Coronavirus antibodies in African bat species.Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome Severe Acute Respiratory Syndrome (SARS) Definition Severe acute respiratory syndrome (SARS) is the first emergent and highly transmissible viral disease to appear during the twenty-first century. (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV. ********** Severe acute respiratory syndrome (SARS) emerged as a newly recognized human disease in the People's Republic of China late in 2002 and spread globally, causing 8,422 infections with 916 (11%) deaths before it was brought under control in 2003 (1). The causative agent was identified as a coronavirus coronavirus /co·ro·na·vi·rus/ (ko-ro´nah-vi?rus) any virus belonging to the family Coronaviridae. Coronavirus /Co·ro·na·vi·rus/ (ko-ro´nah-vi?rus (SARS-CoV) (2-4), and related viruses found in palm civets (Paguma larvata), raccoon dogs (Nycereutesprocyonoides) (5), and insectivorous insectivorous eating insects to the extent that they are significant as a contributor to the patient's diet. bats in Asia cluster phylogenetically phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history: a phylogenetic classification of species. together with SARS-CoV in a putative group 4 (6-10). Farmed food animals such as civets may acquire SARS-like-CoV infection from bats, and adaptation of the viruses to these secondary hosts may occasionally give rise to strains capable of spreading and causing disease in humans (HCoV) (10). The Study Bat serum specimens (n = 705) collected from 1986 through 1999 in South Africa (SA) and the Democratic Republic of the Congo (DRC DRC Democratic Republic of Congo DRC Down (Stage) Right Center DRC Director(ate) of Reserve Components DRC Disability Rights Commission (United Kingdom) ) were tested. The first 248 serum specimens were collected from 1986 through 1989 in the Mpumalanga and Limpopo Provinces of SA for studies on rabies-related viruses, with the approval of the provincial Directorates of Nature Conservation and the Animal Ethics Committee ethics committee A multidisciplinary hospital body composed of a broad spectrum of personnel–eg, physicians, nurses, social workers, priests, and others, which addresses the moral and ethical issues within the hospital. See DNR, Institutional review board. of the University of the Witwatersrand Due to the 1959 Extension of University Education Act the school was only allowed to register a small number of black students for most of the apartheid era, even though several notable black anti-apartheid leaders graduated from the university. . The remaining 457 serum samples were collected in 1995-1999 in the Bandundu and Oriental Provinces of the DRC for studies on Ebola and Marburg viruses, under the auspices of the International Committees for the Control of Ebola hemorrhagic fever Noun 1. Ebola hemorrhagic fever - a severe and often fatal disease in humans and nonhuman primates (monkeys and chimpanzees) caused by the Ebola virus; characterized by high fever and severe internal bleeding; can be spread from person to person; is largely limited to in Kikwit, and Marburg hemorrhagic fever Noun 1. Marburg hemorrhagic fever - a viral disease of green monkeys caused by the Marburg virus; when transmitted to humans it causes serious or fatal illness green monkey disease, Marburg disease in Durba-Watsa, coordinated by the World Health Organization on behalf of the government of DRC. Bats were caught in mist nets, anesthetized a·nes·the·tize also a·naes·the·tize tr.v. a·nes·the·tized, a·nes·the·tiz·ing, a·nes·the·tiz·es To induce anesthesia in. a·nes , and exsanguinated by cardiac puncture. Serum specimens were stored at -70[degrees]C until analyzed. For screening of serum specimens we used the SARS-CoV ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent. ELISA n. kit (EUROIMMUN AG, Lubeck, Germany) with minor modifications. Bat serum samples were tested at a dilution of 1:50, and horseradish horseradish Hardy perennial plant (Armoracia lapathifolia) of the mustard family, native to Mediterranean lands and grown throughout the temperate zones. Its hotly pungent, fleshy root is used as a condiment and is traditionally considered medicinal. peroxidase--labeled goat antibat immunoglobulin (Ig) conjugate conjugate /con·ju·gate/ (kon´jdbobr-gat) 1. paired, or equally coupled; working in unison. 2. a conjugate diameter of the pelvic inlet; used alone usually to denote the true conjugate diameter; see (Bethyl, Montgomery, AL, USA) was used as secondary antibody at a dilution of 1:2,000. Negative bat serum was obtained from a captive-bred Rousettus aegyptiacus at the National Institute for Communicable Diseases, Sandringham, SA. The cutoff was determined as 3x the mean optical density value at 450/605 nm observed in negative control samples. Positive serum samples were retested and their titers determined. To evaluate test specificity and to exclude possible cross-reactivity to other viruses, especially to HCoVs, which have a seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided in humans >90% (11), 662 human serum specimens were screened (online Technical Appendix, available from www.cdc.gov/EID/content/13/9/1367-Techapp.htm), including those from 90 patients with other acute respiratory infections, 70 HCoV-229E--positive serum specimens and 4 HCoV-NL63--positive serum specimens (provided by L. van der Hock hock: see wine. ). A confirmatory Western blot Western blot A technique developed in 1979 that is used to confirm ELISA results. HIV antigen is purified by electrophoresis and attached by blotting to a nylon or nitrocellulose filter. (WB) was done by using protein lysates from Vero E6 cell cultures (American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for [Manassas, VA, USA] CRL CRL - Carnegie Representation Language. Carnegie Group, Inc. Frame language derived from SRL. Written in Common LISP. Used in the product Knowledge Craft. 1586) infected with SARS-CoV Hong Kong isolate 6109 (GenBank accession no. AY278491) and from uninfected Vero E6 cultures. Bat serum specimens were applied at dilutions of 1:500 and 1:2,000. Secondary detection was performed with the SuperSignal West Dura Extended Substrate chemiluminescence chemiluminescence /chemi·lu·mi·nes·cence/ (kem?i-loo?mi-nes´ens) luminescence produced by direct transformation of chemical energy into light energy. detection assay (Pierce Biotechnology, Rockford, IL, USA). The signal intensity of the 150-kDa spike (S), 50-kDa nucleocapsid nucleocapsid /nu·cleo·cap·sid/ (noo?kle-o-kap´sid) a unit of viral structure, consisting of a capsid with the enclosed nucleic acid. nu·cle·o·cap·sid n. (N) proteins was evaluated independently by 2 operators. For a second confirmatory WB, recombinant SARS-CoV proteins were used. For prokaryotic pro·kar·y·ote also pro·car·y·ote n. An organism of the kingdom Monera (or Prokaryotae), comprising the bacteria and cyanobacteria, characterized by the absence of a distinct, membrane-bound nucleus or membrane-bound organelles, and by DNA that expression of recombinant SARS-CoV N protein and a fragment of the S protein (amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. positions 318-510), we followed the instructions of the Champion pET Directional TOPO TOPO Tri-N-Octylphosphine Oxide TOPO Topographic/Topography TOPO Trioctyl-Phosphine Oxide ToPo Torposten (German Military Gate Post) TOPO Tunable Optical Parametric Oscillator Expression kit (Invitrogen, Karlsruhe, Germany) using plasmids pET 101-N and pET 102-Saa318-510. Purification and refolding of the protein on column were done as described previously (12). Purified recombinant protein (15 [micro]g) was resolved by electrophoresis on a discontinuous 12% sodium dodecyl sulfate--polyacrylamide gel. After blotting, nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. strips were incubated with bat serum samples diluted 1:2,500 and 1:5,000. One positive bat serum specimen was used as a reference to exclude variations in experimental procedures and signal intensities. Serum specimens that produced signals at a dilution 1:5,000 were considered positive as none of the control serum specimens showed reactivity at that dilution. To evaluate assay specificity, we tested 19 control serum specimens comprising 12 randomly selected bat serum specimens that were negative by ELISA, 2 SARS-CoV--positive human serum specimens, and 5 SARS-CoV-negative human serum specimens, including 4 HCoV-NL63-positive serum specimens. A commercial indirect immunofluorescence test (SARS-CoV-IFTII kit, EUROIMMUN AG) was carried out as described by the manufacturer, except that bat serum samples were diluted 1:100, and slides were incubated at room temperature for 2 hours. Reactions were detected with goat-antibat immunogolublin (Ig) (Bethyl) at a dilution of 1:1,000 and fluorescein fluorescein /flu·o·res·ce·in/ (fldbobr-res´en) a fluorescing dye; its sodium salt is used as a tracer in retinal angiography and as a diagnostic aid for revealing corneal trauma and fitting contact lenses. isothiocyanate-labeled donkey-antigoat Ig (Dianova, Hamburg, Germany) at a dilution of 1:100. Specificity of the indirect immunofluorescence test (IIFT IIFT Indian Institute of Foreign Trade IIFT Indian Institute of Fashion Technology (Bangalore) ) was determined by screening 572 human serum specimens. The sensitivity and correlation of lIFT versus ELISA were analyzed (online Technical Appendix). In addition, the 19 selected control serum samples were tested. Virus neutralization tests were performed as described elsewhere (13) except for using Vero E6 cells cultured in Dulbecco's modified Eagle medium and SARS-CoV Hong Kong isolate 6109 (3.25 x l07 PFU/mL, diluted 1:5,000). Bat serum dilutions in quadruplicate quad·ru·pli·cate adj. 1. Multiplied by four; quadruple. 2. Fourth in a group of four identical things. n. One of a group of four identical things. tr. & intr.v. ranged from 1:10 to 1:320. After incubation at 37[degrees]C in 5% C[Os.ub.2] for 3 days, the cells were fixed with 8% formaldehyde and results interpreted as described (13). Viral RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was extracted from serum by using a QIAamp viral RNA extraction kit (QIAGEN, Hilden, Germany), and reverse transcription--PCR (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) was performed essentially as described elsewhere (14), with the exception that 140 [micro]L was not available from every bat. In such cases, input volume was reduced and replaced with water. A minimum of 20 [micro]L was usually tested. Antibody activity to SARS-CoV antigen was detected by ELISA in 7 of 26 bat species tested at both collection sites with a seroprevalence of 6.7% (47/705). The highest prevalences were found in the fruit bat Rousettus aegyptiacus (Chiroptera: Pteropodidae) (16.4%) and the insectivorous bat Mops condylurus (Chiroptera: Molossidae) (12.2%) (Table). ELISA titers ranged from 50 (73% of the serum samples) to 800. Confirmatory WB analyses performed by 2 methods on ELISA-positive samples for which sufficient material remained available, were positive in 36 (97.3%) of 37 serum specimens, but IIFT was positive in only 12 (32.4%) of 37 samples (Table; Figures 1, 2; Figure in online Technical Appendix). None of the assays used detected antibodies to other human pathogenic coronaviruses (online Technical Appendix; Figures 1, 2). Neutralizing activity to SARS-CoV was not found in any of the ELISA-positive samples, and RT-PCR did not detect CoV nucleic acid in 262 serum specimens tested (data not shown). [FIGURES 1-2 OMITTED] Conclusions The results of WB analyses support the specificity of the ELISA used in this study. The IF test is known to be less sensitive than ELISA but still provided confirmation in one third of the serum specimens tested. The negative results in the viral neutralization tests are not unexpected because this assay detects only antibodies that interfere with the specific entry mechanism of SARS-CoV, and putative group 4 CoVs from African bats may not use it. Moreover, deletions and mutations found in Asian bat SARS-like--CoV isolates lie in the S protein region essential for binding of SARS-CoV to the cellular receptor, angiotensin-converting enzyme 2, and thus are likely to affect cross-neutralization, as emphasized by conflicting results obtained in Asia (6,7,15). The negative findings obtained in RT-PCR can be explained by the unlikelihood of finding virus nucleic acid in serum. Studies in Asia used rectal swabs instead of serum samples, and the virus likely persists in the enteric enteric /en·ter·ic/ (en-ter´ik) within or pertaining to the small intestine. en·ter·ic adj. 1. Of, relating to, or within the intestine. 2. tract but may not be found in serum at all. Both bat species (R. aegyptiacus and M. condylurus) are widely distributed in Africa but vary in the degree of contact with humans. R. aegyptiacus roosts in caves but forages in orchards, whereas M. condylurus roosts in buildings. The results of this preliminary study suggest that some of the African bat species harbor agents related to putative group 4 CoV, and therefore further investigations should be undertaken to determine potential public health risks. Acknowledgments We thank A. Teichmann for excellent technical assistance. We also thank Lia van der Hoek for providing us with HCoV-NL63-positive serum specimens. References (1.) Chan-Yeung M, Xu RH. SARS: epidemiology. Respirology. 2003;8(Suppl):S9-14. (2.) Peiris JS, Lai ST, Poon poon n. Any of several trees of the genus Calophyllum, of southern Asia, having light hard wood used for masts and spars. [Sinhalese p LL, Guan guan: see curassow. Y, Yam LY, Lim W, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. 2003;361:1319-25. (3.) Drosten C, Gunther S, Preiser W, van der Weft S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003;348:1967-76. (4.) Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348:1953-66. (5.) Guan Y, Zheng B J, He YQ, Liu XL, Zhuang ZX, Cheung CL, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science. 2003;302:276-8. (6.) Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science. 2005;310:676-9. (7.) Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A. 2005; 102:14040-5. (8.) Tang XC, Zhang JX, Zhang SY, Wang P, Fan XH, Li LF, et al. Prevalence and genetic diversity of coronaviruses in bats from China. J Virol. 2006;80:7481-90. (9.) Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19:531-45. (10.) Wang LF, Shi Z, Zhang S, Field H, Daszak P, Eaton BT. Review of bats and SARS. Emerg Infect Dis. 2006;12:1834-40. (11.) Hofmann H, Pyrc K, van der Hock L, Geier M, Berkhout B, Pohlmann S. Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proe Natl Acad Sci U S A. 2005;102:7988-93. (12.) Oganesyan N, Kim SH, Kim R. On-column protein refolding for crystallization Crystallization The formation of a solid from a solution, melt, vapor, or a different solid phase. Crystallization from solution is an important industrial operation because of the large number of materials marketed as crystalline particles. . J Struct Funct Genomics. 2005;6:177-82. (13.) Niedrig M, Lademann M, Emmerich P, Lafrenz M. Assessment of IgG antibodies against yellow fever virus yellow fever virus n. An arbovirus of the genus Flavivirus that causes yellow fever and is transmitted by mosquitoes. after vaccination with 17D by different assays: neutralization test, haemagglutination Noun 1. haemagglutination - agglutination of red blood cells hemagglutination agglutination - a clumping of bacteria or red cells when held together by antibodies (agglutinins) inhibition test, immunofluorescence Immunofluorescence A technique that uses a fluorochrome to indicate the occurrence of a specific antigen-antibody reaction. The fluorochrome labels either an antigen or an antibody. assay and ELISA. Trop Med Int Health. 1999;4:867-71. (14.) de Souza Luna LK, Heiser V, Regamey N, Panning M, Drexler JF, Mulangu S, et al. Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) and nonfluorescent low-density microarray. J Clin Microbiol. 2007;45:1049-52. (15.) Sui J, Li W, Roberts A, Matthews LJ, Murakami A, Vogel L, et al. Evaluation of human monoclonal antibody 80R for immnnoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants. J Virol. 2005;79:5900-6. Address for correspondence: Marcel A. Mailer, Robert Koch-Institut, Center for Biological Safety, ZBS-1, Nordufer 20, 13353 Berlin, Germany; email: mueller.m.a@gmx.de Marcel A. Muller, * Janusz T. Paweska, ([dagger]) Patricia A. Leman lem·an n. Archaic 1. A sweetheart; a lover. 2. A mistress. [Middle English leofman, lemman : leof, dear (from Old English , ([dagger]) Christian Drosten, ([double dagger]) Klaus Grywna, ([double dagger]) Alan Kemp, ([dagger]) Leo Leo, in astronomy Leo [Lat.,=the lion], northern constellation lying S of Ursa Major and on the ecliptic (apparent path of the sun through the heavens) between Cancer and Virgo; it is one of the constellations of the zodiac. Braack, ([section]) Karen Sonnenberg, ([paragraph]) Matthias Niedrig, * and Robert Swanepoel ([dagger]) * Robert Koch-Institut, Berlin, Germany; ([dagger]) National Institute for Communicable Diseases, Sandringham, South Africa; ([double dagger]) Bernhard Nocht Institute for Tropical Medicine Bernhard Nocht Institute for Tropical Medicine; (Bernhard-Nocht-Institut für Tropenmedizin) is a medical institution based in Hamburg, Germany which is dedicated to research, treatment, training and therapy of tropical and infectious diseases, (including HIV). , Hamburg, Germany; ([section]) Conservation International, Cape Town, South Africa; and ([paragraph]) EUROIMMUN AG, Lubeck, Germany Mr Muller is a scientist at the Center for Biological Safety of the Robert Koch-Institut, Berlin. He has been involved in establishing diagnostic tools for SARS-CoV detection, assisted international quality assurance studies on SARS diagnostics, and worked on SARS-CoV susceptibility studies. Currently he is finishing his PhD thesis on expression analysis of coronavirus structural proteins.
Table. Antibody to SARS-CoV in bat sera collected in 1986-1999 at 4
locations in central and southern Africa *
ELISA: positive/tested (%) [dagger]
Limpopo Mpumalanga Oriental
Province, Province, Province,
SA SA DRC
Fruit bats
Casinycteris argynnis
Eidolon helvum
Epomophorus gambianus 0/4 0/6
Epomophorus wahlbergi 0/2
Epomops franqueti
Hypsignathus monstrosus
Lyssonycteris angolensis 1/16
(6.3)
Myonycteris torquata
Rousettus aegyptiacus 11/29 17/142
(37.9) (12.0)
Insect bats
Chaerephon pumila 0/35 0/18
Hipposideros caffer 0/5 0/9
Hipposideros commersoni 0/16
Miniopterus inflatus 1/34
(2.9)
Miniopterus schreibersi 0/1
Mops condylurus 3/19 11/96
(15.8) (11.5)
Mops midas 0/15
Myotis bocagei 0/1
Nycteris argae 0/1
N. thebaica 0/6
Pipistrellus capensis 0/1
Rhinolophus darlingi 0/1
Rhinolophus landed 0/2
Rhinolophus fumigatus 1/204
(0.5)
Scotophilus borbonicus 0/1
S. dinganii 0/5
Taphozous mauritianus 0/1
Totals 14/128 11/120 20/422
(10.9) (9.2) (4.7)
ELISA: positive/
tested (%) [dagger] WB: IIFT:
positive/ positive/
Bandundu tested tested
Province, [double [double
DRC Total dagger] dagger]
Fruit bats
Casinycteris argynnis 0/3 0/3
Eidolon helvum 0/6 0/6
Epomophorus gambianus 0/10
Epomophorus wahlbergi 0/2
Epomops franqueti 0/5 0/5
Hypsignathus monstrosus 1/11 1/11 1/1 0/1
(9.1) (9.1)
Lyssonycteris angolensis 0/2 1/18 1/1 0/1
(5.6)
Myonycteris torquata 1/7 1/7
(14.3) (14.3)
Rousettus aegyptiacus 28/171 26/26 7/26
(16.4)
Insect bats
Chaerephon pumila 0/1 0/54
Hipposideros caffer 0/15
Hipposideros commersoni 0/16
Miniopterus inflatus 1/34
(2.9)
Miniopterus schreibersi 0/1
Mops condylurus 14/115 8/9 5/9
(12.2)
Mops midas 0/15
Myotis bocagei 0/1
Nycteris argae 0/1
N. thebaica 0/6
Pipistrellus capensis 0/1
Rhinolophus darlingi 0/1
Rhinolophus landed 0/2
Rhinolophus fumigatus 1/204
(0.5)
Scotophilus borbonicus 0/1
S. dinganii 0/5
Taphozous mauritianus 0/1
Totals 2/35 47/705 36/37 12/37
(5.7) (6.7)
* SARS-CoV, severe acute respiratory syndrome-associated coronavirus;
SA, South Africa; DRC, Democratic Republic of Congo; WB, Western blot;
IIFT, indirect immunofluorescence test.
([dagger]) Serum specimens were screened for antibody by modification
of a commercially available ELISA kit. Titers ranged from 1:50
to 1:800.
([double dagger]) Confirmatory tests were performed by 2 WB analyses
and IIFT when sufficient sample was available.
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