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Assessment of hepatitis B virus DNA and hepatitis C virus RNA in the common bedbug (Cimex lectularius L.) and kissing bug (Rodnius prolixus)
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2001 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 96, No. 7, 2001 ISSN 0002-9270/01/$20.00 PII S0002-9270(01)02518-7
Assessment of Hepatitis B Virus DNA and Hepatitis C Virus RNA in the Common Bedbug (Cimex lectularius L.) and Kissing Bug (Rodnius prolixus) Ann L. Silverman, M.D., Li Hua Qu, M.D., Jamie Blow, Ph.D., Ian M. Zitron, Ph.D., Stuart C. Gordon, M.D., and Edward D. Walker, Ph.D. Division of Gastroenterology-Hepatology, Department of Medicine, William Beaumont Hospital, Royal Oak, Michigan; Department of Entomology, Michigan State University, East Lansing, Michigan; and 485th Medical Detachment, Fort Polk, Louisiana
OBJECTIVE: Historical clinical studies suggest the potential for insect-borne transmission of human hepatitis viruses. Studies of hepatitis B virus (HBV) persistence in insects were performed before the advent of molecular techniques, and studies to assess possible insect-borne transmission of hepatitis viruses have not yet been performed. The aim of this study was to determine, using molecular techniques, whether HBV and hepatitis C virus (HCV) persist in and are excreted in the feces of the bedbug Cimex lectularius L. and kissing bug Rodnius prolixus after an infectious meal. METHODS: Blood-feeding insects from the insect order Hemiptera (Cimex lectularius L. and Rhodnius prolixus) were fed on blood from infected patients with high titers of HBV, HCV, and control uninfected patients. Insects and insect excrement were collected at weekly intervals and tested for HBV DNA and HCV RNA using the polymerase chain reaction. RESULTS: HBV DNA was detected in bedbugs and excrement up to 6 wk after feeding on an infectious meal. HBV DNA was also detected in most kissing bugs and excrement up to 2 wk after feeding. HCV RNA was not detected in bedbugs at any time after feeding. CONCLUSIONS: We did not detect HCV RNA in bedbugs after feeding on an infectious meal. Our data provide molecular evidence to suggest that HBV may persist in Hemiptera. Additional studies are ongoing to determine whether this viral persistence is capable of infection. (Am J Gastroenterol 2001;96:2194 –2198. © 2001 by Am. Coll. of Gastroenterology)
This paper was presented in part at the 49th Annual Meeting of the American Association for the Study of Liver Diseases; November 7–10, 1998; Chicago, Illinois. The opinions or assertions contained herein are the private views of the authors and are not construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
INTRODUCTION Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major causes of acute and chronic viral hepatitis worldwide. HBV is a unique, partially double-stranded DNA virus that has been classified as a hepadnavirus (1), and HCV is a single-stranded RNA virus in the Flaviviridae family (2). HBV and HCV have been detected in many body fluids and tissues, and are transmitted from person to person via blood products, sexual contact, percutaneous exposure, and perinatal exposure. However, substantial proportions of community-acquired HBV and HCV infections have no identifiable risk factors. A higher prevalence of HBV and HCV infections has been noted in underdeveloped countries as compared with developing countries (3, 4). Although the observed high rates of HBV and HCV infections in underdeveloped countries may partially be explained by poor sanitation practices, failure to test blood products, nonsterile vaccination techniques, and higher rates of perinatal transmission, these areas of the world also have problems with health care delivery and insect control. The potential for insect-borne transmission of human hepatitis viruses has been considered in earlier studies, many of which were performed before the availability of molecular technologies. For example, Dick et al. (5) showed that mosquitoes caught at sites adjacent to areas of high population density and low socioeconomic level were positive for hepatitis B surface antigen (HBsAg), whereas mosquitoes caught in areas of low population density and high socioeconomic level had no detectable HBsAg. Another study from Greece (6) demonstrated a correlation between the mosquito density in two cities and the prevalence of both HBsAg and anti-HBs. Other insects have also been implicated in the transmission of human hepatitis viruses. Studies involving bedbugs, most reported over two decades ago (7–10), found that HBV proteins could be detected in body parts and fecal material of laboratory and wild-caught bedbugs for many weeks after feeding on an infected meal. The findings of these and other epidemiological studies have
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provided the scientific foundation to postulate that insects may serve as potential vectors of human HBV. Currently, no studies have suggested that HCV may be transmitted by insects. The purpose of this study was to determine, using molecular techniques, whether HBV and HCV persist in insects. We used the polymerase chain reaction (PCR) to detect the presence of HBV DNA in the body parts and excrement from bedbugs (Cimex lectularius L.) and kissing bugs (Rodnius prolixus) collected up to 6 wk after infected feedings. The authenticity of HBV sequences in the PCR products was confirmed by Southern blot analysis. We also used reverse transcription (RT)-PCR to evaluate the presence of HCV RNA in bedbugs fed directly on HCV-infected patients and uninfected controls.
MATERIALS AND METHODS Kissing Bugs Rodnius prolixus were obtained from a colony maintained by Dr. John Edman, University of Massachusetts, and raised in cohorts. Eggs were removed from adult jars on a weekly basis and placed in a separate 1-L Nalgene jar with a few older bugs to provide a source of intestinal bacteria for the hatchlings. Colony feeding on Harlan SD rats was done weekly using a protocol approved by the Michigan State University on Animal Use and Care. Rats were used once every 2 wk for feeding. Experimental feedings and some colony maintenance were performed using a Hemotek 5W1 artificial feeding system (Discovery Workshops, England). This membranebased system consisted of five independently-controlled heated feeding chambers, each of which held 5 ml of blood. Three types of feeding membranes (parafilm, latex, and cellulose) were used for colony maintenance. A cellulose membrane was used exclusively during infected feedings. The membrane was stretched across the feeding chamber and secured with an O-ring. The chambers were filled with blood, capped and screwed into the heating element, then placed on the insect jar for feeding. Bovine blood preserved with heparin (Pel-Freez Biologicals, Rogers, AZ) or equine blood (Veterinary Dynamics, Templeton, CA) was used for routine colony maintenance. For experimental feedings, kissing bugs were removed from the container after feeding. One uninfected and five infected kissing bugs were removed at weekly intervals for 6 wk. Filter paper placed on the bottom of the containers to collect kissing bug excrement was changed at weekly intervals. Kissing bugs and filter paper were stored at ⫺80°C until tested for viral sequences by PCR. Bedbugs Cimex lectularius L. were obtained from a colony that was originally collected from a U.S. Army basic training billet at Fort Dix, NJ, on March 20, 1973, and were reared in the laboratory using standard methods. Bugs were raised from
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eggs and placed with a few older bugs to provide a source of intestinal bacteria for the hatchlings. Rearing containers consisted of a one-quart wide-mouth mason jar with a top made of nitex nylon screen. A special area was constructed for routine handling of the bugs, which consisted of a large Plexiglas sheet with a barrier of tanglefoot to prevent escape. Bugs were housed in a separate room designed to prevent escape. Bedbug colonies were fed on laboratoryraised, Harlan SD rats. Bedbugs would not feed consistently on the membrane feeding device for routine colony maintenance. Experimental feedings on humans were performed by placing adult bedbugs in a small Plexiglas rearing jar sealed at the top and covered with a nylon mesh on the top. Infected patients (described below) and uninfected controls placed the flexor surface of their forearm over the membrane and allowed the bugs to feed for approximately 20 –30 min or until most of the bugs were engorged. Bugs were anesthetized by placing the rearing jar in an ice bucket for 5–10 min and unfed bugs were removed. Over the following 6 wk, at least two bugs from the infected and control jars were removed at weekly intervals. Filter paper placed on the bottom of the rearing jar to collect bedbug excrement also was removed at weekly intervals. Bedbugs and filter paper were frozen at ⫺80°C until tested for viral nucleic acids by PCR. Patient Blood and Experimental Infection Blood (5 ml) from HBV- and HCV-infected patients and uninfected volunteers was collected by phlebotomy into tubes containing heparin. The whole blood was transported on ice from William Beaumont Hospital to Michigan State University and fed to the kissing bugs within 6 h of blood collection. The concentration of HBV DNA was determined using a liquid phase molecular hybridization assay (Abbott Laboratories, Abbott Park, IL). Serum HCV RNA was measured using the Superquant assay (National Genetics Institute, Culver City, CA). Authorization to feed the bedbugs directly on humans was obtained from the Human Investigation Committees of William Beaumont Hospital and Michigan State University. For experiments on hepatitis B infection, bedbugs fed on three HBV-infected volunteers with high viral titers (300, 600, ⬎2,000 pg/ml) as measured using the Digene signal amplification method (Viromed Laboratories, Minneapolis, MN). Three HBV-negative volunteers served as controls. For the experiment on hepatitis C infection, bedbugs fed on one HCV-infected patient with a high viral titer (3,800,000 copies/ml) and one HCV-negative volunteer. At least three bugs experimentally exposed to HCV were collected on a weekly basis for 6 wk and stored at ⫺80°C until PCR was performed. HBV DNA and RNA Analysis HBV DNA in bedbugs, kissing bugs, and excrement was evaluated for all time points. Total DNA from 1 l of
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infected heparinized blood, bedbugs, and kissing bugs (ground with a tissue grinder), and from spots of blood or excrement on the filter paper was extracted using the onestep DNAzol method (Molecular Research Center, Cincinnati, OH). PCR for HBV DNA and RNA was performed using single and seminested primers from the highly conserved precore and core region of the viral genome as previously described (11). In the second experiment that was done to detect persistence of hepatitis B in bedbugs, both RNA and DNA were extracted using Trizol (Molecular Research Center). DNA recovery was not as efficient as the DNAzol method, and a seminested PCR was done using the same region of the conserved precore and core region of the HBV genome. Insects fed on uninfected blood and reagents alone served as negative controls, blood from HBV-infected patients served as positive controls for DNA, and HBV-infected liver tissue served as the positive control for HBV RNA. The presence of HBV DNA in the bedbugs and excrement was independently confirmed using the signal amplification assay (Viromed Laboratories). PCR products from the bedbugs and their excrement were transferred to nylon membranes (Magnagraph MSI, Westboro, MA). Authenticity of HBV DNA sequences was confirmed by hybridization with a Digoxigenin-tailed oligonucleotide, the sequence of which was internal to the PCR primers. HCV RNA Analysis The presence of HCV RNA in bedbugs fed on infected material was evaluated by RT-PCR. Serum from an HCVinfected patient served as a positive control, and bedbugs fed on uninfected patients served as negative controls. Total RNA from 20 l of infected liver tissue and bedbugs (ground with a tissue grinder) was extracted using Trizol reagent, and RT-PCR was performed using primers from the 5⬘-untranslated region of the viral genome as previously described (12).
RESULTS Persistence of HBV DNA in Bedbugs Evidence of persistent HBV DNA was found in bedbugs and excrement for up to 6 wk after feeding on an infected meal. As shown in Table 1, HBV DNA was detected by PCR in extracted bodies as early as day 1. Excrement tested positive for HBV DNA within 7 days after feeding. Bedbugs and excrement remained HBV DNA positive throughout the 54 days of testing. All control bedbugs, which fed on uninfected blood, did not have detectable virus in the insect or in the feces during the 54 days of testing (not shown). HBV RNA was not detected in any insects tested, but was detected in HBV-infected liver. The authenticity of HBV sequences in the PCR products from bedbugs and excrement was confirmed by Southern blot analysis. A representative ethidium bromide stained gel
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Table 1. Detection of HBV DNA in Bedbugs (Cimex lectularius L.) and Excrement at Various Time Points After Feeding on an Infected Meal
Day 0 1 3 7 14 21 28 35 42 54
Bedbugs
Excrement
(No. HBV DNA Positive/Total)
(No. HBV DNA Positive/Total)
NT 2/2 2/2 8/8 8/9 9/9 8/9 8/8 5/5 3/3
0/2 0/2 NT 8/8 8/8 8/8 7/8 7/8 6/6 3/3
NT ⫽ not tested.
of the PCR-amplified material from bedbugs and excrement is shown in Figure 1. The major band at 258 bp specifically hybridized with internal digoxigenen-labeled oligonucleotides, indicating that the PCR product was derived from genomic HBV sequences. Signal amplification testing further confirmed the presence of HBV DNA in bedbugs and excrement (data not shown). Persistence of HBV DNA in Kissing Bugs Kissing bugs fed on HBV-infected humans also showed persistent evidence of HBV DNA. As shown in Table 2, HBV DNA was detected by PCR in extracted bodies as early as day 7. Excrement tested positive for HBV DNA within 14 days after feeding. Kissing bugs remained HBV DNA positive throughout 42 days of testing. HBV DNA could not be tested in excrement after day 14 because kissing bugs did not excrete fecal material 2 wk after feeding. No HBV DNA was detected in kissing bugs fed on uninfected volunteers (not shown). Persistence of HCV RNA in Bedbugs In contrast to the results on HBV DNA, no evidence of persistent HCV RNA was found in bedbugs at any time tested after feeding on HCV-infected blood. As shown in Table 3, bedbugs remained HCV RNA negative throughout the 54 days of testing. Accordingly, excreted fecal material from these insects was not tested for HCV RNA.
DISCUSSION Many disease agents are transmitted to humans by insects. Mechanisms of insect-borne transmission include: 1) direct inoculation of the infectious agent into humans after replication in the insect (e.g., malaria or yellow fever); 2) direct contact of the infectious agent from the insect through a break in the skin (e.g., Rocky Mountain Spotted fever); and 3) deposition of the infectious agent on the skin through excreted gut contents (e.g., Chagas disease, epidemic louseborne typhus). However, because hepatitis viruses are host-
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Figure 1. Representative ethidium bromide-stained gel of PCR products from bedbugs collected after feeding on HBV-infected blood. The numbers at the top indicate the time in weeks that the bedbugs were collected after feeding on uninfected and infected blood. The lane on the right represents the serum HBV positive control. The expected 240 bp band was detected after nested PCR. Even with a technique this sensitive, no signal was visible in the insects fed on negative control blood.
specific, relatively few studies of insect-borne transmission of HBV and HCV have been performed. There is little prior evidence to support the notion that HCV persists in insects. We previously showed that HCV RNA does not persist in the mosquito beyond 1 day postfeeding (12). Similarly, in the present study, we found no evidence that HCV RNA persists in bedbugs at any time point up to 6 wk after feeding on HCV-infected blood. Given the ability of HBV to survive under adverse conditions (13), this virus may be resistant to the conditions of the insect gut. Earlier studies assayed HBsAg to investigate possible mechanisms for insect-borne transmission of HBV. In a study by el-Masry and Kotkat (10), the authors detected HBsAg in fed, starved, and nymph bedbugs (Cimex lectularius L.) obtained from sleeping wards of the Central Security forces in Egypt and laboratory animal house, where the HBsAg carrier rate was 3.6% in recruits of the Security force. They speculated that mechanical transmission could Table 2. Detection of HBV DNA in Kissing Bugs (Rhodnius prolixus) and Excrement at Various Time Points After Feeding on an Infected Meal
Day 7 14 21 28 35 42
Kissing Bugs
Excrement
(No. HBV DNA Positive/Total)
(No. HBV DNA Positive/Total)
4/5 4/5 3/5 4/5 2/5 3/5
0/2 2/2 NT NT NT NT
HBV DNA in excrement was not tested after day 14 because kissing bugs did not excrete fecal material for ⬎2 wk after feeding. NT ⫽ not tested.
occur during an interrupted blood meal either from infected mouth-parts or through regurgitation of the blood. Jupp et al. (9) detected HBsAg in the salivary glands and feces of bedbugs, suggesting that mechanical transmission of HBV to humans could occur either by percutaneous exposure to infected feces or by regurgitation after an interrupted feeding. Ogston et al. (8) studied colony-reared bedbugs (Cimex hemipterous), which fed in the laboratory on HBV-infected blood. These investigators detected HBsAg in insect abdomens for as long as 6 wk after a single blood meal. Detection of HBsAg also has been used to explore the potential of other insects as vectors of viral hepatitis, including kissing bugs (14), blackflies (15), and mosquitoes (16). However, a major limitation of all of these studies is that HBsAg may be present in noninfectious particles, which are produced during viral replication. In fact, HBsAg is known to be present in much higher quantities (103–106-fold higher) than infecTable 3. Absence of HCV RNA in Bedbugs (Cimex lectularius) at Various Time Points After Feeding on an Infected Meal Bedbugs Day 0 1 3 7 14 21 28 35 42 54 NT ⫽ not tested.
(No. HCV RNA Positive/Total) NT 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
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tious virus (17). In the present study, we monitored HBV DNA to document viral persistence because HBV DNA is a hallmark of the infectious virion. Our results showed that HBV DNA was uniformly detected at weekly intervals for up to 6 wk after feeding on HBVinfected blood in bedbugs and their excrement. Persistence of HBV DNA does not seem to be caused by viral replication, however, because no HBV RNA was detected in any of the bedbugs after feeding. The fact that HBV can be detected for such a long time after feeding probably reflects persistence in the environment in the gut lumen, as we have no evidence of viral replication. Furthermore, other studies in our laboratory have shown that when HBV-infected blood is injected into the body cavity of the bedbug, the virus becomes undetectable by PCR (J. Blow, unpublished results). We also detected HBV DNA after molting in the immature stages of the bedbugs that fed. The frequency with which bedbugs will feed is closely correlated with egg laying, longevity, and temperature, which affects all other processes including digestion rate. The fact that HBV DNA was still present in bedbugs after molting from one immature stage to the next is important, as bedbugs still have evidence of the virus between feedings (i.e., rules out surface contamination). Although HBV DNA is part of the virion, the presence of HBV DNA in bedbugs and excrement does not necessarily imply infectious virus. Studies are now in progress in an animal model to determine whether virus isolated from bedbugs after feeding is infectious. In contrast to our results on bedbugs, HBV DNA was not uniformly detected in kissing bugs after 1-wk postfeeding. This lack of HBV DNA persistence was observed despite the fact that kissing bugs ingested a larger meal than bedbugs. Kissing bugs transmit Trypanasoma cruzi by depositing the organism in the feces. The organism is then rubbed into the inflamed area of the bite. This potential mechanism is possible for transmission of viral hepatitis if the virus in the feces remains infectious. In summary, the purpose of this study was to use molecular techniques to determine whether human hepatitis viruses persist in insect vectors. We have shown that HBV DNA persists for long periods of time in both bedbugs and kissing bugs after feeding on an infectious meal, and is excreted in the feces. In contrast, we found that HCV RNA is undetectable in bedbugs after feeding on blood from a patient with a high viral titer. Studies are in progress to determine whether the detected viral nucleic acid is capable of causing infection. Our laboratories are also pursuing studies to determine whether HCV RNA persists in kissing bugs.
ACKNOWLEDGMENTS Supported by William Beaumont Hospital Research Institute, Royal Oak, Michigan. We thank Suzy Abileah and
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David Ternes for technical assistance. We also thank Linda Wuestehube, Ph.D., for editorial assistance. Reprint requests and correspondence: Ann L. Silverman, M.D., Division of Gastroenterology-Hepatology, William Beaumont Hospital, 3601 W. 13 Mile Road, Royal Oak, MI 48073. Received Oct. 11, 2000; accepted Feb. 7, 2001.
REFERENCES 1. Lau JY, Wright TL. Molecular virology and pathogenesis of hepatitis B. Lancet 1993;342:1335– 40. 2. Miller RH, Purcell RH. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci USA 1990;87:2057– 61. 3. Shafritz DA, Sherman M, Ran T-K. Hepatitis B virus persistence. In: Zakim D, Boyer TD, eds. Hepatology. Philadelphia: WB Saunders, 1990:945– 8. 4. Mansell CJ, Locarnini SA. Epidemiology of hepatitis C in the East. Semin Liver Dis 1995;15:15–32. 5. Dick SJ, Tamburro CH, Leevy CM. Hepatitis B antigen in urban-caught mosquitoes. JAMA 1974;229:1627–9. 6. Papaevangelou G, Kourea-Kremastinou T. Role of mosquitoes in transmission of hepatitis B virus infection. J Infect Dis 1974;130:78 – 80. 7. Wills W, Larouze B, London WT, et al. Hepatitis-B virus in bedbugs (Cimex hemipterus) from Senegal. Lancet 1977;2: 217–9. 8. Ogston CW, Wittenstein FS, London WT, et al. Persistence of hepatitis B surface antigen in the bedbug Cimex hemipterus (Fabr.). J Infect Dis 1979;140:411– 4. 9. Jupp PG, McElligott SE, Lecatsas G. The mechanical transmission of hepatitis B virus by the common bedbug (Cimex lectularius L.) in South Africa. S Afr Med J 1983;63:77– 81. 10. el-Masry SA, Kotkat AM. Hepatitis B surface antigen in Cimex lectularius. J Egypt Public Health Assoc 1990;65:229 –36. 11. Kaneko S, Miller RH, Di Bisceglie AM, et al. Detection of hepatitis B virus DNA in serum by polymerase chain reaction. Application for clinical diagnosis. Gastroenterology 1990;99: 799 – 804. 12. Silverman AL, McCray DG, Gordon SC, et al. Experimental evidence against replication or dissemination of hepatitis C virus in mosquitoes (Diptera:Culicidae) using detection by reverse transcriptase polymerase chain reaction. J Med Entomol 1996;33:398 – 401. 13. Bond WW, Favero MS, Petersen NJ, et al. Survival of hepatitis B virus after drying and storage for one week. Lancet 1981;1:550 –1 (letter). 14. Villarejos VM, Zuniga A, Gutierrez DA. Possible role of haematophagous insects in the transmission of Type B hepatitis. Trans R Soc Trop Med Hyg 1975;69:368 –9 (letter). 15. Chanteau S, Sechan Y, Moulia-Pelat JP, et al. The blackfly Simulium buissoni and infection by hepatitis B virus on a holoendemic island of the Marquesas archipelago in French Polynesia. Am J Trop Med Hyg 1993;48:763–70. 16. Hyams KC. Mosquito transmission of hepatitis B. Trop Geogr Med 1989;41:185–9. 17. Fields BN, Knipe DM, Howley PM, et al. Hepadnaviridae and their replication. In: Fields BN, Knipe DM, Howley PM, et al., eds. Field’s virology. Philadelphia: Lippincott-Raven, 1996: 2704 –5.
Vol. 96, No. 7, 2001 ISSN 0002-9270/01/$20.00 PII S0002-9270(01)02518-7
Assessment of Hepatitis B Virus DNA and Hepatitis C Virus RNA in the Common Bedbug (Cimex lectularius L.) and Kissing Bug (Rodnius prolixus) Ann L. Silverman, M.D., Li Hua Qu, M.D., Jamie Blow, Ph.D., Ian M. Zitron, Ph.D., Stuart C. Gordon, M.D., and Edward D. Walker, Ph.D. Division of Gastroenterology-Hepatology, Department of Medicine, William Beaumont Hospital, Royal Oak, Michigan; Department of Entomology, Michigan State University, East Lansing, Michigan; and 485th Medical Detachment, Fort Polk, Louisiana
OBJECTIVE: Historical clinical studies suggest the potential for insect-borne transmission of human hepatitis viruses. Studies of hepatitis B virus (HBV) persistence in insects were performed before the advent of molecular techniques, and studies to assess possible insect-borne transmission of hepatitis viruses have not yet been performed. The aim of this study was to determine, using molecular techniques, whether HBV and hepatitis C virus (HCV) persist in and are excreted in the feces of the bedbug Cimex lectularius L. and kissing bug Rodnius prolixus after an infectious meal. METHODS: Blood-feeding insects from the insect order Hemiptera (Cimex lectularius L. and Rhodnius prolixus) were fed on blood from infected patients with high titers of HBV, HCV, and control uninfected patients. Insects and insect excrement were collected at weekly intervals and tested for HBV DNA and HCV RNA using the polymerase chain reaction. RESULTS: HBV DNA was detected in bedbugs and excrement up to 6 wk after feeding on an infectious meal. HBV DNA was also detected in most kissing bugs and excrement up to 2 wk after feeding. HCV RNA was not detected in bedbugs at any time after feeding. CONCLUSIONS: We did not detect HCV RNA in bedbugs after feeding on an infectious meal. Our data provide molecular evidence to suggest that HBV may persist in Hemiptera. Additional studies are ongoing to determine whether this viral persistence is capable of infection. (Am J Gastroenterol 2001;96:2194 –2198. © 2001 by Am. Coll. of Gastroenterology)
This paper was presented in part at the 49th Annual Meeting of the American Association for the Study of Liver Diseases; November 7–10, 1998; Chicago, Illinois. The opinions or assertions contained herein are the private views of the authors and are not construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
INTRODUCTION Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major causes of acute and chronic viral hepatitis worldwide. HBV is a unique, partially double-stranded DNA virus that has been classified as a hepadnavirus (1), and HCV is a single-stranded RNA virus in the Flaviviridae family (2). HBV and HCV have been detected in many body fluids and tissues, and are transmitted from person to person via blood products, sexual contact, percutaneous exposure, and perinatal exposure. However, substantial proportions of community-acquired HBV and HCV infections have no identifiable risk factors. A higher prevalence of HBV and HCV infections has been noted in underdeveloped countries as compared with developing countries (3, 4). Although the observed high rates of HBV and HCV infections in underdeveloped countries may partially be explained by poor sanitation practices, failure to test blood products, nonsterile vaccination techniques, and higher rates of perinatal transmission, these areas of the world also have problems with health care delivery and insect control. The potential for insect-borne transmission of human hepatitis viruses has been considered in earlier studies, many of which were performed before the availability of molecular technologies. For example, Dick et al. (5) showed that mosquitoes caught at sites adjacent to areas of high population density and low socioeconomic level were positive for hepatitis B surface antigen (HBsAg), whereas mosquitoes caught in areas of low population density and high socioeconomic level had no detectable HBsAg. Another study from Greece (6) demonstrated a correlation between the mosquito density in two cities and the prevalence of both HBsAg and anti-HBs. Other insects have also been implicated in the transmission of human hepatitis viruses. Studies involving bedbugs, most reported over two decades ago (7–10), found that HBV proteins could be detected in body parts and fecal material of laboratory and wild-caught bedbugs for many weeks after feeding on an infected meal. The findings of these and other epidemiological studies have
AJG – July, 2001
provided the scientific foundation to postulate that insects may serve as potential vectors of human HBV. Currently, no studies have suggested that HCV may be transmitted by insects. The purpose of this study was to determine, using molecular techniques, whether HBV and HCV persist in insects. We used the polymerase chain reaction (PCR) to detect the presence of HBV DNA in the body parts and excrement from bedbugs (Cimex lectularius L.) and kissing bugs (Rodnius prolixus) collected up to 6 wk after infected feedings. The authenticity of HBV sequences in the PCR products was confirmed by Southern blot analysis. We also used reverse transcription (RT)-PCR to evaluate the presence of HCV RNA in bedbugs fed directly on HCV-infected patients and uninfected controls.
MATERIALS AND METHODS Kissing Bugs Rodnius prolixus were obtained from a colony maintained by Dr. John Edman, University of Massachusetts, and raised in cohorts. Eggs were removed from adult jars on a weekly basis and placed in a separate 1-L Nalgene jar with a few older bugs to provide a source of intestinal bacteria for the hatchlings. Colony feeding on Harlan SD rats was done weekly using a protocol approved by the Michigan State University on Animal Use and Care. Rats were used once every 2 wk for feeding. Experimental feedings and some colony maintenance were performed using a Hemotek 5W1 artificial feeding system (Discovery Workshops, England). This membranebased system consisted of five independently-controlled heated feeding chambers, each of which held 5 ml of blood. Three types of feeding membranes (parafilm, latex, and cellulose) were used for colony maintenance. A cellulose membrane was used exclusively during infected feedings. The membrane was stretched across the feeding chamber and secured with an O-ring. The chambers were filled with blood, capped and screwed into the heating element, then placed on the insect jar for feeding. Bovine blood preserved with heparin (Pel-Freez Biologicals, Rogers, AZ) or equine blood (Veterinary Dynamics, Templeton, CA) was used for routine colony maintenance. For experimental feedings, kissing bugs were removed from the container after feeding. One uninfected and five infected kissing bugs were removed at weekly intervals for 6 wk. Filter paper placed on the bottom of the containers to collect kissing bug excrement was changed at weekly intervals. Kissing bugs and filter paper were stored at ⫺80°C until tested for viral sequences by PCR. Bedbugs Cimex lectularius L. were obtained from a colony that was originally collected from a U.S. Army basic training billet at Fort Dix, NJ, on March 20, 1973, and were reared in the laboratory using standard methods. Bugs were raised from
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eggs and placed with a few older bugs to provide a source of intestinal bacteria for the hatchlings. Rearing containers consisted of a one-quart wide-mouth mason jar with a top made of nitex nylon screen. A special area was constructed for routine handling of the bugs, which consisted of a large Plexiglas sheet with a barrier of tanglefoot to prevent escape. Bugs were housed in a separate room designed to prevent escape. Bedbug colonies were fed on laboratoryraised, Harlan SD rats. Bedbugs would not feed consistently on the membrane feeding device for routine colony maintenance. Experimental feedings on humans were performed by placing adult bedbugs in a small Plexiglas rearing jar sealed at the top and covered with a nylon mesh on the top. Infected patients (described below) and uninfected controls placed the flexor surface of their forearm over the membrane and allowed the bugs to feed for approximately 20 –30 min or until most of the bugs were engorged. Bugs were anesthetized by placing the rearing jar in an ice bucket for 5–10 min and unfed bugs were removed. Over the following 6 wk, at least two bugs from the infected and control jars were removed at weekly intervals. Filter paper placed on the bottom of the rearing jar to collect bedbug excrement also was removed at weekly intervals. Bedbugs and filter paper were frozen at ⫺80°C until tested for viral nucleic acids by PCR. Patient Blood and Experimental Infection Blood (5 ml) from HBV- and HCV-infected patients and uninfected volunteers was collected by phlebotomy into tubes containing heparin. The whole blood was transported on ice from William Beaumont Hospital to Michigan State University and fed to the kissing bugs within 6 h of blood collection. The concentration of HBV DNA was determined using a liquid phase molecular hybridization assay (Abbott Laboratories, Abbott Park, IL). Serum HCV RNA was measured using the Superquant assay (National Genetics Institute, Culver City, CA). Authorization to feed the bedbugs directly on humans was obtained from the Human Investigation Committees of William Beaumont Hospital and Michigan State University. For experiments on hepatitis B infection, bedbugs fed on three HBV-infected volunteers with high viral titers (300, 600, ⬎2,000 pg/ml) as measured using the Digene signal amplification method (Viromed Laboratories, Minneapolis, MN). Three HBV-negative volunteers served as controls. For the experiment on hepatitis C infection, bedbugs fed on one HCV-infected patient with a high viral titer (3,800,000 copies/ml) and one HCV-negative volunteer. At least three bugs experimentally exposed to HCV were collected on a weekly basis for 6 wk and stored at ⫺80°C until PCR was performed. HBV DNA and RNA Analysis HBV DNA in bedbugs, kissing bugs, and excrement was evaluated for all time points. Total DNA from 1 l of
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infected heparinized blood, bedbugs, and kissing bugs (ground with a tissue grinder), and from spots of blood or excrement on the filter paper was extracted using the onestep DNAzol method (Molecular Research Center, Cincinnati, OH). PCR for HBV DNA and RNA was performed using single and seminested primers from the highly conserved precore and core region of the viral genome as previously described (11). In the second experiment that was done to detect persistence of hepatitis B in bedbugs, both RNA and DNA were extracted using Trizol (Molecular Research Center). DNA recovery was not as efficient as the DNAzol method, and a seminested PCR was done using the same region of the conserved precore and core region of the HBV genome. Insects fed on uninfected blood and reagents alone served as negative controls, blood from HBV-infected patients served as positive controls for DNA, and HBV-infected liver tissue served as the positive control for HBV RNA. The presence of HBV DNA in the bedbugs and excrement was independently confirmed using the signal amplification assay (Viromed Laboratories). PCR products from the bedbugs and their excrement were transferred to nylon membranes (Magnagraph MSI, Westboro, MA). Authenticity of HBV DNA sequences was confirmed by hybridization with a Digoxigenin-tailed oligonucleotide, the sequence of which was internal to the PCR primers. HCV RNA Analysis The presence of HCV RNA in bedbugs fed on infected material was evaluated by RT-PCR. Serum from an HCVinfected patient served as a positive control, and bedbugs fed on uninfected patients served as negative controls. Total RNA from 20 l of infected liver tissue and bedbugs (ground with a tissue grinder) was extracted using Trizol reagent, and RT-PCR was performed using primers from the 5⬘-untranslated region of the viral genome as previously described (12).
RESULTS Persistence of HBV DNA in Bedbugs Evidence of persistent HBV DNA was found in bedbugs and excrement for up to 6 wk after feeding on an infected meal. As shown in Table 1, HBV DNA was detected by PCR in extracted bodies as early as day 1. Excrement tested positive for HBV DNA within 7 days after feeding. Bedbugs and excrement remained HBV DNA positive throughout the 54 days of testing. All control bedbugs, which fed on uninfected blood, did not have detectable virus in the insect or in the feces during the 54 days of testing (not shown). HBV RNA was not detected in any insects tested, but was detected in HBV-infected liver. The authenticity of HBV sequences in the PCR products from bedbugs and excrement was confirmed by Southern blot analysis. A representative ethidium bromide stained gel
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Table 1. Detection of HBV DNA in Bedbugs (Cimex lectularius L.) and Excrement at Various Time Points After Feeding on an Infected Meal
Day 0 1 3 7 14 21 28 35 42 54
Bedbugs
Excrement
(No. HBV DNA Positive/Total)
(No. HBV DNA Positive/Total)
NT 2/2 2/2 8/8 8/9 9/9 8/9 8/8 5/5 3/3
0/2 0/2 NT 8/8 8/8 8/8 7/8 7/8 6/6 3/3
NT ⫽ not tested.
of the PCR-amplified material from bedbugs and excrement is shown in Figure 1. The major band at 258 bp specifically hybridized with internal digoxigenen-labeled oligonucleotides, indicating that the PCR product was derived from genomic HBV sequences. Signal amplification testing further confirmed the presence of HBV DNA in bedbugs and excrement (data not shown). Persistence of HBV DNA in Kissing Bugs Kissing bugs fed on HBV-infected humans also showed persistent evidence of HBV DNA. As shown in Table 2, HBV DNA was detected by PCR in extracted bodies as early as day 7. Excrement tested positive for HBV DNA within 14 days after feeding. Kissing bugs remained HBV DNA positive throughout 42 days of testing. HBV DNA could not be tested in excrement after day 14 because kissing bugs did not excrete fecal material 2 wk after feeding. No HBV DNA was detected in kissing bugs fed on uninfected volunteers (not shown). Persistence of HCV RNA in Bedbugs In contrast to the results on HBV DNA, no evidence of persistent HCV RNA was found in bedbugs at any time tested after feeding on HCV-infected blood. As shown in Table 3, bedbugs remained HCV RNA negative throughout the 54 days of testing. Accordingly, excreted fecal material from these insects was not tested for HCV RNA.
DISCUSSION Many disease agents are transmitted to humans by insects. Mechanisms of insect-borne transmission include: 1) direct inoculation of the infectious agent into humans after replication in the insect (e.g., malaria or yellow fever); 2) direct contact of the infectious agent from the insect through a break in the skin (e.g., Rocky Mountain Spotted fever); and 3) deposition of the infectious agent on the skin through excreted gut contents (e.g., Chagas disease, epidemic louseborne typhus). However, because hepatitis viruses are host-
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HBV and HCV in Insects
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Figure 1. Representative ethidium bromide-stained gel of PCR products from bedbugs collected after feeding on HBV-infected blood. The numbers at the top indicate the time in weeks that the bedbugs were collected after feeding on uninfected and infected blood. The lane on the right represents the serum HBV positive control. The expected 240 bp band was detected after nested PCR. Even with a technique this sensitive, no signal was visible in the insects fed on negative control blood.
specific, relatively few studies of insect-borne transmission of HBV and HCV have been performed. There is little prior evidence to support the notion that HCV persists in insects. We previously showed that HCV RNA does not persist in the mosquito beyond 1 day postfeeding (12). Similarly, in the present study, we found no evidence that HCV RNA persists in bedbugs at any time point up to 6 wk after feeding on HCV-infected blood. Given the ability of HBV to survive under adverse conditions (13), this virus may be resistant to the conditions of the insect gut. Earlier studies assayed HBsAg to investigate possible mechanisms for insect-borne transmission of HBV. In a study by el-Masry and Kotkat (10), the authors detected HBsAg in fed, starved, and nymph bedbugs (Cimex lectularius L.) obtained from sleeping wards of the Central Security forces in Egypt and laboratory animal house, where the HBsAg carrier rate was 3.6% in recruits of the Security force. They speculated that mechanical transmission could Table 2. Detection of HBV DNA in Kissing Bugs (Rhodnius prolixus) and Excrement at Various Time Points After Feeding on an Infected Meal
Day 7 14 21 28 35 42
Kissing Bugs
Excrement
(No. HBV DNA Positive/Total)
(No. HBV DNA Positive/Total)
4/5 4/5 3/5 4/5 2/5 3/5
0/2 2/2 NT NT NT NT
HBV DNA in excrement was not tested after day 14 because kissing bugs did not excrete fecal material for ⬎2 wk after feeding. NT ⫽ not tested.
occur during an interrupted blood meal either from infected mouth-parts or through regurgitation of the blood. Jupp et al. (9) detected HBsAg in the salivary glands and feces of bedbugs, suggesting that mechanical transmission of HBV to humans could occur either by percutaneous exposure to infected feces or by regurgitation after an interrupted feeding. Ogston et al. (8) studied colony-reared bedbugs (Cimex hemipterous), which fed in the laboratory on HBV-infected blood. These investigators detected HBsAg in insect abdomens for as long as 6 wk after a single blood meal. Detection of HBsAg also has been used to explore the potential of other insects as vectors of viral hepatitis, including kissing bugs (14), blackflies (15), and mosquitoes (16). However, a major limitation of all of these studies is that HBsAg may be present in noninfectious particles, which are produced during viral replication. In fact, HBsAg is known to be present in much higher quantities (103–106-fold higher) than infecTable 3. Absence of HCV RNA in Bedbugs (Cimex lectularius) at Various Time Points After Feeding on an Infected Meal Bedbugs Day 0 1 3 7 14 21 28 35 42 54 NT ⫽ not tested.
(No. HCV RNA Positive/Total) NT 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
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tious virus (17). In the present study, we monitored HBV DNA to document viral persistence because HBV DNA is a hallmark of the infectious virion. Our results showed that HBV DNA was uniformly detected at weekly intervals for up to 6 wk after feeding on HBVinfected blood in bedbugs and their excrement. Persistence of HBV DNA does not seem to be caused by viral replication, however, because no HBV RNA was detected in any of the bedbugs after feeding. The fact that HBV can be detected for such a long time after feeding probably reflects persistence in the environment in the gut lumen, as we have no evidence of viral replication. Furthermore, other studies in our laboratory have shown that when HBV-infected blood is injected into the body cavity of the bedbug, the virus becomes undetectable by PCR (J. Blow, unpublished results). We also detected HBV DNA after molting in the immature stages of the bedbugs that fed. The frequency with which bedbugs will feed is closely correlated with egg laying, longevity, and temperature, which affects all other processes including digestion rate. The fact that HBV DNA was still present in bedbugs after molting from one immature stage to the next is important, as bedbugs still have evidence of the virus between feedings (i.e., rules out surface contamination). Although HBV DNA is part of the virion, the presence of HBV DNA in bedbugs and excrement does not necessarily imply infectious virus. Studies are now in progress in an animal model to determine whether virus isolated from bedbugs after feeding is infectious. In contrast to our results on bedbugs, HBV DNA was not uniformly detected in kissing bugs after 1-wk postfeeding. This lack of HBV DNA persistence was observed despite the fact that kissing bugs ingested a larger meal than bedbugs. Kissing bugs transmit Trypanasoma cruzi by depositing the organism in the feces. The organism is then rubbed into the inflamed area of the bite. This potential mechanism is possible for transmission of viral hepatitis if the virus in the feces remains infectious. In summary, the purpose of this study was to use molecular techniques to determine whether human hepatitis viruses persist in insect vectors. We have shown that HBV DNA persists for long periods of time in both bedbugs and kissing bugs after feeding on an infectious meal, and is excreted in the feces. In contrast, we found that HCV RNA is undetectable in bedbugs after feeding on blood from a patient with a high viral titer. Studies are in progress to determine whether the detected viral nucleic acid is capable of causing infection. Our laboratories are also pursuing studies to determine whether HCV RNA persists in kissing bugs.
ACKNOWLEDGMENTS Supported by William Beaumont Hospital Research Institute, Royal Oak, Michigan. We thank Suzy Abileah and
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David Ternes for technical assistance. We also thank Linda Wuestehube, Ph.D., for editorial assistance. Reprint requests and correspondence: Ann L. Silverman, M.D., Division of Gastroenterology-Hepatology, William Beaumont Hospital, 3601 W. 13 Mile Road, Royal Oak, MI 48073. Received Oct. 11, 2000; accepted Feb. 7, 2001.
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