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Neotropical primary bat cell lines show restricted dengue virus replication
Comparative Immunology, Microbiology and Infectious Diseases 50 (2017) 101–105
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Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Neotropical primary bat cell lines show restricted dengue virus replication Andrés Moreira-Soto, Claudio Soto-Garita, Eugenia Corrales-Aguilar ∗ Virology-CIET (Research Center for Tropical Diseases), Faculty of Microbiology, University of Costa Rica, 11501-2060 San José, Costa Rica
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Article history: Received 17 June 2016 Received in revised form 6 December 2016 Accepted 19 December 2016 Keywords: Dengue Viral infection in vitro studies Bats
a b s t r a c t Dengue is the most widespread arboviral disease affecting humans. Bats are recognized carriers of emerging viral zoonoses and have been proposed as dengue reservoirs, since RNA/NS1 and/or antiviral antibodies have been detected. Yet, experimental inoculation of Artibeus bats failed to show virus replication. This conflicting results prevent drawing further conclusions of whether bats sustain dengue infection. To test bat cellular permissivity to dengue infection, we established primary bat embryonic cells from diverse organs and tissues of Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus. We observed a limited serotype-, organ-, and bat species- specific dengue susceptibility. Only some Molossusderived primary cells sustained poorly initial Dengue serotype-1 replication, though it was latter absent. To elucidate if Molossus bats may play a role in dengue replication, ecological or in vivo experiments must be performed. Taken together our results show that Dengue did not replicate efficiently in cell lines derived from Neotropical bat species. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Dengue is the most widespread arboviral disease that affects humans worldwide and a major cause of disease in tropical endemic countries [1]. Dengue virus (DENV) is divided into four distinct but genetically related serotypes [2]. Dengue causes a wide disease spectra in humans, which can go from a clinically unapparent infection to hemorrhagic fever and death [1]. The World Health Organization (WHO) estimates around 50–100 million human cases per year [2]. DENV in the old world is transmitted and maintained between two distinct sylvatic and urban cycles. The sylvatic cycle is well documented in Africa and Asia, where the virus is maintained between old world primates and Aedes mosquitoes [3,4]. Even though a definite animal host has not yet been identified in America, several research groups found evidence of infection in different mammals [5–7]. Within mammals, bats have been proposed as carriers of emerging viral zoonoses [8,9]. Furthermore, several groups have shown presence of viral nucleic acid and antibodies against dengue [5–7,10–14] suggesting a puta-
Abbreviations: DENV, dengue virus; MOI, multiplicity of infection; FBS, fetal bovine serum; RT-PCR, retro transcription-polymerase chain reaction. ∗ Corresponding author at: Virology, Microbiology, University of Costa Rica, Ciudad Universitaria Rodrigo Facio, San Pedro 1150-2060 San José, Costa Rica. E-mail addresses: [email protected] (A. Moreira-Soto), [email protected] (C. Soto-Garita), [email protected] (E. Corrales-Aguilar). http://dx.doi.org/10.1016/j.cimid.2016.12.004 0147-9571/© 2016 Elsevier Ltd. All rights reserved.
tive role as DENV reservoirs or hosts. Additionally, experimental infection studies in controlled laboratory settings showed no evidence of pathology or infection [15,16]. In one study, 35% of Artibeus intermedius bats infected with an excess of DENV-2 displayed seroconversion (even at low level) and one bat kidney as well as many sera were DENV RNA positive [16]. Nevertheless these results may be due to an excessively high viral dose for infection, suggesting that these bats are not suitable hosts for DENV-2 [16]. Similar results were found in systematic experimental inoculation of DENV serotypes 1 and 4 in Artibeus jamaicensis [15]. Other more recent study, suggested that American bats may not be reservoirs or amplification hosts for DENV infection since they were not able to detect virus RNA in liver or spleen tissue [17]. Overall results so far are conflicting and no conclusions can be withdrawn regarding the role of bats in DENV transmission and epidemiology. In vitro, one study using a bat cell line TB.1 Lu (derived from Tadarida brasiliensis) showed only DENV-1 infection by RT- PCR [18]. Another study in India failed to detect DENV-2 replication in a Pipistrellus embryonic cell line [19]. Most of these studies face limitations due to the bats species and organs used for generating cell lines. Therefore, studies about the susceptibility to in vitro infection by 4 DENV serotypes in different bat cells derived from several species are needed in order to assess if bat cells from different bat species or organs may be permissive to viral entry and be able to produce viral progeny in vitro. Here, we report on the susceptibility of different Neotropical primary bat cell cultures to the 4 DENV serotypes infection.
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2.1. Bats capturing and processing Pregnant individuals of three bat species presenting different trophic guilds (Artibeus jamaicensis-frugivorous, Molossus sinaloae-insectivorous, and Desmodus rotundus-hematophagous) were captured. Permission for bat capturing and processing was obtained from the Institutional Committee of Care and Use of Animals of the University of Costa Rica (CICUA-124-12) according to international animal welfare standards and by University of Costa Rica project B4-656. The individuals were captured using mist nets and immediately transported to the laboratory. Bats were euthanized using a combination of Ketamine (1 mg/ml) and Xylazine (1 mg/ml). 2.2. Embryonic primary cell cultures The excision of fetal tissues and isolation of primary bat cells were made as described elsewhere [20]. Briefly, after a 1- or 2h trypsinization of fetal tissues (mesenchymal fibroblasts of the whole embryonic body without internal organs or the head) or whole dissected organs, cells were allowed to adhere and grow in a 5% CO2 atmosphere in D-MEM supplemented with 10% FBS (All from Gibco, USA). We therefore established primary embryonic bat cultures resulting from different organs (kidney, liver, intestine) disaggregation and mesenchymal fibroblasts from the three bat species. Cultures derived from bat organs may include a mixture of different cell types consisting of epithelial, endothelial and organ characteristic (such as hepatocytes for the liver) cells. Primary bat cultures were screened for Mycoplasma sp. and Rabies virus by PCR (negative results, data not shown). No primary embryonic bat culture was used over passage 4 after sub-culturing to lessen cell differentiation or loss and to heighten culture homogeneity. 2.3. Detection of DENV virus infection through plaque quantification and immunofluorescence assays
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Artibeus kidney
Artibeus intestine
2. Materials and methods
DENV-4
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Fig 1. Viral growth kinetics of DENV serotype 1 in 2 Desmodus derived bat primary embryonic cells and LLC-MK2 cells. Cells were infected with DENV-1 at low MOI (0.1), absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 24 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. One representative experiment of many is shown. Each value depicted represents the mean of triplicates (n = 3).
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Fig. 2. Viral growth kinetics of DENV serotypes 1–4 in different bat primary embryonic cell lines derived from different bat species and LLC-MK2 cells. Cells were infected with DENV at an MOI of 2–3, absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 48 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. One representative experiment of three is shown. Each value depicted represents the mean of triplicates (n = 3).
For viral growth kinetics, 2 different Desmodus derived bat primary embryonic cells and LLC-MK2 cells were infected with DENV-1 at a low MOI (0.1), adsorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 24 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. For determination of permissivity for the 4 DENV serotypes, different bat primary embryonic cell cultures derived from different bat species and LLC-MK2 cells (as positive controls) were infected with DENV serotypes at a higher virus dose (MOI of 2–3), since lack of virus production was observed with a low virus dose, absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected and infectious virus titer was determined by a standard plaque assay in LLC-MK2 cells at 48 and 96 h post infection. Primary embryonic bat cells were infected with DENV-1 and DENV-2 (MOI of 2) respectively and viral infection was monitored by immunofluorescence. After 24 h of infection, cells were fixed with methanol, incubated for 15 min with a permeabilization solution (PBS 1%, Saponin 0.1% and FBS 1%) and stained using an anti-NS3 protein antibody (GENETEX 124252), an anti- DENV 1–4 antibody (recognizing E and other structural proteins) (GENETEX 29202), and secondary antibody goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen) or goat anti-mouse IgG FITC (Sigma-Aldrich), respectively. Nuclei were counterstained with DAPI. Images were taken using a Cytation 3 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc., USA). An image analysis pipeline was developed using Cell Profiler software 2.1.1 (http://www.cellprofiler.org/ citations.shtml). 3. Results
Dengue reference viruses DENV-1 Angola (D1/AO/XX/1988), DENV-2 Jamaica (D2/JM/1409/1983), DENV-3 Nicaragua (D3/NI/30-94/1994), and DENV-4 Dominica (D4/DM/814669/1981) stocks were produced in C6/36 cells and titrated in LLC-MK2 using standard plaque assays.
3.1. Primary bat cell lines show restricted dengue virus replication To analyze DENV virus infection of primary bat cells, first we assessed if 2 different cell lines sustained viral replication by
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Fig. 3. NS3 staining after Dengue infection. Primary embryonic bat cells were infected with the two DENV serotypes and viral infection was monitored by immunofluorescence using an anti-NS3 protein antibody, an anti- DENV 1–4 antibody (recognizing E and other structural proteins), and secondary antibodies goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen) and goat anti-mouse IgG FITC (Sigma-Aldrich), respectively (green). Nuclei were counterstained with DAPI (blue). Images were taken using a Cytation 3 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc., USA). An image analysis pipeline was developed using Cell Profiler software 2.1.1 (http://www.cellprofiler.org/ citations.shtml) to eliminate background unspecific staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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performing low MOI (0.1) virus growth curves using Desmodus fibroblasts and kidney cells. We used DENV-1 since previous studies have detected it by RT-PCR in infected bat cell lines [18]. Supernatants were collected every 24 h and then titrated in LLC-MK2 cells using a standard plaque assay. As shown in Fig. 1, no viral growth in Desmodus fibroblasts and kidney cells is observed in comparison to a growth curve using a susceptible and permissive cell line (LLC-MK2) widely used for DENV experiments. To evaluate permissivity for infection of DENV serotype- and cell type- specificity as described elsewhere [18], higher virus dose growth curves using 11 different bat cells of distinct organs or origin (kidney, liver, intestine and fibroblasts) and bat species (Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus) were done (Fig. 2). Cells were infected with the 4 DENV serotypes at a higher MOI (2–3), adsorbed during 1 h and incubated until 96 h post infection. Supernatants were collected every 48 h and then titrated in LLC-MK2 cells using a standard plaque assay. Fig. 2 shows a serotype and cell type specificity since only DENV-1 is able to replicate in a limited manner in Molossus fibroblasts, liver, kidney, intestine cells; and in Artibeus fibroblasts. Nevertheless, viral replication diminishes 48 h later in all cases to an almost negative level. Furthermore, minimal DENV-4 replication is observed in some Molossus cells. To discard lack of virus production caused by apoptosis and/or cell death, cells were analyzed in flow cytometry staining with Propidium Iodide and Annexin V at 96 hpi. Percentages of apoptotic and dead cells were not significantly higher in comparison to LLC-MK2 cells in order to cause disruption of virus replication (Supplementary Fig. 1). These results suggest a limited, differentiated cell susceptibility to infection depending on the DENV serotype and the specific bat cell type and species. 3.2. Dengue NS3 detection is primary bat cell lines show initial replication in some organ- and bat species-derived cells Afterwards, we measured expression of a non-structural protein (NS3) in cells infected with DENV to analyze if lack of virus production was caused by disruption in the virus early steps of replication after infection (Fig. 3). The presence of a non-structural protein such as NS3 [21] in some of the primary embryonic bat cells infected with DENV-1 (white arrows) and to a minimal extent with DENV-2 (red arrows) confirmed that dengue may infect and replicate in some of these cells in a serotype specific manner. Noteworthy is the pattern of staining found in some cells presenting limited DENV virus replication. The NS3 protein was stained in a punctuated pattern in the nucleus and cytoplasm (e.g. Molossus DENV-2 staining) (red arrows) whereas a broad distribution throughout the cytoplasm is observed not only in the permissive LLC-MK2 cells but also in the Molossus DENV-1 staining (white arrows). The significance of this unexplained cell type-specific variation in NS3 staining may represent differences in its subcellular localization [22] or a possible disruption of an infectious cycle. 4. Discussion and conclusions In our results, we observe a serotype-, cell origin- and bat species- specific DENV permissivity of infection, though virus replication seems to be limited. Therefore DENV virus appears to replicate unproductively and inefficiently in some bat cells and consequently, bats from some of these species (Artibeus and Desmodus) may unlikely serve as DENV reservoirs. Nevertheless, we detected a limited DENV replication in cells derived from Molossus bats. Molossus bats are known to share roosting sites with humans in human-made structures [23], and their insectivorous trophic guild might suggest more contact with mosquitoes or mosquito-eating insects. Therefore, a close proximity in the urban or peri-urban set-
tings where humans and mosquitoes endure DENV infection may involve these bat species in a putative transmission cycle. However, to elucidate if these bats play a role in DENV replication and transmission as reservoirs more studies have to be performed. Here we detect that bat primary cells from solely one bat species (Molossus) possess low permissiveness to DENV serotype 1, though these cells when infected sustain inefficiently virus replication. All primary cells obtained from the 2 other Neotropical bats show low to null permissiveness to the 4 DENV serotypes. Taking into account that DENV might have originated in Africa or Southeast Asia [4], it would be interesting to test cell lines from African or Asian bat species to assess if virus exposure for a longer time span might represent a differential DENV susceptibility. Whether bat cells clear DENV infection by their innate immune response mechanisms or lack cellular mediators like proteins e.g. enzymes needed for the virus replication cycle to be efficient (abortive infection) is still unclear. In humans, peripheral blood monocytes and macrophages show high susceptibility to DENV infection [24,25] and the role of DENV primary infection of dendritic cells in the pathogenesis of diseases has been well established [26,27]. Assessing susceptibility to DENV infection in bat monocytes, macrophages, and/or dendritic cells therefore is from high interest, inferring that DENV well-described receptors (such as DC-SIGN [27] or others [28]) are expressed by these bat cells. It cannot be excluded from our in vitro experiments, that these particular cell types are though susceptible to DENV infection and the reason why some bats show evidence of DENV exposure (presence of neutralizing antibodies) [14,16]. Several groups have suggested a putative role of bats as hosts in DENV ecology [5–7,10–14]. Cross-reactivity among the members of the members of the viral family Flaviviridae [29] may explain the serological data found by many of these groups, but the detection of viral RNA or NS1 in blood of wild bats suggests that bats could serve as hosts. To elucidate if bats play a role in DENV replication, ecological or in vivo experiments must be performed using an appropriate bat specie and the suitable DENV serotype. Yet, further experiments such as successful virus isolation in a susceptible cell culture system directly from bat samples, demonstration of infection by Aedes aegypti or other mosquitoes bites with subsequent viremia, or metagenomic analysis may enlighten the putative, if existent, role of bats in a dengue virus cycle. Funding This work was supported by the University of Costa Rica Project VI-803-B2-285 and FEES-CONARE VI-803-B4-656. The authors declare no conflicts of interest. Acknowledgments We thank Francisco Vega for valuable technical assistance. We thank Dr. Victor Hugo Sancho from the Rabies Control Program from the Department of Agriculture of Costa Rica (MAG). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cimid.2016.12. 004. References [1] S. Yacoub, J. Mongkolsapaya, G. Screaton, Recent advances in understanding dengue, F1000Research 5 (2016), http://dx.doi.org/10.12688/f1000research. 6233.1.
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Neotropical primary bat cell lines show restricted dengue virus replication Andrés Moreira-Soto, Claudio Soto-Garita, Eugenia Corrales-Aguilar ∗ Virology-CIET (Research Center for Tropical Diseases), Faculty of Microbiology, University of Costa Rica, 11501-2060 San José, Costa Rica
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Article history: Received 17 June 2016 Received in revised form 6 December 2016 Accepted 19 December 2016 Keywords: Dengue Viral infection in vitro studies Bats
a b s t r a c t Dengue is the most widespread arboviral disease affecting humans. Bats are recognized carriers of emerging viral zoonoses and have been proposed as dengue reservoirs, since RNA/NS1 and/or antiviral antibodies have been detected. Yet, experimental inoculation of Artibeus bats failed to show virus replication. This conflicting results prevent drawing further conclusions of whether bats sustain dengue infection. To test bat cellular permissivity to dengue infection, we established primary bat embryonic cells from diverse organs and tissues of Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus. We observed a limited serotype-, organ-, and bat species- specific dengue susceptibility. Only some Molossusderived primary cells sustained poorly initial Dengue serotype-1 replication, though it was latter absent. To elucidate if Molossus bats may play a role in dengue replication, ecological or in vivo experiments must be performed. Taken together our results show that Dengue did not replicate efficiently in cell lines derived from Neotropical bat species. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Dengue is the most widespread arboviral disease that affects humans worldwide and a major cause of disease in tropical endemic countries [1]. Dengue virus (DENV) is divided into four distinct but genetically related serotypes [2]. Dengue causes a wide disease spectra in humans, which can go from a clinically unapparent infection to hemorrhagic fever and death [1]. The World Health Organization (WHO) estimates around 50–100 million human cases per year [2]. DENV in the old world is transmitted and maintained between two distinct sylvatic and urban cycles. The sylvatic cycle is well documented in Africa and Asia, where the virus is maintained between old world primates and Aedes mosquitoes [3,4]. Even though a definite animal host has not yet been identified in America, several research groups found evidence of infection in different mammals [5–7]. Within mammals, bats have been proposed as carriers of emerging viral zoonoses [8,9]. Furthermore, several groups have shown presence of viral nucleic acid and antibodies against dengue [5–7,10–14] suggesting a puta-
Abbreviations: DENV, dengue virus; MOI, multiplicity of infection; FBS, fetal bovine serum; RT-PCR, retro transcription-polymerase chain reaction. ∗ Corresponding author at: Virology, Microbiology, University of Costa Rica, Ciudad Universitaria Rodrigo Facio, San Pedro 1150-2060 San José, Costa Rica. E-mail addresses: [email protected] (A. Moreira-Soto), [email protected] (C. Soto-Garita), [email protected] (E. Corrales-Aguilar). http://dx.doi.org/10.1016/j.cimid.2016.12.004 0147-9571/© 2016 Elsevier Ltd. All rights reserved.
tive role as DENV reservoirs or hosts. Additionally, experimental infection studies in controlled laboratory settings showed no evidence of pathology or infection [15,16]. In one study, 35% of Artibeus intermedius bats infected with an excess of DENV-2 displayed seroconversion (even at low level) and one bat kidney as well as many sera were DENV RNA positive [16]. Nevertheless these results may be due to an excessively high viral dose for infection, suggesting that these bats are not suitable hosts for DENV-2 [16]. Similar results were found in systematic experimental inoculation of DENV serotypes 1 and 4 in Artibeus jamaicensis [15]. Other more recent study, suggested that American bats may not be reservoirs or amplification hosts for DENV infection since they were not able to detect virus RNA in liver or spleen tissue [17]. Overall results so far are conflicting and no conclusions can be withdrawn regarding the role of bats in DENV transmission and epidemiology. In vitro, one study using a bat cell line TB.1 Lu (derived from Tadarida brasiliensis) showed only DENV-1 infection by RT- PCR [18]. Another study in India failed to detect DENV-2 replication in a Pipistrellus embryonic cell line [19]. Most of these studies face limitations due to the bats species and organs used for generating cell lines. Therefore, studies about the susceptibility to in vitro infection by 4 DENV serotypes in different bat cells derived from several species are needed in order to assess if bat cells from different bat species or organs may be permissive to viral entry and be able to produce viral progeny in vitro. Here, we report on the susceptibility of different Neotropical primary bat cell cultures to the 4 DENV serotypes infection.
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2.1. Bats capturing and processing Pregnant individuals of three bat species presenting different trophic guilds (Artibeus jamaicensis-frugivorous, Molossus sinaloae-insectivorous, and Desmodus rotundus-hematophagous) were captured. Permission for bat capturing and processing was obtained from the Institutional Committee of Care and Use of Animals of the University of Costa Rica (CICUA-124-12) according to international animal welfare standards and by University of Costa Rica project B4-656. The individuals were captured using mist nets and immediately transported to the laboratory. Bats were euthanized using a combination of Ketamine (1 mg/ml) and Xylazine (1 mg/ml). 2.2. Embryonic primary cell cultures The excision of fetal tissues and isolation of primary bat cells were made as described elsewhere [20]. Briefly, after a 1- or 2h trypsinization of fetal tissues (mesenchymal fibroblasts of the whole embryonic body without internal organs or the head) or whole dissected organs, cells were allowed to adhere and grow in a 5% CO2 atmosphere in D-MEM supplemented with 10% FBS (All from Gibco, USA). We therefore established primary embryonic bat cultures resulting from different organs (kidney, liver, intestine) disaggregation and mesenchymal fibroblasts from the three bat species. Cultures derived from bat organs may include a mixture of different cell types consisting of epithelial, endothelial and organ characteristic (such as hepatocytes for the liver) cells. Primary bat cultures were screened for Mycoplasma sp. and Rabies virus by PCR (negative results, data not shown). No primary embryonic bat culture was used over passage 4 after sub-culturing to lessen cell differentiation or loss and to heighten culture homogeneity. 2.3. Detection of DENV virus infection through plaque quantification and immunofluorescence assays
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2. Materials and methods
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Fig 1. Viral growth kinetics of DENV serotype 1 in 2 Desmodus derived bat primary embryonic cells and LLC-MK2 cells. Cells were infected with DENV-1 at low MOI (0.1), absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 24 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. One representative experiment of many is shown. Each value depicted represents the mean of triplicates (n = 3).
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Fig. 2. Viral growth kinetics of DENV serotypes 1–4 in different bat primary embryonic cell lines derived from different bat species and LLC-MK2 cells. Cells were infected with DENV at an MOI of 2–3, absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 48 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. One representative experiment of three is shown. Each value depicted represents the mean of triplicates (n = 3).
For viral growth kinetics, 2 different Desmodus derived bat primary embryonic cells and LLC-MK2 cells were infected with DENV-1 at a low MOI (0.1), adsorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected every 24 h. Titers of infectious virus were determined by a standard plaque assay in LLC-MK2 cells. For determination of permissivity for the 4 DENV serotypes, different bat primary embryonic cell cultures derived from different bat species and LLC-MK2 cells (as positive controls) were infected with DENV serotypes at a higher virus dose (MOI of 2–3), since lack of virus production was observed with a low virus dose, absorbed at 37 ◦ C during 1 h and incubated at 37 ◦ C 5% CO2 . Supernatants were collected and infectious virus titer was determined by a standard plaque assay in LLC-MK2 cells at 48 and 96 h post infection. Primary embryonic bat cells were infected with DENV-1 and DENV-2 (MOI of 2) respectively and viral infection was monitored by immunofluorescence. After 24 h of infection, cells were fixed with methanol, incubated for 15 min with a permeabilization solution (PBS 1%, Saponin 0.1% and FBS 1%) and stained using an anti-NS3 protein antibody (GENETEX 124252), an anti- DENV 1–4 antibody (recognizing E and other structural proteins) (GENETEX 29202), and secondary antibody goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen) or goat anti-mouse IgG FITC (Sigma-Aldrich), respectively. Nuclei were counterstained with DAPI. Images were taken using a Cytation 3 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc., USA). An image analysis pipeline was developed using Cell Profiler software 2.1.1 (http://www.cellprofiler.org/ citations.shtml). 3. Results
Dengue reference viruses DENV-1 Angola (D1/AO/XX/1988), DENV-2 Jamaica (D2/JM/1409/1983), DENV-3 Nicaragua (D3/NI/30-94/1994), and DENV-4 Dominica (D4/DM/814669/1981) stocks were produced in C6/36 cells and titrated in LLC-MK2 using standard plaque assays.
3.1. Primary bat cell lines show restricted dengue virus replication To analyze DENV virus infection of primary bat cells, first we assessed if 2 different cell lines sustained viral replication by
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Fig. 3. NS3 staining after Dengue infection. Primary embryonic bat cells were infected with the two DENV serotypes and viral infection was monitored by immunofluorescence using an anti-NS3 protein antibody, an anti- DENV 1–4 antibody (recognizing E and other structural proteins), and secondary antibodies goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen) and goat anti-mouse IgG FITC (Sigma-Aldrich), respectively (green). Nuclei were counterstained with DAPI (blue). Images were taken using a Cytation 3 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc., USA). An image analysis pipeline was developed using Cell Profiler software 2.1.1 (http://www.cellprofiler.org/ citations.shtml) to eliminate background unspecific staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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performing low MOI (0.1) virus growth curves using Desmodus fibroblasts and kidney cells. We used DENV-1 since previous studies have detected it by RT-PCR in infected bat cell lines [18]. Supernatants were collected every 24 h and then titrated in LLC-MK2 cells using a standard plaque assay. As shown in Fig. 1, no viral growth in Desmodus fibroblasts and kidney cells is observed in comparison to a growth curve using a susceptible and permissive cell line (LLC-MK2) widely used for DENV experiments. To evaluate permissivity for infection of DENV serotype- and cell type- specificity as described elsewhere [18], higher virus dose growth curves using 11 different bat cells of distinct organs or origin (kidney, liver, intestine and fibroblasts) and bat species (Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus) were done (Fig. 2). Cells were infected with the 4 DENV serotypes at a higher MOI (2–3), adsorbed during 1 h and incubated until 96 h post infection. Supernatants were collected every 48 h and then titrated in LLC-MK2 cells using a standard plaque assay. Fig. 2 shows a serotype and cell type specificity since only DENV-1 is able to replicate in a limited manner in Molossus fibroblasts, liver, kidney, intestine cells; and in Artibeus fibroblasts. Nevertheless, viral replication diminishes 48 h later in all cases to an almost negative level. Furthermore, minimal DENV-4 replication is observed in some Molossus cells. To discard lack of virus production caused by apoptosis and/or cell death, cells were analyzed in flow cytometry staining with Propidium Iodide and Annexin V at 96 hpi. Percentages of apoptotic and dead cells were not significantly higher in comparison to LLC-MK2 cells in order to cause disruption of virus replication (Supplementary Fig. 1). These results suggest a limited, differentiated cell susceptibility to infection depending on the DENV serotype and the specific bat cell type and species. 3.2. Dengue NS3 detection is primary bat cell lines show initial replication in some organ- and bat species-derived cells Afterwards, we measured expression of a non-structural protein (NS3) in cells infected with DENV to analyze if lack of virus production was caused by disruption in the virus early steps of replication after infection (Fig. 3). The presence of a non-structural protein such as NS3 [21] in some of the primary embryonic bat cells infected with DENV-1 (white arrows) and to a minimal extent with DENV-2 (red arrows) confirmed that dengue may infect and replicate in some of these cells in a serotype specific manner. Noteworthy is the pattern of staining found in some cells presenting limited DENV virus replication. The NS3 protein was stained in a punctuated pattern in the nucleus and cytoplasm (e.g. Molossus DENV-2 staining) (red arrows) whereas a broad distribution throughout the cytoplasm is observed not only in the permissive LLC-MK2 cells but also in the Molossus DENV-1 staining (white arrows). The significance of this unexplained cell type-specific variation in NS3 staining may represent differences in its subcellular localization [22] or a possible disruption of an infectious cycle. 4. Discussion and conclusions In our results, we observe a serotype-, cell origin- and bat species- specific DENV permissivity of infection, though virus replication seems to be limited. Therefore DENV virus appears to replicate unproductively and inefficiently in some bat cells and consequently, bats from some of these species (Artibeus and Desmodus) may unlikely serve as DENV reservoirs. Nevertheless, we detected a limited DENV replication in cells derived from Molossus bats. Molossus bats are known to share roosting sites with humans in human-made structures [23], and their insectivorous trophic guild might suggest more contact with mosquitoes or mosquito-eating insects. Therefore, a close proximity in the urban or peri-urban set-
tings where humans and mosquitoes endure DENV infection may involve these bat species in a putative transmission cycle. However, to elucidate if these bats play a role in DENV replication and transmission as reservoirs more studies have to be performed. Here we detect that bat primary cells from solely one bat species (Molossus) possess low permissiveness to DENV serotype 1, though these cells when infected sustain inefficiently virus replication. All primary cells obtained from the 2 other Neotropical bats show low to null permissiveness to the 4 DENV serotypes. Taking into account that DENV might have originated in Africa or Southeast Asia [4], it would be interesting to test cell lines from African or Asian bat species to assess if virus exposure for a longer time span might represent a differential DENV susceptibility. Whether bat cells clear DENV infection by their innate immune response mechanisms or lack cellular mediators like proteins e.g. enzymes needed for the virus replication cycle to be efficient (abortive infection) is still unclear. In humans, peripheral blood monocytes and macrophages show high susceptibility to DENV infection [24,25] and the role of DENV primary infection of dendritic cells in the pathogenesis of diseases has been well established [26,27]. Assessing susceptibility to DENV infection in bat monocytes, macrophages, and/or dendritic cells therefore is from high interest, inferring that DENV well-described receptors (such as DC-SIGN [27] or others [28]) are expressed by these bat cells. It cannot be excluded from our in vitro experiments, that these particular cell types are though susceptible to DENV infection and the reason why some bats show evidence of DENV exposure (presence of neutralizing antibodies) [14,16]. Several groups have suggested a putative role of bats as hosts in DENV ecology [5–7,10–14]. Cross-reactivity among the members of the members of the viral family Flaviviridae [29] may explain the serological data found by many of these groups, but the detection of viral RNA or NS1 in blood of wild bats suggests that bats could serve as hosts. To elucidate if bats play a role in DENV replication, ecological or in vivo experiments must be performed using an appropriate bat specie and the suitable DENV serotype. Yet, further experiments such as successful virus isolation in a susceptible cell culture system directly from bat samples, demonstration of infection by Aedes aegypti or other mosquitoes bites with subsequent viremia, or metagenomic analysis may enlighten the putative, if existent, role of bats in a dengue virus cycle. Funding This work was supported by the University of Costa Rica Project VI-803-B2-285 and FEES-CONARE VI-803-B4-656. The authors declare no conflicts of interest. Acknowledgments We thank Francisco Vega for valuable technical assistance. We thank Dr. Victor Hugo Sancho from the Rabies Control Program from the Department of Agriculture of Costa Rica (MAG). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cimid.2016.12. 004. References [1] S. Yacoub, J. Mongkolsapaya, G. Screaton, Recent advances in understanding dengue, F1000Research 5 (2016), http://dx.doi.org/10.12688/f1000research. 6233.1.
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