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Hepatitis E virus Infection in Work Horses in Egypt
Infection, Genetics and Evolution 7 (2007) 368–373 www.elsevier.com/locate/meegid
Hepatitis E virus Infection in Work Horses in Egypt§ Magdi D. Saad a,*, Hussein A. Hussein b, Moustafa M. Bashandy c, Hamdy H. Kamel d, K.C. Earhart a, David J. Fryauff a, Mary Younan a, Amira H. Mohamed c a
Virology Research Program, United States Naval Medical Research Unit No. 3, Cairo, Egypt b Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt c Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt d Department of Clinical Pathology, Faculty of Veterinary Medicine, Beni-Suef, Egypt Received 27 February 2006; received in revised form 6 July 2006; accepted 20 July 2006 Available online 12 October 2006
Abstract Hepatitis E virus (HEV) is an important cause of hepatitis among young Egyptian adults with high seroprevalence rates seen in both rural areas of the Nile Delta and in suburban Cairo. Because natural antibodies to HEV have been detected in animals and zoonotic transmission is postulated, we surveyed work horses in Cairo for evidence of HEVexposure and viremia. Sera from 200 Cairo work horses were tested by ELISA for the presence of IgG anti-HEVantibody revealed a seropositivity of 13%. Among 100 samples processed for detection of viral genome by means of nested polymerase chain reaction (N-PCR), 4% were positive and indicative of viremia. Viremic animals were less than 1 year old. Relative to PCR-negative horses, PCRpositive animals demonstrated significant elevation of AST ( p = 0.03). Phylogenetic analysis of a 253-bp fragment, in the ORF-1,2,3 overlap region of the HEV genome from the viremic animals showed that three of these viral strains to be identical, and closely related (97–100% nucleotide identity) to two human isolates from Egypt, and distant (78–96%) from 16 other HEV isolates from human and animals and shared 99.6% NI with the fourth strain. The consensus sequence of the four strains was origin obtained elsewhere. These data indicated that horses acquire HEV infection and suggest that cross-species transmission may occur. Whether horses play a role in the transmission of HEV needs further investigation. # 2006 Published by Elsevier B.V. Keywords: HEV; Horse; Egypt; Infection; PCR; Phylogenetic
1. Introduction Hepatitis E virus (HEV) is an important cause of viral hepatitis in young adults mainly in developing countries. HEV transmission occurs in two forms, epidemic and sporadic. The primary risk factors are drinking water and food sources contaminated with feces from HEV-infected persons (Smith, 2001). Investigation into the role of animals as reservoirs of human HEV strains or closely related viruses was crowned by the discovery of swine hepatitis E virus in 1997 (Meng et al., 1997). In several studies on the possible zoonotic nature of this virus, natural antibodies to HEV have been detected in numerous animal species (swine, sheep, goat, cattle, cats and deer) in countries where HEV is endemic or sporadic. In addition, recent § The GenBank accession numbers of the sequences reported in this paper are AY963777–AY963780. * Corresponding author. Tel.: +20 2348 0369; fax: +20 2342 7121. E-mail address: [email protected] (M.D. Saad).
1567-1348/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.meegid.2006.07.007
molecular epidemiological and virological development on HEV supported the detection of HEV RNA in wild boars and deer and zoonotic food-borne transmission by ingestion of meats from these animals in Japan (Clayson et al., 1995; Tei et al., 2003; Matsuda et al., 2003; Okamoto et al., 2004; Tamada et al., 2004; Takahashi et al., 2004; Nishizawa et al., 2005; Li et al., 2005). Disease caused by HEV is mostly sporadic in Egypt and studies have focused primarily on human infections (Divizia et al., 1999; El Zimaity et al., 1993; Gomatos et al., 1996; Hyams et al., 1992; Kamel et al., 1995). Despite the potential for zoonotic transmission of HEV, frequent and close contact between humans and animals in rural Egypt, and the high human HEV seropositivity detected among these rural populations (Darwish et al., 1996; Fix et al., 2000; Miller, 1997), no study has focused on the role of domestic animals in Egypt as a source of HEV infection. Horses are widely used throughout rural and suburban Egypt for pulling carts. They live and work in close contact with humans. Therefore, we investigated HEV infection in Egyptian
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work horses in an effort to determine their possible role in the transmission of HEV. 2. Materials and methods 2.1. Blood and serum samples Blood samples were collected in 1999 by sterile vein-puncture from 100 privately owned work horses in suburban areas of Cairo, particularly ‘‘Old Cairo’’, where most are used as work horses in the hide industry. Owners of these animals are of low socioeconomic status and most animals were in poor health. Blood was permitted to clot, centrifuged at 800 g for 10 min. They were used for identification of HEV specific antibodies using ELISA. After finding evidence for HEV natural antibodies in this set of samples, an additional 100 horses from the same area were bled and tested for HEV antibodies, viremia, and selected serum biochemistry parameters. Sera from this group were separated, and aliquots were kept at 70 -C. Aliquots were used for measuring serum alanin aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin anti-HEV IgG antibody by enzyme immunoassay (EIA) and viral genome detection by reverse transcriptase polymerase chain reaction nested system (nRT-PCR). 2.2. Enzyme immunoassay Horse anti-HEV IgG antibodies were determined using the Hepatitis E [rDNA] Antigen Abbott HEV EIA kit; (Abbott GmbH, Wiesbaden-Delkenheim, Germany) according to (Yoo et al., 2001) with slight modification. Briefly, horse serum was diluted in phosphate-buffered saline at 1:100 and incubated for 60 min at 40 8C with a polystyrene bead coated with the recombinant HEV antigen provided with the kit. The bead was washed four times with 4 ml of distilled water and incubated for 1 h at 40 8C with a 1:5000 dilution of goat anti-horse antibody coupled with horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The bead was washed four times again with 4 ml of distilled water and transferred to a 5ml tube provided with the kit. Substrate was prepared by adding one tablet of o-phenylenediamine–2HCl per 5 ml of substrate buffer containing 0.02% hydrogen peroxide. Three hundred microliters of the substrate solution was added per sample. The reaction mixture was incubated for 30 min in the dark to allow color development. The reaction was stopped by adding 1 ml of 1N sulfuric acid (supplied with the kit) and wells were read at 492.600 nm wavelengths using the Quantum II reader (Abbott Laboratories, USA). Human Anti-HEV IgG positive and negative controls from the kit were tested in parallel to validate reaction conditions and reagents. Cut-off value was calculated according to Yoo et al. (2001).
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Tsarev et al. (1999). Briefly, the first step was an RT-PCR reaction in which 5 ml of the eluted nucleic acid was added to 20 ml reaction solution containing 0.25 ml of each HEV primer (forward and reverse) from a 20 pmol working solution and RTPCR reaction mix (ACCESS RT-PCR System, Promega, Madison, USA) containing 5 ml of 5 reaction buffer, 2 ml of MgSO4 25 mM solution, 0.5 ml of dNTP mix from a 10 mM stock solution, 1 unit AMV RT enzyme, and 0.2 units Tfl DNA in a 0.2 ml PCR tube (Applied Biosystems, Foster City, CA, USA). In the second PCR step, 5 ml from the RT-PCR product was used as a template in a 25 ml reaction solution containing 2.5 ml of 10 reaction buffer, 1.5 ml of 25 mM MgCl2 solution, 0.5 ml of dNTP mix from a 10 mM mix, 0.63 ml of each second round HEV primer from a 20 pmol solution, and 1.25 units Taq DNA polymerase in 0.2 ml PCR tube. Thermal cycling parameters of the RT-PCR step were as follows: a RT step of 45 min at 48 8C, followed by heating to 94 8C for 2 min and PCR amplification of 48 cycles at 94 8C for 30 s, 60 8C for 1 min and 68 8C for 2 min and a final extension at 68 8C for 7 min. The cycling parameters of the nested PCR step were as follows: an initial three cycles of PCR amplification at 94 8C for 1 min, 55 8C for 1 min and 72 8C for 1 min, followed by 32 cycles at 94 8C for 15 s, 55 8C for 45 s, and 72 8C for 1 min. Degenerate primers used for the first-round PCR and the nested PCR flanked a 330 bp and a 310 bp, in first round and nested step, respectively, of the ORF-1,2,3 overlap region of the HEV genome. The primers used for the RT-PCR were outer sense (nt 4988–5007; 50 -GGDCTBGTTCATAACCTGAT-30 and outer antisense (nt 5325–5305; 50 -GGTTGGTTGGATGAATATAGG30 ). The primers for the nested PCR were inner sense (nt 4994– 5016; 50 -GTTCATAACCTGATWGGYATGCT-30 ) and inner antisense (nt 5304–5282; 50 -GGATTGCGAAGGGCTGAGAATCA-30 ) primers locations were according to Genbank accession# AF444003. Amplified products of the nested PCR reaction were analyzed on a 2% agarose gel electrophoresis. Gel was stained with ethidium bromide and visualized on a UV box. Quality control measures were followed to avoid DNA carry over contamination through using one separate room for each step of the molecular detection of HEV. Negative (water) and positive controls (10% human stool suspension positive for HEV RNA from Egypt) were used in each PCR reaction to validate test performance. 2.4. Serum biochemistry and statistical analysis The 100 sera from horses that had been screened by EIA, and PCR were tested for ALT, AST, ALP, and total bilirubin using standard methods on a SmartSpec 3000 spectrophotometer (BioRad, USA). Student t-test was used to analyze serum biochemistry results using SPSS v10 (Lead Technologies, USA). 2.5. DNA sequence analysis
2.3. Viral RNA extraction and RT-PCR Viral nucleic acid (VNA) was extracted from 100 ml of horse serum using silica beads as previously described (Boom et al., 1990). Detection of HEV viral RNA was performed according to
To confirm the positive RT-PCR results, DNA sequencing was performed on the nested PCR product. The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The purified DNA was sequenced on the
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3100 Genetic Analyzer (Applied Biosystems, Foster City, CA USA) using the BigDye terminator chemistries v3.0 and the identified sequences were compared to other HEV sequences in GenBank.
Table 1 DNA sequence similarity between the Egyptian horse HEV (consensus of the four PCR positive samples) and other HEV isolates used in the phylogenetic analysis Isolate name
Genbank accession no.
Source
Nucleotide identity (%)
Egypt-94 Egypt-93 Myanmar Burma India-2 China-1 India-1 Nepal Pakistan USA-2 USA-swine Mexico China-IV JAK-Sai JAK-Sap swJ13-1
AF051352 AF051351 D10330 M32400 X99441 D11092 X98292 AF051830 AF185822 AF060669 AF082843 M74506 AJ272108 AB074915 AB074917 AB097811
Human Human Human Human Human Human Human Human Human Human Swine Human Human Human Human Swine
100 97 96 96 95 94 95 94 94 84 83 85 78 81 79 78
2.6. Phylogenetic analysis A sequence database search was performed using the BLASTn program (BLAST, version 2.0; National Center for Biochemistry Information [NCBI], Bethesda, MD). ClustalX program for Windows (Jeanmougin et al., 1998) was used for sequence alignment, GENEDOC version 2.6.02 (Nicholas, 1997) for visual inspection and editing of alignment, and TREECONW version 1.3b (Van de and De Wachter, 1994) for phylogenetic analyses. The sequences for the Egyptian HEV isolates from horses obtained in this study were compared to sequences of 16 HEV strains isolated from different geographic areas (Asia, Africa, and North America), which are available from GenBank and represent the four major genotypes of HEV. Genetic distance was estimated using the Kimura 2-parameter model and an evolutionary tree was constructed using neighbor-joining (Aye et al., 1992) phylogeny with bootstrap resampling of 500 replicates. The tree was rooted using the single-sequence forced method with the Mexican strain used as the outgroup. The graphic output of the phylogenetic trees was created with the TREECON program. The 16 HEV sequences used for phylogenetic studies are listed with their GenBank accession numbers in Table 1 (Rizzetto, 1997). 2.7. Nucleotide sequence accession number The HEV sequences from work horses reported in this paper have been deposited in GenBank under accession numbers (AY963777–AY963780). 3. Results Of the 200 horses studied, 65% (130/200) and 35% (70/200) were females. Mean age was 3.8 year (range: 0.2–12 year). Twenty-six (13%) of the 200 serum samples tested positive by IgG ELISA for naturally occurring antibodies to HEV. Twelve of these 26 positives (46%) had relatively high OD values, 2– 4.5 times greater than the cut-off value.
Among 100 sera processed for viral RNA detection by RTPCR, four (4%) were PCR-positive for HEV viral genome. All PCR-positive samples were from the ELISA-negative animals and their mean age was 0.3 year, whereas none of the ELISApositive animals had detectable viral RNA in their sera. There was no significant difference between ELISA-positive and ELISA-negative animals for any of the four biochemical parameters tested (Table 2). There was, however, a positive correlation between elevated AST enzyme activity (mean value) and PCR-positive cases (n = 4), p = 0.03 (Table 2). Three of the four sequenced HEV strains (253 bp size fragment each) from work horses were identical and shared 99.6% nucleotide identity with the fourth strain. The consensus of the four strains shared 97–100% nucleotide identity (NI) with the sequences of human HEV isolates recovered from Egypt in 1993 and 1994, respectively. The Egyptian horse HEV sequences were found to have less similarity (78–96% NI) with other selected human and swine HEV isolates (Torresi et al., 1999). Based on the PCR fragments obtained, a phylogenetic tree constructed from the Egyptian horse HEV isolates and 16 other HEV strains (based on the PCR fragment obtained in this work) showed HEV sequences to be divided into four genotypes as
Table 2 Comparison of selected serum biochemistries between HEV-positive and negative animals ELISA Positive (n = 13)
PCR Negative (n = 83)
AST ALT ALP
82.4 15.9 (48.0–110.5) 9.0 2.1 (7.0–14.0) 61.9 26.6 (32.5–135.6)
89.5 24 (44.6–168.1) 10.5 2.7 (7.0–21.0) 59.1 14.6 (32.6–104.6)
Total bilirubina
33.8 25.8 (15.4–111.1)
30.2 15.0 (1.7–70.1)
Enzyme levels (U/l) are expressed as mean + S.D. a mmol/l. * p = 0.03.
Positive (n = 4) *
Negative (n = 96)
114.8 22.1 (82.4–131.0) 10.9 2.9 (7.2–13.5) 62.2 12.5 (44.0–71.5)
88.4 23.1 (44.6–168.1) 10.3 2.6 (7.0–21.0) 59.5 16.5 (32.5–135.6)
38.0 1.6 (35.9–39.3)
30.7 16.7 (1.7–111.2)
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Fig. 1. Neighbor-joining phylogenetic tree of 253 bp of the ORF-1,2,3 overlap region of the HEV genome from Egyptian work horses (preceded by bullets) with other human and animal HEV strains. A genetic distance of 0.02 substitutions per site is shown by the scale bar. The significance of the nodes defining clusters was assessed by bootstrap anlysis. Bootstrap values >70% are indicated at the appropriate nodes.
previously described (Schlauder et al., 1999): an Asian-African genotype 1, a Mexican genotype 2, a U.S. genotype 3 and the Chinese genotype 4. Our results demonstrated that the horse HEV isolates and the two human HEV isolates from Egypt comprise a single cluster within the Asian-African genotype (Fig. 1), implying these viruses originated from a common ancestor. 4. Discussion The isolation of swine HEV in 1997 (Meng et al., 1997), from a country with no record of endemic HEV, which was closely related to the human HEV strain isolated from a patient with no history of travel outside the country, raised the issue of possible zoonotic transmission of this virus. Subsequently, other studies have addressed animal transmission of HEV disease in HEV-endemic and non-endemic countries and have identified HEV antibodies in a variety of animal species (Meng et al., 1999). Our study is the first to investigate the possibility of animals acquiring natural HEV infections in Egypt and the first to document both the seropositivity and HEV infection rate among work horses in suburban Cairo. HEV is widely endemic in Egypt, with a seroprevalence ranging from 17.2 to 60% in adults (Darwish et al., 1996; Fix et al., 2000; Gomatos et al., 1996; Kamel et al., 1995). High seroprevalence of HEV has been recorded in communities in
the Nile Delta and other geographic regions of Egypt. These are agrarian communities of lower socioeconomic status with some marginal sanitary practices that encourage the spread of enteric pathogens. Domestic animals of different species are raised, often within household. Such frequent and close contact easily facilitates the passage of zoonotic animal infections to humans. There are no data published on seroprevalence of HEV in animals in rural areas in Egypt. Such data if available will emphasize cross species transmission and better understanding of the HEV pattern of transmission in Egypt. In this study, the detection of natural antibodies to HEV virus in 13% of horses tested suggests they may acquire HEV infection and may be either an accidental host or serve as a reservoir. Based upon HEV infection rates and seropositivity among humans living in Cairo, the high seropositivity seen in our sample of horses from suburban Cairo, and, in particular, their relatively high infection rate was surprising. We are led to speculate that HEV seropositivity and viral carriage rates may be much higher among animals in rural and suburban communities. This is an area of interest that will be investigated. Based on similar findings reported for swine with anti-HEV antibodies, we anticipated no abnormalities in serum ALT, AST, or bilirubin in seropositive horses. The elevated AST levels that were seen in the four viremic animals were not unexpected, and may be due either in part or wholly to the HEV
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infection. The AST is often used to detect liver disease in large animals, but this enzyme is not liver-specific because it is also increased in myocardial and skeletal muscle diseases. Extensive replication of the virus in organs besides that of the liver has been documented in other animal species (Williams et al., 2001) and is a factor that may account for elevation of AST alone. The detection of the HEV genome in sera of some of the horses tested (n = 4) suggests that viremia occurs after infection. The presence of viral RNA was only detected in ELISA-negative young animals, which may indicate that the duration of viremia is brief and that the virus disappears from blood before the appearance of anti-HEV IgG antibodies. Low level viremia in serum, below the detectable limits of the PCR system we employed, may further explain our inability to detect virus in seropositive animals. It is notable that in other animal species such as swine (Clayson et al., 1995; Hsieh et al., 1999; Meng et al., 1997) HEV RNA was detected in the presence of anti-HEV antibodies and ELISA-positive animals, suggesting a prolonged viremia. Sequence analysis and phylogenetic comparison of the 253 bp fragment from the HEV strains, we detected in horses, to other HEV strains of both human and animal origin showed that three of the Egyptian horse strains were identical (AY963777, AY963778, AY963779) while sharing only 99.6% NI with the fourth (AY963780). The consensus sequence of all four isolates shared 97–100% NI with the sequences of two human HEV strains isolated from Egypt in 1993 and 1994, respectively. We are confident that the HEV sequences reported in this study were not due to contamination, considering the extreme measures and good laboratory practice followed during conducting the laboratory work. Within such short fragment such very close similarity among isolates collected from the same geographic region and similar epidemiological circumstances could be possible. Similar findings were observed among some human HEV strains from Tanedfou, Algeria in an outbreak in 1995 along a small fragment of ORF2 (GenBank Accession numbers AF228535, AF228537, AF228530). The fact that the three identical HEV strains from horses (three of the four strains detected in this study) were also identical to the human strain from Egypt in 1994 (GenBank accession number AF051352) could be due to a low level replication of HEV in infected horses in addition to being infected from one source, which could be true in light of the fact that these working horses, at rest time, drink from one common water trough at the designated working area (the hide industry is located in Old Cairo). In addition, the fact that HEV transmission among human in Egypt is sporadic, puts low level pressure on the virus evolution which may lead to the circulation of very closely related strains within one geographic location. Moreover, the actual genetic heterogeneity of Egyptian HEV strains has not yet been studied, carrying the possibility that identical or very closely related strains of HEV may exit and circulate among hosts living within confined geographic locations and exposed to the same source of infection. The horse and human HEV strains constitute a single cluster within the Asian-African genotype, which is further
divided into African (represented in the Egyptian isolates) and Asian subgenotypes, as previously described (Schlauder et al., 1999). This suggests that horses may acquire HEV infection and that they may play a role in the transmission of sporadic HEV infection to humans. This hypothesis will be greatly strengthened if future investigations show HEV excretion in horse feces. In conclusion, this study presents the first evidence of HEV infection and viremia in horses and their possible role in Egypt as reservoirs or accidental hosts for human HEV. Further studies are warranted to investigate viremia and pathogenesis of HEV in horses and to study HEV transmission among equines and other animals as it may relate to sporadic HEV infection in humans in Egypt. Acknowledgments This work was supported by the DoD GEIS funds Work Unit #847705.82000.25GB.E0018. In conducting the research described in this report (Animal care and use protocol #9904), all aspects involving animal use were conducted in accordance with the Animal Welfare Act (9 CFR, Subchapter A, Parts 1-3), Department of Defense regulations and recognized standards relating to the humane care and use of laboratory animals such as the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and Guide for the Care and Use of Laboratory Animals. The Naval Medical Research Unit No. 3 is an AAALAC International accredited facility. This work was presented at the 11th International Workshop on Virus Evolution and Molecular Epidemiology website: http:// www.kuleuven.be/aidslab/veme.htm. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. I am a military service member (or employee of the U.S. Government). This work was prepared as part of my official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. References Aye, T.T., Uchida, T., Ma, X.Z., Iida, F., Shikata, T., Zhuang, H., Win, K.M., 1992. Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986–1988) of China. Nucl. Acids Res. 20, 3512. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., van der, N.J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503. Clayson, E.T., Innis, B.L., Myint, K.S., Narupiti, S., Vaughn, D.W., Giri, S., Ranabhat, P., Shrestha, M.P., 1995. Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am. J. Trop. Med. Hyg. 53, 228–232. Darwish, M.A., Faris, R., Clemens, J.D., Rao, M.R., Edelman, R., 1996. High seroprevalence of hepatitis A, B, C, and E viruses in residents in an Egyptian village in The Nile Delta: a pilot study. Am. J. Trop. Med. Hyg. 54, 554–558 see comments.
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Hepatitis E virus Infection in Work Horses in Egypt§ Magdi D. Saad a,*, Hussein A. Hussein b, Moustafa M. Bashandy c, Hamdy H. Kamel d, K.C. Earhart a, David J. Fryauff a, Mary Younan a, Amira H. Mohamed c a
Virology Research Program, United States Naval Medical Research Unit No. 3, Cairo, Egypt b Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt c Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt d Department of Clinical Pathology, Faculty of Veterinary Medicine, Beni-Suef, Egypt Received 27 February 2006; received in revised form 6 July 2006; accepted 20 July 2006 Available online 12 October 2006
Abstract Hepatitis E virus (HEV) is an important cause of hepatitis among young Egyptian adults with high seroprevalence rates seen in both rural areas of the Nile Delta and in suburban Cairo. Because natural antibodies to HEV have been detected in animals and zoonotic transmission is postulated, we surveyed work horses in Cairo for evidence of HEVexposure and viremia. Sera from 200 Cairo work horses were tested by ELISA for the presence of IgG anti-HEVantibody revealed a seropositivity of 13%. Among 100 samples processed for detection of viral genome by means of nested polymerase chain reaction (N-PCR), 4% were positive and indicative of viremia. Viremic animals were less than 1 year old. Relative to PCR-negative horses, PCRpositive animals demonstrated significant elevation of AST ( p = 0.03). Phylogenetic analysis of a 253-bp fragment, in the ORF-1,2,3 overlap region of the HEV genome from the viremic animals showed that three of these viral strains to be identical, and closely related (97–100% nucleotide identity) to two human isolates from Egypt, and distant (78–96%) from 16 other HEV isolates from human and animals and shared 99.6% NI with the fourth strain. The consensus sequence of the four strains was origin obtained elsewhere. These data indicated that horses acquire HEV infection and suggest that cross-species transmission may occur. Whether horses play a role in the transmission of HEV needs further investigation. # 2006 Published by Elsevier B.V. Keywords: HEV; Horse; Egypt; Infection; PCR; Phylogenetic
1. Introduction Hepatitis E virus (HEV) is an important cause of viral hepatitis in young adults mainly in developing countries. HEV transmission occurs in two forms, epidemic and sporadic. The primary risk factors are drinking water and food sources contaminated with feces from HEV-infected persons (Smith, 2001). Investigation into the role of animals as reservoirs of human HEV strains or closely related viruses was crowned by the discovery of swine hepatitis E virus in 1997 (Meng et al., 1997). In several studies on the possible zoonotic nature of this virus, natural antibodies to HEV have been detected in numerous animal species (swine, sheep, goat, cattle, cats and deer) in countries where HEV is endemic or sporadic. In addition, recent § The GenBank accession numbers of the sequences reported in this paper are AY963777–AY963780. * Corresponding author. Tel.: +20 2348 0369; fax: +20 2342 7121. E-mail address: [email protected] (M.D. Saad).
1567-1348/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.meegid.2006.07.007
molecular epidemiological and virological development on HEV supported the detection of HEV RNA in wild boars and deer and zoonotic food-borne transmission by ingestion of meats from these animals in Japan (Clayson et al., 1995; Tei et al., 2003; Matsuda et al., 2003; Okamoto et al., 2004; Tamada et al., 2004; Takahashi et al., 2004; Nishizawa et al., 2005; Li et al., 2005). Disease caused by HEV is mostly sporadic in Egypt and studies have focused primarily on human infections (Divizia et al., 1999; El Zimaity et al., 1993; Gomatos et al., 1996; Hyams et al., 1992; Kamel et al., 1995). Despite the potential for zoonotic transmission of HEV, frequent and close contact between humans and animals in rural Egypt, and the high human HEV seropositivity detected among these rural populations (Darwish et al., 1996; Fix et al., 2000; Miller, 1997), no study has focused on the role of domestic animals in Egypt as a source of HEV infection. Horses are widely used throughout rural and suburban Egypt for pulling carts. They live and work in close contact with humans. Therefore, we investigated HEV infection in Egyptian
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work horses in an effort to determine their possible role in the transmission of HEV. 2. Materials and methods 2.1. Blood and serum samples Blood samples were collected in 1999 by sterile vein-puncture from 100 privately owned work horses in suburban areas of Cairo, particularly ‘‘Old Cairo’’, where most are used as work horses in the hide industry. Owners of these animals are of low socioeconomic status and most animals were in poor health. Blood was permitted to clot, centrifuged at 800 g for 10 min. They were used for identification of HEV specific antibodies using ELISA. After finding evidence for HEV natural antibodies in this set of samples, an additional 100 horses from the same area were bled and tested for HEV antibodies, viremia, and selected serum biochemistry parameters. Sera from this group were separated, and aliquots were kept at 70 -C. Aliquots were used for measuring serum alanin aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin anti-HEV IgG antibody by enzyme immunoassay (EIA) and viral genome detection by reverse transcriptase polymerase chain reaction nested system (nRT-PCR). 2.2. Enzyme immunoassay Horse anti-HEV IgG antibodies were determined using the Hepatitis E [rDNA] Antigen Abbott HEV EIA kit; (Abbott GmbH, Wiesbaden-Delkenheim, Germany) according to (Yoo et al., 2001) with slight modification. Briefly, horse serum was diluted in phosphate-buffered saline at 1:100 and incubated for 60 min at 40 8C with a polystyrene bead coated with the recombinant HEV antigen provided with the kit. The bead was washed four times with 4 ml of distilled water and incubated for 1 h at 40 8C with a 1:5000 dilution of goat anti-horse antibody coupled with horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The bead was washed four times again with 4 ml of distilled water and transferred to a 5ml tube provided with the kit. Substrate was prepared by adding one tablet of o-phenylenediamine–2HCl per 5 ml of substrate buffer containing 0.02% hydrogen peroxide. Three hundred microliters of the substrate solution was added per sample. The reaction mixture was incubated for 30 min in the dark to allow color development. The reaction was stopped by adding 1 ml of 1N sulfuric acid (supplied with the kit) and wells were read at 492.600 nm wavelengths using the Quantum II reader (Abbott Laboratories, USA). Human Anti-HEV IgG positive and negative controls from the kit were tested in parallel to validate reaction conditions and reagents. Cut-off value was calculated according to Yoo et al. (2001).
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Tsarev et al. (1999). Briefly, the first step was an RT-PCR reaction in which 5 ml of the eluted nucleic acid was added to 20 ml reaction solution containing 0.25 ml of each HEV primer (forward and reverse) from a 20 pmol working solution and RTPCR reaction mix (ACCESS RT-PCR System, Promega, Madison, USA) containing 5 ml of 5 reaction buffer, 2 ml of MgSO4 25 mM solution, 0.5 ml of dNTP mix from a 10 mM stock solution, 1 unit AMV RT enzyme, and 0.2 units Tfl DNA in a 0.2 ml PCR tube (Applied Biosystems, Foster City, CA, USA). In the second PCR step, 5 ml from the RT-PCR product was used as a template in a 25 ml reaction solution containing 2.5 ml of 10 reaction buffer, 1.5 ml of 25 mM MgCl2 solution, 0.5 ml of dNTP mix from a 10 mM mix, 0.63 ml of each second round HEV primer from a 20 pmol solution, and 1.25 units Taq DNA polymerase in 0.2 ml PCR tube. Thermal cycling parameters of the RT-PCR step were as follows: a RT step of 45 min at 48 8C, followed by heating to 94 8C for 2 min and PCR amplification of 48 cycles at 94 8C for 30 s, 60 8C for 1 min and 68 8C for 2 min and a final extension at 68 8C for 7 min. The cycling parameters of the nested PCR step were as follows: an initial three cycles of PCR amplification at 94 8C for 1 min, 55 8C for 1 min and 72 8C for 1 min, followed by 32 cycles at 94 8C for 15 s, 55 8C for 45 s, and 72 8C for 1 min. Degenerate primers used for the first-round PCR and the nested PCR flanked a 330 bp and a 310 bp, in first round and nested step, respectively, of the ORF-1,2,3 overlap region of the HEV genome. The primers used for the RT-PCR were outer sense (nt 4988–5007; 50 -GGDCTBGTTCATAACCTGAT-30 and outer antisense (nt 5325–5305; 50 -GGTTGGTTGGATGAATATAGG30 ). The primers for the nested PCR were inner sense (nt 4994– 5016; 50 -GTTCATAACCTGATWGGYATGCT-30 ) and inner antisense (nt 5304–5282; 50 -GGATTGCGAAGGGCTGAGAATCA-30 ) primers locations were according to Genbank accession# AF444003. Amplified products of the nested PCR reaction were analyzed on a 2% agarose gel electrophoresis. Gel was stained with ethidium bromide and visualized on a UV box. Quality control measures were followed to avoid DNA carry over contamination through using one separate room for each step of the molecular detection of HEV. Negative (water) and positive controls (10% human stool suspension positive for HEV RNA from Egypt) were used in each PCR reaction to validate test performance. 2.4. Serum biochemistry and statistical analysis The 100 sera from horses that had been screened by EIA, and PCR were tested for ALT, AST, ALP, and total bilirubin using standard methods on a SmartSpec 3000 spectrophotometer (BioRad, USA). Student t-test was used to analyze serum biochemistry results using SPSS v10 (Lead Technologies, USA). 2.5. DNA sequence analysis
2.3. Viral RNA extraction and RT-PCR Viral nucleic acid (VNA) was extracted from 100 ml of horse serum using silica beads as previously described (Boom et al., 1990). Detection of HEV viral RNA was performed according to
To confirm the positive RT-PCR results, DNA sequencing was performed on the nested PCR product. The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The purified DNA was sequenced on the
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3100 Genetic Analyzer (Applied Biosystems, Foster City, CA USA) using the BigDye terminator chemistries v3.0 and the identified sequences were compared to other HEV sequences in GenBank.
Table 1 DNA sequence similarity between the Egyptian horse HEV (consensus of the four PCR positive samples) and other HEV isolates used in the phylogenetic analysis Isolate name
Genbank accession no.
Source
Nucleotide identity (%)
Egypt-94 Egypt-93 Myanmar Burma India-2 China-1 India-1 Nepal Pakistan USA-2 USA-swine Mexico China-IV JAK-Sai JAK-Sap swJ13-1
AF051352 AF051351 D10330 M32400 X99441 D11092 X98292 AF051830 AF185822 AF060669 AF082843 M74506 AJ272108 AB074915 AB074917 AB097811
Human Human Human Human Human Human Human Human Human Human Swine Human Human Human Human Swine
100 97 96 96 95 94 95 94 94 84 83 85 78 81 79 78
2.6. Phylogenetic analysis A sequence database search was performed using the BLASTn program (BLAST, version 2.0; National Center for Biochemistry Information [NCBI], Bethesda, MD). ClustalX program for Windows (Jeanmougin et al., 1998) was used for sequence alignment, GENEDOC version 2.6.02 (Nicholas, 1997) for visual inspection and editing of alignment, and TREECONW version 1.3b (Van de and De Wachter, 1994) for phylogenetic analyses. The sequences for the Egyptian HEV isolates from horses obtained in this study were compared to sequences of 16 HEV strains isolated from different geographic areas (Asia, Africa, and North America), which are available from GenBank and represent the four major genotypes of HEV. Genetic distance was estimated using the Kimura 2-parameter model and an evolutionary tree was constructed using neighbor-joining (Aye et al., 1992) phylogeny with bootstrap resampling of 500 replicates. The tree was rooted using the single-sequence forced method with the Mexican strain used as the outgroup. The graphic output of the phylogenetic trees was created with the TREECON program. The 16 HEV sequences used for phylogenetic studies are listed with their GenBank accession numbers in Table 1 (Rizzetto, 1997). 2.7. Nucleotide sequence accession number The HEV sequences from work horses reported in this paper have been deposited in GenBank under accession numbers (AY963777–AY963780). 3. Results Of the 200 horses studied, 65% (130/200) and 35% (70/200) were females. Mean age was 3.8 year (range: 0.2–12 year). Twenty-six (13%) of the 200 serum samples tested positive by IgG ELISA for naturally occurring antibodies to HEV. Twelve of these 26 positives (46%) had relatively high OD values, 2– 4.5 times greater than the cut-off value.
Among 100 sera processed for viral RNA detection by RTPCR, four (4%) were PCR-positive for HEV viral genome. All PCR-positive samples were from the ELISA-negative animals and their mean age was 0.3 year, whereas none of the ELISApositive animals had detectable viral RNA in their sera. There was no significant difference between ELISA-positive and ELISA-negative animals for any of the four biochemical parameters tested (Table 2). There was, however, a positive correlation between elevated AST enzyme activity (mean value) and PCR-positive cases (n = 4), p = 0.03 (Table 2). Three of the four sequenced HEV strains (253 bp size fragment each) from work horses were identical and shared 99.6% nucleotide identity with the fourth strain. The consensus of the four strains shared 97–100% nucleotide identity (NI) with the sequences of human HEV isolates recovered from Egypt in 1993 and 1994, respectively. The Egyptian horse HEV sequences were found to have less similarity (78–96% NI) with other selected human and swine HEV isolates (Torresi et al., 1999). Based on the PCR fragments obtained, a phylogenetic tree constructed from the Egyptian horse HEV isolates and 16 other HEV strains (based on the PCR fragment obtained in this work) showed HEV sequences to be divided into four genotypes as
Table 2 Comparison of selected serum biochemistries between HEV-positive and negative animals ELISA Positive (n = 13)
PCR Negative (n = 83)
AST ALT ALP
82.4 15.9 (48.0–110.5) 9.0 2.1 (7.0–14.0) 61.9 26.6 (32.5–135.6)
89.5 24 (44.6–168.1) 10.5 2.7 (7.0–21.0) 59.1 14.6 (32.6–104.6)
Total bilirubina
33.8 25.8 (15.4–111.1)
30.2 15.0 (1.7–70.1)
Enzyme levels (U/l) are expressed as mean + S.D. a mmol/l. * p = 0.03.
Positive (n = 4) *
Negative (n = 96)
114.8 22.1 (82.4–131.0) 10.9 2.9 (7.2–13.5) 62.2 12.5 (44.0–71.5)
88.4 23.1 (44.6–168.1) 10.3 2.6 (7.0–21.0) 59.5 16.5 (32.5–135.6)
38.0 1.6 (35.9–39.3)
30.7 16.7 (1.7–111.2)
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Fig. 1. Neighbor-joining phylogenetic tree of 253 bp of the ORF-1,2,3 overlap region of the HEV genome from Egyptian work horses (preceded by bullets) with other human and animal HEV strains. A genetic distance of 0.02 substitutions per site is shown by the scale bar. The significance of the nodes defining clusters was assessed by bootstrap anlysis. Bootstrap values >70% are indicated at the appropriate nodes.
previously described (Schlauder et al., 1999): an Asian-African genotype 1, a Mexican genotype 2, a U.S. genotype 3 and the Chinese genotype 4. Our results demonstrated that the horse HEV isolates and the two human HEV isolates from Egypt comprise a single cluster within the Asian-African genotype (Fig. 1), implying these viruses originated from a common ancestor. 4. Discussion The isolation of swine HEV in 1997 (Meng et al., 1997), from a country with no record of endemic HEV, which was closely related to the human HEV strain isolated from a patient with no history of travel outside the country, raised the issue of possible zoonotic transmission of this virus. Subsequently, other studies have addressed animal transmission of HEV disease in HEV-endemic and non-endemic countries and have identified HEV antibodies in a variety of animal species (Meng et al., 1999). Our study is the first to investigate the possibility of animals acquiring natural HEV infections in Egypt and the first to document both the seropositivity and HEV infection rate among work horses in suburban Cairo. HEV is widely endemic in Egypt, with a seroprevalence ranging from 17.2 to 60% in adults (Darwish et al., 1996; Fix et al., 2000; Gomatos et al., 1996; Kamel et al., 1995). High seroprevalence of HEV has been recorded in communities in
the Nile Delta and other geographic regions of Egypt. These are agrarian communities of lower socioeconomic status with some marginal sanitary practices that encourage the spread of enteric pathogens. Domestic animals of different species are raised, often within household. Such frequent and close contact easily facilitates the passage of zoonotic animal infections to humans. There are no data published on seroprevalence of HEV in animals in rural areas in Egypt. Such data if available will emphasize cross species transmission and better understanding of the HEV pattern of transmission in Egypt. In this study, the detection of natural antibodies to HEV virus in 13% of horses tested suggests they may acquire HEV infection and may be either an accidental host or serve as a reservoir. Based upon HEV infection rates and seropositivity among humans living in Cairo, the high seropositivity seen in our sample of horses from suburban Cairo, and, in particular, their relatively high infection rate was surprising. We are led to speculate that HEV seropositivity and viral carriage rates may be much higher among animals in rural and suburban communities. This is an area of interest that will be investigated. Based on similar findings reported for swine with anti-HEV antibodies, we anticipated no abnormalities in serum ALT, AST, or bilirubin in seropositive horses. The elevated AST levels that were seen in the four viremic animals were not unexpected, and may be due either in part or wholly to the HEV
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infection. The AST is often used to detect liver disease in large animals, but this enzyme is not liver-specific because it is also increased in myocardial and skeletal muscle diseases. Extensive replication of the virus in organs besides that of the liver has been documented in other animal species (Williams et al., 2001) and is a factor that may account for elevation of AST alone. The detection of the HEV genome in sera of some of the horses tested (n = 4) suggests that viremia occurs after infection. The presence of viral RNA was only detected in ELISA-negative young animals, which may indicate that the duration of viremia is brief and that the virus disappears from blood before the appearance of anti-HEV IgG antibodies. Low level viremia in serum, below the detectable limits of the PCR system we employed, may further explain our inability to detect virus in seropositive animals. It is notable that in other animal species such as swine (Clayson et al., 1995; Hsieh et al., 1999; Meng et al., 1997) HEV RNA was detected in the presence of anti-HEV antibodies and ELISA-positive animals, suggesting a prolonged viremia. Sequence analysis and phylogenetic comparison of the 253 bp fragment from the HEV strains, we detected in horses, to other HEV strains of both human and animal origin showed that three of the Egyptian horse strains were identical (AY963777, AY963778, AY963779) while sharing only 99.6% NI with the fourth (AY963780). The consensus sequence of all four isolates shared 97–100% NI with the sequences of two human HEV strains isolated from Egypt in 1993 and 1994, respectively. We are confident that the HEV sequences reported in this study were not due to contamination, considering the extreme measures and good laboratory practice followed during conducting the laboratory work. Within such short fragment such very close similarity among isolates collected from the same geographic region and similar epidemiological circumstances could be possible. Similar findings were observed among some human HEV strains from Tanedfou, Algeria in an outbreak in 1995 along a small fragment of ORF2 (GenBank Accession numbers AF228535, AF228537, AF228530). The fact that the three identical HEV strains from horses (three of the four strains detected in this study) were also identical to the human strain from Egypt in 1994 (GenBank accession number AF051352) could be due to a low level replication of HEV in infected horses in addition to being infected from one source, which could be true in light of the fact that these working horses, at rest time, drink from one common water trough at the designated working area (the hide industry is located in Old Cairo). In addition, the fact that HEV transmission among human in Egypt is sporadic, puts low level pressure on the virus evolution which may lead to the circulation of very closely related strains within one geographic location. Moreover, the actual genetic heterogeneity of Egyptian HEV strains has not yet been studied, carrying the possibility that identical or very closely related strains of HEV may exit and circulate among hosts living within confined geographic locations and exposed to the same source of infection. The horse and human HEV strains constitute a single cluster within the Asian-African genotype, which is further
divided into African (represented in the Egyptian isolates) and Asian subgenotypes, as previously described (Schlauder et al., 1999). This suggests that horses may acquire HEV infection and that they may play a role in the transmission of sporadic HEV infection to humans. This hypothesis will be greatly strengthened if future investigations show HEV excretion in horse feces. In conclusion, this study presents the first evidence of HEV infection and viremia in horses and their possible role in Egypt as reservoirs or accidental hosts for human HEV. Further studies are warranted to investigate viremia and pathogenesis of HEV in horses and to study HEV transmission among equines and other animals as it may relate to sporadic HEV infection in humans in Egypt. Acknowledgments This work was supported by the DoD GEIS funds Work Unit #847705.82000.25GB.E0018. In conducting the research described in this report (Animal care and use protocol #9904), all aspects involving animal use were conducted in accordance with the Animal Welfare Act (9 CFR, Subchapter A, Parts 1-3), Department of Defense regulations and recognized standards relating to the humane care and use of laboratory animals such as the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and Guide for the Care and Use of Laboratory Animals. The Naval Medical Research Unit No. 3 is an AAALAC International accredited facility. This work was presented at the 11th International Workshop on Virus Evolution and Molecular Epidemiology website: http:// www.kuleuven.be/aidslab/veme.htm. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. I am a military service member (or employee of the U.S. Government). This work was prepared as part of my official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. References Aye, T.T., Uchida, T., Ma, X.Z., Iida, F., Shikata, T., Zhuang, H., Win, K.M., 1992. Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986–1988) of China. Nucl. Acids Res. 20, 3512. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., van der, N.J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503. Clayson, E.T., Innis, B.L., Myint, K.S., Narupiti, S., Vaughn, D.W., Giri, S., Ranabhat, P., Shrestha, M.P., 1995. Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am. J. Trop. Med. Hyg. 53, 228–232. Darwish, M.A., Faris, R., Clemens, J.D., Rao, M.R., Edelman, R., 1996. High seroprevalence of hepatitis A, B, C, and E viruses in residents in an Egyptian village in The Nile Delta: a pilot study. Am. J. Trop. Med. Hyg. 54, 554–558 see comments.
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