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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both CHIKV and ONNV can cause febrile illness in humans. Clinically, the symptoms of chikungunya are difficult to distinguish from those of dengue fever and so may be misdiagnosed.4–6 CHIKV infections are characterized by fever, headache, nausea, vomiting, myalgia, rash, and arthralgia.7 The clinical features of ONNV infections include a low-grade fever, symmetrical polyarthralgia, lymphadenopathy, generalized papular or maculopapular exanthema, and joint pain.5,8
CHIKV was first isolated in 1953 in Tanzania from the serum of a febrile human.9 There have been numerous isolates made from both humans and mosquitoes in central, western, and southern Africa as well as in Asia.10 CHIKV is believed to have originated in Africa where it is maintained in a sylvatic cycle involving wild primates and forest-dwelling mosquitoes such as Aedes furcifer. It was subsequently introduced into Asia where, as in the African urban cycle, it is transmitted by Ae. aegypti mosquitoes.3 CHIKV has been the cause of substantial epidemics in both Southeast Asia and in Africa.11,12,13
ONNV, which is the most closely related virus to CHIKV, was initially isolated in northern Uganda from anopheline mosquitoes and human serum during a 1959 epidemic.4,7 This virus has been associated with relatively few but large-scale epidemics.14 A major epidemic involving more than 2 million cases, with no recorded fatalities, was caused by ONNV in 1959 in East Africa.15,16 After an absence of 35 years another epidemic, in 1996, occurred in southern Uganda.17 A more recent epidemic (2002) was reported near the shores of Lake Wamala in the Mubende District, central Uganda (Julius Lutwama, personal communication). ONNV is transmitted by anopheline mosquitoes such as Anopheles gambiae, the most important vector for human malaria, and An. funestus.15,18 Humans may be the only natural host of ONNV, as other vertebrate reservoirs have not been identified. Igbo Ora virus was initially thought to be a separate but closely related virus based on cross-complement fixation tests.19 However, sequence analysis of this virus indicates that it is a strain of ONNV.17
Johnson suggested that mutations in CHIKV led to the emergence of ONNV and its ability to be transmitted by anopheline mosquitoes.16 More recent sequence analysis by Lanciotti and others and Powers and others indicate that CHIKV and ONNV are "genetically distinct."7,16,17 Lanciotti and others examined the complete nucleotide sequence of five alphaviruses: 3 ONNV (SG650, Gulu, and Igbo Ora), Semliki Forest (SFV), and Ross River virus (RRV). A comparison of the coding structural region of the viruses was also made, capsid through E1 genes, of six alphavirus isolates: 3 ONNV (SG650, Gulu, and Igbo Ora), CHIK, SFV, and RRV.17 Powers and others examined the areas of the E1 envelope glycoprotein and 3' noncoding region from 24 alphavirus isolates: 18 CHIKV, 3 ONNV, 2 Sindbis, and 1 SFV.7 Both of these studies confirmed that CHIKV and ONNV are separate but related viruses in the Semliki Forest antigenic complex. Powers and others revealed two CHIKV lineages7; one lineage contained all isolates from western Africa, and the second contained all southern and eastern African strains and the Asian isolates. Data were interpreted as indicating an African origin for CHIKV with divergence of ONNV and CHIKV from a common ancestor thousands of years ago.7
Here we report results of a comparison of these viruses and strains in vitro and in vivo. The three strains of ONNV and one strain of CHIKV were grown on anopheline (Mos 55) and Aedes (C6/36) derived cell culture and two vertebrate cell lines (BHK-21 and Vero) to analyze their ability to grow in vitro. The viruses were also compared for their ability to infect and disseminate in An. gambiae and Ae. aegypti mosquitoes. This new information will provide the foundation for studies on the basis/mechanism of viral-vector species specificity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stock viruses were produced after a single passage in Vero cells maintained at 37°C in Leibovitz L-15 medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cell supernatants were harvested when 75% of the cells showed cytopathic effect (3 + CPE), aliquoted, and stored at –80°C for use in all experiments.
In vitro growth kinetics of viruses. Two vertebrate-derived, BHK-21 (hamster kidney) and Vero (green monkey kidney), and two mosquito-derived, C6/36 (Ae. albopictus) and MOS-55 (An. gambiae), cell lines were used for these studies. All cells were maintained in Leibovitz L-15 medium with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Vertebrate and mosquito cells were maintained at 37°C and 28°C, respectively. For each virus, confluent cell monolayers in 25 cm2 flasks were infected with a standard 1 mL inoculum by rocking at room temperature for 1 hour. The inoculum was then removed and after three washes with 5 mL L-15, 5.5 mL of medium was added per flask. A sample of 0.5 mL was removed immediately. Additional 0.5 mL samples were collected at 24-hour intervals and replaced with 0.5 mL of fresh medium. Samples were stored at –80°C until titrated. Experiments were repeated three times. Data represents virus production for a standardized monolayer area (25 cm2). Due to a difference in the size of individual cells, the multiplicity of infection varied for the different cell lines, for example, 0.25 for C6/36 cells and 0.63 for BHK-21 cells.
Mosquitoes. The white-eyed Higgs variant of the Rexville D strain of Ae. aegypti and the G3 strain of An. gambiae were reared at 27°C and 80% relative humidity under a 16-hour light:8-hour dark photoperiod, as previously described.23–25 Adults were supplied with a cotton wool pad soaked in a 10% sucrose solution ad libitum and fed on anesthetized hamsters once per week for egg production.
Virus infections of mosquitoes. Four-day-old adult female Ae. aegypti or An. gambiae mosquitoes were fed a blood meal containing one of the three strains of ONNV or the CHIKV to be analyzed. Fresh virus was grown from stock and harvested from Vero cells when 75% of the cells showed CPE. The viral supernatant was mixed with an equal volume of defibrinated sheep blood (Colorado Serum Company, Denver, CO). As a phagostimulant, adenosine triphosphate at a final concentration of 2 mM was added to the blood meal. Mosquitoes were fed using an isolation glove box located in a Biosafety Level 3 insectary. Infectious blood was heated to 37°C and placed in a Hemotek feeding apparatus (Discovery Workshops, Accrington, Lancashire, UK), and mosquitoes were allowed to feed for 1 hour.26 Fully engorged females were separated from unfed females and were placed into new cartons. Three to eight mosquitoes were removed for titration on Days 0, 1, 2, 3, 7, and 14 postinfection (p.i.) and were stored at –80°C. Day 0 samples, collected immediately after feeding, were used to determine the titer of virus imbibed and to evaluate continuity between experiments.
Titrations. Viral samples harvested from cell culture and in mosquitoes were quantified as tissue culture infectious dose 50 end-point titers (log10 TCID50/mL) using a standardized procedure.27 Briefly, 100 µL samples of cell culture supernatant/mosquito triturate were pipetted into wells of the first column of a 96-well plate, serially diluted in a 10-fold series, seeded with Vero cells, and incubated at 37°C for 7 days.27 Prior to titration, each mosquito was triturated in 1 mL of L-15 medium and filtered through a 0.22-µM syringe filter (Millipore, Carrigwohill, Cork, Ireland).27 Differences in viral infection rates between Ae. aegypti and An. gambiae were tested for significance by Fisher’s exact test using SPSS version 11.5 (SPSS Inc. Chicago, IL).
Immunofluorescence assay. Salivary glands were dissected from 7- and 14-day p.i. mosquitoes for analysis to determine dissemination rates. The mosquitoes were dissected on glass microscope slides in phosphate-buffered saline. Salivary glands were air dried, fixed in cold acetone for 10 minutes, and stained using a cross-reactive mouse hyperimmune ascitic fluid raised against CHIKV as the primary antibody and amplifying the signal using indirect immunofluorescence assay (IFA) protocols previously described.27–29
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RESULTS |
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). In contrast, CHIKV strain 37997 failed to infect the An. gambiae cell line, MOS-55, but was able to infect the two vertebrate cell lines, Vero and BHK, and Ae. albopictus cells, C6/36 (Figure 1D). Cytopathic effect developed rapidly within 48 hours p.i. in both vertebrate cell lines, with subsequent total monolayer destruction, but was not observed in either of the mosquito cell lines.
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). The second analyzed the differences in infection rates for either Ae. aegypti or An. gambiae mosquitoes between the three strains of ONNV (Table 1). No significant difference (P > 0.05) was found in the infection rates for both species of mosquitoes on Days 0 and 1 p.i. for all three ONNV strains and CHIKV (Table 1). Therefore, differences in infection rates observed at later time points p.i. cannot be attributed to the initial titer of virus imbibed.
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). There was decline in viral titer on Days 2 and 3 p.i., which was then followed by an increase in the titer and the number of mosquitoes infected; by Day 14 p.i., the mean titer was 4.3 and 3.8 log10 TCID50/mosquito for Ae. aegypti and An. gambiae, respectively. One hundred percent of the mosquitoes were infected on 14 days p.i. (Table 1).
Although Gulu replicated in both mosquito species, the number of infected mosquitoes and the titer of the individual mosquitoes decreased steadily from days 0 to 14 p.i. A comparison of infection rates between Gulu and SG650 Day-14 p.i. revealed a significantly lower rate in the Gulu infection rates in both Ae. aegypti and An. gambiae mosquitoes (Table 1). A significant difference was also found for this virus between the two mosquito species on Days 7 and 14 p.i. (Table 1). On Day 14 p.i., 10% of the Ae. aegypti and 44% of the An. gambiae mosquitoes were infected.
Comparison of infection rates of Igbo Ora using Fisher’s exact test indicated that there was not a significant difference (P > 0.05) in the replication of this strain in the two mosquitoes tested until Day 14 p.i. (Table 1). At 14 days p.i., 25% of Ae. aegypti mosquitoes and 75% of An. gambiae mosquitoes were infected (Table 1). Igbo Ora infection rates on Day 14 p.i. did not show a significant difference from the Gulu infection rates in Ae. aegypti or An. gambiae. However, both Gulu and Igbo Ora produced significantly lower infection rates than SG650 on Day 14 p.i. (Table 1).
The most significant difference in infection rates was found between Ae. aegypti and An. gambiae infected with CHIKV strain 37997. The blood meal titers were identical, 7.95 log10 TCID50/mL of CHIKV, and the initial amount of virus imbibed were similar for both Ae. aegypti and An. gambiae (Table 1). However, the infection rates on Days 2, 3, 7, and 14 p.i. for Ae. aegypti and An. gambiae with CHIKV varied significantly (Table 1). On Day 14 p.i. 100% of Ae. aegypti were infected, whereas none of the An. gambiae mosquitoes were infected (Table 1).
In vivo infection and dissemination.
Salivary glands of individual mosquitoes were analyzed by IFA on Days 7 and 14 p.i. to determine dissemination rates for the two species of mosquitoes with the three strains of ONNV (Table 2). ONNV strains SG650, Gulu, and Igbo Ora disseminated in both species of mosquitoes by Day 14 p.i. (Table 2). For each strain of ONNV, there was not a significant difference in dissemination rates between Ae. aegypti and An. gambiae (P < 0.05), and no difference was found in Ae. aegypti between strains. However, there was a significant difference when virus strains were compared with each other in An. gambiae. SG650 and Igbo Ora disseminated in significantly more mosquitoes than the Gulu strain in An. gambiae (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous experimental growth curves with ONNV and CHIKV have used various cell lines. Vertebrate lines that have been infected include BHK,30–32 HeLa cells,33 Vero cells,34,35 LLC-MK2,34 and XTC-2 cells.36 Numerous arthropod-derived lines have also been tested, and it seems that those derived from Aedes are more sensitive than those from Culex.37 Buckley used Ae. aegypti and Ae. albopictus cell lines and concluded that although both viruses replicated in Ae. albopictus cells, CHIKV replicated in Ae. aegypti cells whereas ONNV did not.31 Unpublished data cited by Karabatsos reported infection of several cells lines including An. stephensi.1 Igbo Ora virus behaved similarly to ONNV, as might be predicted based on the recent phylogenetic analysis of this isolate. Igarashi described the sensitivity of the C6/36 clone of Singh’s Aedes albopictus cells (SAAL) to CHIK infection.38 A peak titer of approximately 8.5 logs occurred at 2 days p.i., with a gradual decline to 7 logs by 6.5 days p.i. Some CPE was reported in these cloned cells but not in the un-cloned (SAAR) line.38
Differential growth of ONNV and CHIKV in mosquito cells has been documented.39 Both viruses grew in the SAAL, AM-60 (Aedes malayensis) and AP-61 cell lines (Aedes pseudoscutellaris), whereas only CHIKV grew in AA-20A (Ae. aegypti) and Singh’s Ae. aegypti (S.AA) lines. By raising the incubation temperature from 28°C to 32°C, these workers observed plaque formation by ONNV in AP-61 cells. Ironically, the An. gambiae cell line AG-55 (= MOS-55) was included in these studies but not tested for susceptibility to ONNV or CHIKV. The original publication describing the generation of this cell line commented that "The cells were infected with O’nyong-nyong virus (which An. gambiae transmits in nature) without any cytopathic effect" but provides no supporting data.40 Both viruses replicate in the RA243 cell line from the tick Rhipicephalus appendiculatus.41
Our data are the first to compare the growth of ONNV and CHIKV in the MOS-55 cell line, which previous analysis has confirmed to be genotypically authentic with An. gambiae.42 All three strains of the ONNV replicated in the four cell lines, including MOS-55; however, CHIKV strain 37997 failed to replicate in MOS-55. The more rapid decline in viral titers from the vertebrate lines probably reflects less sustained virion production due to cell death. Our observations demonstrate that the vector specificity of CHIKV, which has been found in nature and confirmed in our in vivo experiments, are apparently retained in in vitro systems. CHIKV failed to infect the An. gambiae derived cell line, in contrast with ONNV, which can infect and replicate in both of the Ae. albopictus and An. gambiae derived cell lines.
Our experiments demonstrated that all three of the ONNV strains used were able to infect and replicate in both An. gambiae and Ae. aegypti, but strain variations were apparent. Although both the individual mosquito viral titers and the proportion of infected mosquitoes declined over time for the Gulu and Igbo Ora strains, dissemination data indicates that the viruses replicated in both species of mosquito. Based on titration, significantly higher rates of infection were observed with ONNV SG650 in Ae. aegypti and An. gambiae when compared with the Gulu and Igbo Ora strains. The dissemination rates between these three strains were not found to be significantly different in Ae. aegypti; however, the dissemination rate of Gulu in An. gambiae was significantly lower than for the other two ONNV strains. In An. gambiae, SG650 titers remained relatively high even early during infection, without the obvious eclipse phase that was observed in Ae. aegypti on Days 2 and 3 p.i. A possible explanation for the observed ONNV strain variation is that since they were first isolated, both Gulu and Igbo Ora have been passaged repeatedly through suckling mice. Selection for neurovirulence may therefore have compromised infectivity for mosquitoes. Because strain SG650 had only been passed once on Vero cells, one would speculate that it is more likely to have retained its natural phenotype. Of the three ONNV strains, it replicated most efficiently. The Gulu strain has been passed more frequently than the Igbo Ora strain (14 mouse-brain passes versus 6 passes), replicated relatively poorly in mosquitoes, and was associated with significantly lower infection rates in both species of mosquito when compared with SG650 and a significantly lower dissemination rate in An. gambiae when compared with SG650 and Igbo Ora.
Our data for CHIKV clearly demonstrated that this virus is infectious to Ae. aegypti but not An. gambiae. In Ae. aegypti, we observed that viral titers remained high throughout the 14-day experimental period with 100% infection rates and 38% dissemination rates by Day 7. Early laboratory experiments concluded that An. gambiae and An. stephensi are barely susceptible to CHIKV and although An. albimanus can be infected, it cannot transmit the virus.43–46 Our data for An. gambiae are supportive of these observations, and although we found some mosquitoes to be still infected at 7 days p.i., titers were extremely low. By Day 14 p.i., none of the An. gambiae mosquitoes remained infected, and there was no dissemination into the salivary glands.
In conclusion, both our in vitro and in vivo data support and extend previous work that has demonstrated the unusual capability of ONNV to infect both anopheline and culicine mosquitoes. Infection and dissemination rates were found to vary between the three strains of ONNV analyzed and between the two species of mosquitoes analyzed. Data from in vitro and in vivo infection with CHIKV strain 37997 determined that this strain will only infect and disseminate in Ae. aegypti mosquitoes and not in An. gambiae mosquitoes.
Received September 2, 2004. Accepted for publication October 29, 2004.
Acknowledgments: We would like to thank Jing Haung, Patricia Hamilton, Amanda Knoblock, and Roberto H. Nussenzveig for their expert technical assistance with this project. We would also like to thank Dr. Robert Tesh and Hilda Guzman for providing the viruses used in this study. This project was funded by NIH grant no. AI47877 and DARPA grant cooperative agreement NOO178-02-2-9002 with the Chemical Biological Radiological Sciences & Technology Defense Branch of the Naval Surface Warfare Center, Dahlgren, Virginia. Kate McElroy was supported by a CDC Fellowship Training Program in Vector-Borne Infectious Diseases T01/CCT622892, and Roberto H. Nussenzveig was supported by NIH T32 training grant A107536.
Authors’ addresses: Dana L. Vanlandingham, Chao Hong, Kimberly Klingler, Konstantin Tsetsarkin, Kate L. McElroy, and Stephen Higgs, Keiller 2.104, L 20762, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0609, Telephone: 409-747-2426, Fax: 409-772-2511. Ann M. Powers, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521, Telephone: (970) 266-3535, Fax: (970) 221-6476. Michael J. Lehane, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK, Telephone: 0151 705 3316 (office), 0151 705 3231 (lab), Fax: 0151 705 3369.
Reprint requests: Stephen Higgs, Department of Pathology, Keiller 2.104, 301 University Blvd., Galveston, TX 77555-0609, Telephone: 409-747-2426, Fax: 409-772-2511, E-mail: sthiggs{at}utmb.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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