate to a Hybond-N membrane (Amersham/Pharmacia) using a Minifold II Slot–Blot Apparatus (Schleicher and Schuell, Inc.). After filtration, the DNA was alkalidenatured in situ and probed to the HindIII D fragment of VACV strain WR genome [10] following nick-translation labelling with [32 P]␣dCTP as described previously [1,10]. The blots were exposed to radiographic films.
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Fig. 1c confirmed that CTGV was more sensitive to CDV treatment than VACV-IOC, showing EC50 values of 3.8 ± 0.42 M and 9.84 ± 0.33 M, respectively. These values generated a SI of >166.3 for CTGV and >64.2 for VACV-IOC. However, inhibition of virus yield has not been regularly used to analyse the antiviral activity of CDV. Therefore, no correlation with previous published data could be inferred. The highest concentration tested generated levels of yield inhibition >99% for both viruses, therefore 47.4 M CDV was chosen for subsequent kinetic studies of intracellular progeny production and virus protein accumulation in the presence of the drug. As observed in Fig. 1d, the production of CTGV and VACV-IOC infectious particles was inhibited early during infection, reaching >90% reduction at 18 h in the presence of 47.4 M CDV. This effect was clearly enhanced after 24 h of infection. Inhibition of virus yield was accompanied by a severe reduction in the accumulation of virus structural proteins, as detected by Western blot analysis of infected cells treated with 47.4 M CDV (Fig. 1e). As mentioned previously, VACV-like infections have been frequently reported in distinct states of Brazil since CTGV isolation in 1999 [2–5]. Some isolates have been identified as CTGV-like viruses by molecular analysis [5]. We were interested in determining whether other CTGV-like isolates collected in distinct outbreaks were equally sensitive to CDV. Three samples isolated in different states of Brazil from 2001 to 2007 [5] were evaluated by plaque reduction assay following 48 h of infection. As shown in Fig. 2, an inhibition of plaque number by nearly 100% was observed when 27 M CDV was added at zero time of infection for the three isolates. These results indicate that the isolates responded to CDV similarly to CTGV, reinforcing their identification as CTGV-like viruses [5]. It has been reported that pre-treatment of Vero cells with CDV induces an efficient antiviral state [12]. To evaluate the drug efficacy
3. Results and discussion Cytotoxicity was evaluated first. The results shown in Fig. 1a indicate that CDV was not toxic to BSC-40 cells at concentrations of up to 632 M after 48 h post-treatment (50% cytotoxic concentration (CC50 ) >632 M). In an initial screening for antiviral activity, the formation of virus plaques in BSC-40 cells was evaluated. As shown in Fig. 1b, CDV activity against both viruses was dose-dependent. Cidofovir at 32 M already led to nearly 100% inhibition of CTGV plaque numbers whereas VACV-IOC was inhibited by ca. 80%. The EC50 was 7.68 ± 1.35 M for CTGV and 9.66 ± 0.94 M for VACV-IOC. Taking into account these values and the CC50 , we estimated the selective index (SI) (CC50 /EC50 ) to be >82.3 for CTGV and >65.4 for VACV-IOC. Previous studies also using plaque reduction assays, but in HFF cells, reported EC50 values of 10.1 M and 13.4 M for VACV-NYCBH and VACV-IHD, respectively. VACV strains Elstree, WR and Copenhagen and cowpox virus strain Brighton Red, however, presented higher EC50 values ranging from 41.6 M to 46.2 M [11]. Inhibition of virus plaque numbers was also accompanied by a significant reduction in plaque size (data not shown). Therefore, to avoid miscalculation due to the very small plaques that may not have been visualised, the effect of CDV on the yield of intracellular virus produced during infection was determined. BSC-40 cells were infected with CTGV and VACV-IOC in the presence of CDV concentrations ranging from 3.2 M to 47.4 M and virus yield was evaluated after 24 h post-infection. The results in
Fig. 2. Antiviral effect of cidofovir (CDV) on different vaccinia virus field isolates. Cells were infected with 100 plaque-forming units of the following field isolates: VI03 (county of Vieiras, Minas Gerais State, 2001); ALE-H1 (county of Alegre, Espírito Santo State, 2006); and PO-01 (county of Porciúncula, Rio de Janeiro State, 2007). CDV at 27 M was added at zero time of infection and viral plaques were visualised after 48 h. A representative assay is shown.
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D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79
Fig. 3. Pre-treatment of cells with cidofovir (CDV). (a) Cells were pre-treated with 32 M CDV for the indicated times and then infected with Cantagalo virus (CTGV) or the vaccinia virus smallpox vaccine strain IOC (VACV-IOC) (200 plaque-forming units (PFU)). After 48 h in the absence of CDV, viral plaques were counted. (b) Cells were pre-treated (Pre) with 32 M or 48 M CDV for 24 h and then infected with CTGV or VACV-IOC (5000 PFU), respectively. With no pre-treatment (Post), cells were treated with 32 M or 48 M CDV at zero time of infection. After 24 h, the cells were harvested for evaluation of viral DNA accumulation or determination of virus yield. M, mock-infected cells; C, untreated, infected cells. The values represent the mean of at least two independent assays. In (b), a representative DNA assay is shown.
in our virus–cell system, we pre-incubated cells with 32 M CDV for 0 h, 1 h, 3 h, 5 h, 14 h, 17 h, 20 h and 24 h prior to infection. The cells were then infected in the absence of CDV and the plaque number was determined at 48 h post-infection. As observed in Fig. 3a, pre-treatment with CDV for 1 h induced a mild antiviral action. Nevertheless, when the drug was added at 17 h prior to infection, CTGV plaque number was reduced by 80%. VACV-IOC was more resistant than CTGV and achieved only 72% inhibition when pre-treatment was performed for 24 h. This result indicated that CDV was able to establish effectively an antiviral state in BSC40 cells, and no addition of CDV during infection was required to inhibit severely CTGV and VACV-IOC replication. This effect was probably due to the long intracellular half-life of CDV metabolites in BSC-40 cells, as has been previously demonstrated to occur in Vero cells [12]. Pre-treatment with CDV for 24 h also reduced virus yields without requiring the presence of the drug during the subsequent 24 h of infection when the cells were harvested for virus titration. To equalise the inhibition levels to ca. 92%, CDV concentrations were adjusted to 32 M and 48 M for CTGV and VACV-IOC, respectively (Fig. 3b). However, using these CDV concentrations during the infection period without pre-treatment, inhibition rates of 99.9% were observed for both viruses. Under similar experimental conditions, virus DNA replication was analysed and proved to be severely repressed (Fig. 3b), thus confirming the efficacy of the CDV-induced antiviral state. The results obtained in this study demonstrate that CDV presents a potent antiviral activity against the smallpox vaccine strain IOC and mainly against the VACV field strain CTGV. The EC50 values found for CTGV and VACV-IOC were lower than the numbers reported for other VACV strains using a similar strategy to evaluate virus replication [11]. VACV-like infections have been persistently reported in Brazil with an increasing number of human cases [1–5]. This emerging viral zoonosis results in significant agricultural and occupational effects, and the search for an effective antiviral therapy is a major concern. Moreover, the possibility of resuming smallpox vaccine production in Brazil also supports the need for an effective anti-VACV therapy. So far, FK506 has been the only drug reported to present antiviral activity against VAVC-IOC and, to a lesser extent, CTGV [10]. Nevertheless, this macrolide is a potent immunosuppressive drug and its antiviral effectiveness in vivo has not been determined. Therefore, our data on CDV efficacy against CTGV and VACVIOC replication are extremely encouraging. However, the poor oral bioavailability of CDV may limit its use in a major episode [6]. Nevertheless, it is worthy of noting that CDV topical administration
has been successful in treating cutaneous lesions in animals infected with VACV-WR, cowpox and orf viruses as well as humans infected with molluscum contagiosum virus, revealing long-lasting response levels [13–15]. Therefore, topical formulations of CDV for treating CTGV-like lesions on dairy cattle and humans may be seriously considered as a promising antiviral therapy to restrain the spread of this emerging zoonosis. Acknowledgments The authors thank Gilead Sciences, Inc., Foster City, CA, for providing cidofovir, and Ademilson Bizerra for technical assistance. Funding: This work was supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), Faperj (Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil) and IFS (International Foundation for Science, Sweden). Competing interests: None declared. Ethical approval: Not required. References [1] Damaso CR, Esposito JJ, Condit RC, Moussatché N. An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 2000;277:439–49. [2] Trindade GS, da Fonseca FG, Marques JT, Nogueira ML, Mendes LC, Borges AS, et al. Arac¸atuba virus: a vaccinialike virus associated with infection in humans and cattle. Emerg Infect Dis 2003;9:155–60. [3] Nagasse-Sugahara TK, Kisieliu JJ, Ueda-Ito M, Curti SP, Figueiredo CA, Cruz AS, et al. Human vaccinia-like virus outbreaks in São Paulo and Goiás states, Brazil: virus detection, isolation and identification. Rev Inst Med Trop Sao Paulo 2004;46:315–22. [4] Lobato ZI, Trindade GS, Frois MC, Ribeiro EB, Dias GR, Teixeira BM, et al. Outbreak of exantemal disease caused by vaccinia virus in human and cattle in Zona da Mata region, Minas Gerais. Arq Bras Med Vet Zootech 2005;57:423–9. [5] Damaso CR, Reis SA, Jesus DM, Lima PS, Moussatché N. A PCR-based assay for detection of emerging vaccinia-like viruses isolated in Brazil. Diagn Microbiol Infect Dis 2007;57:39–46. [6] Prichard MN, Kern ER. Orthopoxvirus targets for the development of antiviral therapies. Curr Drug Targets Infect Disord 2005;5:17–28. [7] Parrino J, Graham BS. Smallpox vaccines: past, present, and future. J Allergy Clin Immunol 2006;118:1320–6. [8] Borges MB, Kato SE, Damaso CR, Moussatché N, da Silva Freire M, Lambert Passos SR, et al. Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies. Biologicals 2007;36:105–10. [9] Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988. [10] Reis SA, Moussatche N, Damaso CR. FK506 a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against orthopoxvirus infection in cell culture. J Appl Microbiol 2006;100:1373–80. [11] Kern ER, Hartline C, Harden E, Keith K, Rodriguez N, Beadle JR, et al. Enhanced inhibition of orthopoxvirus replication in vitro by alkoxyalkyl esters of cidofovir and cyclic cidofovir. Antimicrob Agents Chemother 2002;46:991–5.
D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79 [12] Aduma P, Connelly MC, Srinivas RV, Fridland A. Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates. Mol Pharmacol 1995;47: 816–22. [13] Quenelle DC, Collins DJ, Kern ER. Cutaneous infections of mice with vaccinia or cowpox viruses and efficacy of cidofovir. Antiviral Res 2004;63: 33–40.
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[14] Scagliarini A, McInnes CJ, Gallina L, Dal Pozzo F, Scagliarini L, Snoeck R, et al. Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs. Antiviral Res 2007;73:169–74. [15] Calista D. Topical cidofovir for severe cutaneous human papillomavirus and molluscum contagiosum infections in patients with HIV/AIDS. A pilot study. J Eur Acad Dermatol Venereol 2000;14:484–8.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
In vitro activity of cidofovir against the emerging Cantagalo virus and the smallpox vaccine strain IOC Desyreé Murta Jesus, Nissin Moussatché, Clarissa R. Damaso ∗ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - CCS, Ilha do Fundão, Rio de Janeiro 21941-590, Brazil
a r t i c l e
i n f o
Article history: Received 21 March 2008 Accepted 17 July 2008 Keywords: Cantagalo virus Smallpox vaccine Cidofovir Antiviral agent Vaccinia virus Poxvirus
a b s t r a c t The antiviral effect of cidofovir was evaluated against two strains of vaccinia virus: the field strain Cantagalo virus (CTGV) and the smallpox vaccine IOC. The drug severely inhibited virus replication, revealing an EC50 (drug concentration required to inhibit 50% of virus replication) of 7.68 M and 9.66 M, respectively, for CTGV and vaccine strain IOC. Similarly, other field isolates of Cantagalo-like viruses recently collected in distinct outbreaks were equally sensitive to the drug. Pre-treatment of cells prior to infection effectively established an antiviral state, inhibiting virus replication by >90% after 24 h in the absence of cidofovir. CTGV infections represent an emerging zoonosis, and outbreaks have been frequently reported in several states of Brazil. Also, the possibility of resuming the manufacture of smallpox vaccine supports the need to evaluate the effect of antiviral drugs on the Brazilian vaccine strain IOC. As there is no currently approved antipoxvirus therapy, our data are extremely encouraging. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Cantagalo virus (CTGV) is a strain of vaccinia virus (VACV) isolated during an outbreak of a pustular disease in dairy farms in Brazil [1]. Subsequent episodes of VACV-like infections have frequently been reported during the last decade [2–5]. The economical losses and occupational aspects of this emerging disorder are quite alarming, primarily affecting small rural properties [4,5]. None the less, detection of new cases is usually not managed adequately and milkers do not seek health care promptly, leading to delayed notification of the episodes. The spread of VACV-like infections in Brazil is also distressing because no antiviral therapy is currently licensed for poxvirus-associated diseases [6]. In fact, the concern is widespread because these disorders also include the adverse events following exposure of individuals to smallpox vaccine. There is a general debate worldwide regarding whether smallpox vaccination should be resumed, the choice of the population groups to receive pre-event vaccination and the safest VACV strains to be used [7]. In Brazil, the health authorities have firmly discussed whether to re-start the manufacture of smallpox vaccine [8]. The vaccine used in the Brazilian systematic smallpox vaccination campaign for several decades was produced at the Instituto Oswaldo Cruz, Rio de Janeiro, and generated primary take rates usually >90% [9]. This VACV strain has been previously charac-
∗ Corresponding author. Tel.: +55 21 2562 6510; fax: +55 21 2280 8193. E-mail address: [email protected] (C.R. Damaso).
terised and named IOC [1]. In the last few years, several studies have addressed the immunogenicity and efficacy of distinct VACV strains [7] and, most importantly, their response to antiviral drugs [6]. Nevertheless, the sensitivity of VACV-IOC and CTGV to antiviral compounds remains largely unknown, except for the reported antiviral action of the immunosuppressive drug FK506 [10]. Cidofovir (CDV) is an acyclic nucleoside phosphonate with potent activity against several DNA viruses, including the poxviruses. It is one of the most promising and effective antipoxvirus drugs studied so far [6,11]. However, CDV is licensed for clinical use only against human cytomegalovirus retinitis in acquired immune deficiency syndrome (AIDS) patients [11]. In the present work, we evaluated the antiviral efficacy of CDV on the replication of the VACV field strain Cantagalo and the smallpox vaccine strain IOC. 2. Materials and methods 2.1. Cells and viruses BSC-40 cells (African green monkey kidney) were propagated in monolayer cultures at 37 ◦ C in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 2% heat-inactivated foetal bovine serum, as described previously [10]. CTGV isolates CM-01 [1], VI03, ALE-H1 and PO-01 [5] and vaccinia virus strain IOC clone A11-1 [1] were available in the laboratory’s collection and routinely propagated and titered by plaque assay in BSC-40 cells, as described previously [1,10].
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.07.015
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D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79
2.2. Cidofovir CDV was a generous gift from Gilead Sciences, Inc. (Foster City, CA). The drug was dissolved in phosphate-buffered saline and stored at −20 ◦ C as a 5 mg/mL stock solution. 2.3. Cytotoxicity assay Cell viability was evaluated following incubation of confluent monolayers in a 96-well plate for 48 h at 37 ◦ C with CDV concentrations ranging from 3 M to 632 M. The neutral red uptake assay was performed as described previously [10]. All measurements are expressed as the average of three assays performed in eight replicates. 2.4. Plaque reduction assay For plaque reduction assays, monolayers of BSC-40 cells grown in six-well plates were infected with 100 plaque-forming units (PFU) or 200 PFU of each virus. After a 90 min adsorption period,
viral inocula were removed (zero time of infection) and the cells were incubated with fresh medium in the absence or presence of different concentrations of CDV ranging from 3.2 M to 47.4 M. After 48 h the cells were stained with 0.1% crystal violet in 10% formaldehyde and the viral plaques were counted manually [10]. In some assays, 32 M CDV was added to the cells at 1 h, 3 h, 5 h, 14 h, 17 h, 20 h or 24 h prior to infection without subsequent addition of the drug during the 48 h infection period. The percentage of inhibition of plaque formation was calculated as follows: 100 − [(mean number of plaques in test × 100)/(mean number of plaques in control)]. The EC50 values (drug concentration required to inhibit 50% of virus replication) were estimated from the plots. 2.5. Determination of the production of intracellular virus Analysis of intracellular virus yield was done as described previously [10]. BSC-40 monolayers were infected with 1000 PFU or 5000 PFU of each virus and at zero time of infection the cells were incubated in the absence or presence of CDV at concentrations ranging from 3.2 M to 47.4 M. After 24 h the monolayers
Fig. 1. Cytotoxicity and antiviral effect of cidofovir (CDV). (a) The indicated concentrations of CDV were added to BSC-40 cells and cytotoxicity was evaluated after 48 h incubation. (b) Plaque number reduction assay. Cells were infected with 100 plaque-forming units (PFU) of Cantagalo virus (CTGV) or the vaccinia virus smallpox vaccine strain IOC (VACV-IOC) in presence of the indicated concentrations of CDV. Viral plaques were counted after 48 h. (c) Dose-dependent inhibition of virus yield. Cells were infected with CTGV or VACV-IOC (1000 PFU) in the presence of the indicated concentrations of CDV. Virus yield was determined at 24 h post-infection. The values represent the mean of at least three independent assays. (d) Time-course analysis of virus yield. Infection proceeded for 48 h in the absence or presence of 47.4 M CDV and virus yield was determined at the indicated time points. (e) Inhibition of virus protein accumulation. The cells were infected and treated or not with 47.4 M CDV. Samples were collected at 24 h, 30 h and 48 h post-infection and analysed by Western blot using antibodies against VACV structural proteins. M, mock-infected cells. Molecular weight markers (kDa) are shown on the right.
D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79
were harvested for yield determination by plaque assay in BSC-40 cells, as described previously [1,10]. For time-course analysis, the infection was carried out in the absence or presence of 47.4 M CDV and the samples were harvested for yield determination at 0 h, 6 h, 18 h, 24 h, 30 h and 48 h post-infection. In some assays, CDV at 32 M (CTGV) or 48 M (VACV-IOC) was added 24 h prior to infection without addition of the drug after zero time. The percentage of yield inhibition and the EC50 values were determined essentially as described in Section 2.4. 2.6. Detection of virus protein accumulation by Western blot BSC-40 cells in 35-mm dishes were infected with 5000 PFU of each virus and treated with 47.4 M CDV at zero time of infection. After 24 h, 30 h and 48 h post-infection, the cells were collected in sodium dodecyl sulphate (SDS)-containing sample buffer and processed for SDS-polyacrylamide gel electrophoresis analysis, followed by Western blot detection of virus structural proteins (anti-total VACV proteins), essentially as described previously [1,5,10]. 2.7. Measurement of viral DNA accumulation by slot–blot hybridisation BSC-40 cells in 35 mm dishes were infected with 5000 PFU of each virus and treated with CDV at 32 M (CTGV) or 48 M (VACV-IOC) at zero time of infection. Alternatively, the cells were pre-treated with CDV 24 h prior to infection, and at zero time fresh medium without drug was added to the infected cells. At 24 h post-infection, cell extracts were obtained essentially as described previously [10] and applied in triplicate to a Hybond-N membrane (Amersham/Pharmacia) using a Minifold II Slot–Blot Apparatus (Schleicher and Schuell, Inc.). After filtration, the DNA was alkalidenatured in situ and probed to the HindIII D fragment of VACV strain WR genome [10] following nick-translation labelling with [32 P]␣dCTP as described previously [1,10]. The blots were exposed to radiographic films.
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Fig. 1c confirmed that CTGV was more sensitive to CDV treatment than VACV-IOC, showing EC50 values of 3.8 ± 0.42 M and 9.84 ± 0.33 M, respectively. These values generated a SI of >166.3 for CTGV and >64.2 for VACV-IOC. However, inhibition of virus yield has not been regularly used to analyse the antiviral activity of CDV. Therefore, no correlation with previous published data could be inferred. The highest concentration tested generated levels of yield inhibition >99% for both viruses, therefore 47.4 M CDV was chosen for subsequent kinetic studies of intracellular progeny production and virus protein accumulation in the presence of the drug. As observed in Fig. 1d, the production of CTGV and VACV-IOC infectious particles was inhibited early during infection, reaching >90% reduction at 18 h in the presence of 47.4 M CDV. This effect was clearly enhanced after 24 h of infection. Inhibition of virus yield was accompanied by a severe reduction in the accumulation of virus structural proteins, as detected by Western blot analysis of infected cells treated with 47.4 M CDV (Fig. 1e). As mentioned previously, VACV-like infections have been frequently reported in distinct states of Brazil since CTGV isolation in 1999 [2–5]. Some isolates have been identified as CTGV-like viruses by molecular analysis [5]. We were interested in determining whether other CTGV-like isolates collected in distinct outbreaks were equally sensitive to CDV. Three samples isolated in different states of Brazil from 2001 to 2007 [5] were evaluated by plaque reduction assay following 48 h of infection. As shown in Fig. 2, an inhibition of plaque number by nearly 100% was observed when 27 M CDV was added at zero time of infection for the three isolates. These results indicate that the isolates responded to CDV similarly to CTGV, reinforcing their identification as CTGV-like viruses [5]. It has been reported that pre-treatment of Vero cells with CDV induces an efficient antiviral state [12]. To evaluate the drug efficacy
3. Results and discussion Cytotoxicity was evaluated first. The results shown in Fig. 1a indicate that CDV was not toxic to BSC-40 cells at concentrations of up to 632 M after 48 h post-treatment (50% cytotoxic concentration (CC50 ) >632 M). In an initial screening for antiviral activity, the formation of virus plaques in BSC-40 cells was evaluated. As shown in Fig. 1b, CDV activity against both viruses was dose-dependent. Cidofovir at 32 M already led to nearly 100% inhibition of CTGV plaque numbers whereas VACV-IOC was inhibited by ca. 80%. The EC50 was 7.68 ± 1.35 M for CTGV and 9.66 ± 0.94 M for VACV-IOC. Taking into account these values and the CC50 , we estimated the selective index (SI) (CC50 /EC50 ) to be >82.3 for CTGV and >65.4 for VACV-IOC. Previous studies also using plaque reduction assays, but in HFF cells, reported EC50 values of 10.1 M and 13.4 M for VACV-NYCBH and VACV-IHD, respectively. VACV strains Elstree, WR and Copenhagen and cowpox virus strain Brighton Red, however, presented higher EC50 values ranging from 41.6 M to 46.2 M [11]. Inhibition of virus plaque numbers was also accompanied by a significant reduction in plaque size (data not shown). Therefore, to avoid miscalculation due to the very small plaques that may not have been visualised, the effect of CDV on the yield of intracellular virus produced during infection was determined. BSC-40 cells were infected with CTGV and VACV-IOC in the presence of CDV concentrations ranging from 3.2 M to 47.4 M and virus yield was evaluated after 24 h post-infection. The results in
Fig. 2. Antiviral effect of cidofovir (CDV) on different vaccinia virus field isolates. Cells were infected with 100 plaque-forming units of the following field isolates: VI03 (county of Vieiras, Minas Gerais State, 2001); ALE-H1 (county of Alegre, Espírito Santo State, 2006); and PO-01 (county of Porciúncula, Rio de Janeiro State, 2007). CDV at 27 M was added at zero time of infection and viral plaques were visualised after 48 h. A representative assay is shown.
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Fig. 3. Pre-treatment of cells with cidofovir (CDV). (a) Cells were pre-treated with 32 M CDV for the indicated times and then infected with Cantagalo virus (CTGV) or the vaccinia virus smallpox vaccine strain IOC (VACV-IOC) (200 plaque-forming units (PFU)). After 48 h in the absence of CDV, viral plaques were counted. (b) Cells were pre-treated (Pre) with 32 M or 48 M CDV for 24 h and then infected with CTGV or VACV-IOC (5000 PFU), respectively. With no pre-treatment (Post), cells were treated with 32 M or 48 M CDV at zero time of infection. After 24 h, the cells were harvested for evaluation of viral DNA accumulation or determination of virus yield. M, mock-infected cells; C, untreated, infected cells. The values represent the mean of at least two independent assays. In (b), a representative DNA assay is shown.
in our virus–cell system, we pre-incubated cells with 32 M CDV for 0 h, 1 h, 3 h, 5 h, 14 h, 17 h, 20 h and 24 h prior to infection. The cells were then infected in the absence of CDV and the plaque number was determined at 48 h post-infection. As observed in Fig. 3a, pre-treatment with CDV for 1 h induced a mild antiviral action. Nevertheless, when the drug was added at 17 h prior to infection, CTGV plaque number was reduced by 80%. VACV-IOC was more resistant than CTGV and achieved only 72% inhibition when pre-treatment was performed for 24 h. This result indicated that CDV was able to establish effectively an antiviral state in BSC40 cells, and no addition of CDV during infection was required to inhibit severely CTGV and VACV-IOC replication. This effect was probably due to the long intracellular half-life of CDV metabolites in BSC-40 cells, as has been previously demonstrated to occur in Vero cells [12]. Pre-treatment with CDV for 24 h also reduced virus yields without requiring the presence of the drug during the subsequent 24 h of infection when the cells were harvested for virus titration. To equalise the inhibition levels to ca. 92%, CDV concentrations were adjusted to 32 M and 48 M for CTGV and VACV-IOC, respectively (Fig. 3b). However, using these CDV concentrations during the infection period without pre-treatment, inhibition rates of 99.9% were observed for both viruses. Under similar experimental conditions, virus DNA replication was analysed and proved to be severely repressed (Fig. 3b), thus confirming the efficacy of the CDV-induced antiviral state. The results obtained in this study demonstrate that CDV presents a potent antiviral activity against the smallpox vaccine strain IOC and mainly against the VACV field strain CTGV. The EC50 values found for CTGV and VACV-IOC were lower than the numbers reported for other VACV strains using a similar strategy to evaluate virus replication [11]. VACV-like infections have been persistently reported in Brazil with an increasing number of human cases [1–5]. This emerging viral zoonosis results in significant agricultural and occupational effects, and the search for an effective antiviral therapy is a major concern. Moreover, the possibility of resuming smallpox vaccine production in Brazil also supports the need for an effective anti-VACV therapy. So far, FK506 has been the only drug reported to present antiviral activity against VAVC-IOC and, to a lesser extent, CTGV [10]. Nevertheless, this macrolide is a potent immunosuppressive drug and its antiviral effectiveness in vivo has not been determined. Therefore, our data on CDV efficacy against CTGV and VACVIOC replication are extremely encouraging. However, the poor oral bioavailability of CDV may limit its use in a major episode [6]. Nevertheless, it is worthy of noting that CDV topical administration
has been successful in treating cutaneous lesions in animals infected with VACV-WR, cowpox and orf viruses as well as humans infected with molluscum contagiosum virus, revealing long-lasting response levels [13–15]. Therefore, topical formulations of CDV for treating CTGV-like lesions on dairy cattle and humans may be seriously considered as a promising antiviral therapy to restrain the spread of this emerging zoonosis. Acknowledgments The authors thank Gilead Sciences, Inc., Foster City, CA, for providing cidofovir, and Ademilson Bizerra for technical assistance. Funding: This work was supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), Faperj (Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil) and IFS (International Foundation for Science, Sweden). Competing interests: None declared. Ethical approval: Not required. References [1] Damaso CR, Esposito JJ, Condit RC, Moussatché N. An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 2000;277:439–49. [2] Trindade GS, da Fonseca FG, Marques JT, Nogueira ML, Mendes LC, Borges AS, et al. Arac¸atuba virus: a vaccinialike virus associated with infection in humans and cattle. Emerg Infect Dis 2003;9:155–60. [3] Nagasse-Sugahara TK, Kisieliu JJ, Ueda-Ito M, Curti SP, Figueiredo CA, Cruz AS, et al. Human vaccinia-like virus outbreaks in São Paulo and Goiás states, Brazil: virus detection, isolation and identification. Rev Inst Med Trop Sao Paulo 2004;46:315–22. [4] Lobato ZI, Trindade GS, Frois MC, Ribeiro EB, Dias GR, Teixeira BM, et al. Outbreak of exantemal disease caused by vaccinia virus in human and cattle in Zona da Mata region, Minas Gerais. Arq Bras Med Vet Zootech 2005;57:423–9. [5] Damaso CR, Reis SA, Jesus DM, Lima PS, Moussatché N. A PCR-based assay for detection of emerging vaccinia-like viruses isolated in Brazil. Diagn Microbiol Infect Dis 2007;57:39–46. [6] Prichard MN, Kern ER. Orthopoxvirus targets for the development of antiviral therapies. Curr Drug Targets Infect Disord 2005;5:17–28. [7] Parrino J, Graham BS. Smallpox vaccines: past, present, and future. J Allergy Clin Immunol 2006;118:1320–6. [8] Borges MB, Kato SE, Damaso CR, Moussatché N, da Silva Freire M, Lambert Passos SR, et al. Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies. Biologicals 2007;36:105–10. [9] Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva, Switzerland: World Health Organization; 1988. [10] Reis SA, Moussatche N, Damaso CR. FK506 a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against orthopoxvirus infection in cell culture. J Appl Microbiol 2006;100:1373–80. [11] Kern ER, Hartline C, Harden E, Keith K, Rodriguez N, Beadle JR, et al. Enhanced inhibition of orthopoxvirus replication in vitro by alkoxyalkyl esters of cidofovir and cyclic cidofovir. Antimicrob Agents Chemother 2002;46:991–5.
D.M. Jesus et al. / International Journal of Antimicrobial Agents 33 (2009) 75–79 [12] Aduma P, Connelly MC, Srinivas RV, Fridland A. Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates. Mol Pharmacol 1995;47: 816–22. [13] Quenelle DC, Collins DJ, Kern ER. Cutaneous infections of mice with vaccinia or cowpox viruses and efficacy of cidofovir. Antiviral Res 2004;63: 33–40.
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[14] Scagliarini A, McInnes CJ, Gallina L, Dal Pozzo F, Scagliarini L, Snoeck R, et al. Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs. Antiviral Res 2007;73:169–74. [15] Calista D. Topical cidofovir for severe cutaneous human papillomavirus and molluscum contagiosum infections in patients with HIV/AIDS. A pilot study. J Eur Acad Dermatol Venereol 2000;14:484–8.