Journal of Medical Virology 38:27-31 (1992)
Prevalence of Serum Antibodies Against Lymphocytic Choriomeningitis Virus in Selected Populations From Two U.S. Cities Charles B. Stephensen, Sharon R. Blount, Robert E. Lanford, Kathryn V. Holmes, Richard J. Montali, Michael E. Fleenor, and Joseph F.E. Shaw Division of International Health, Department of Public Health Sciences, School of Public Health, University of Alabama at Birmingham (C.B.S., S.R.B.) and Bureau of Disease Control, Jefferson County Department of Health (M.E.F., J.F.E.S.), Birmingham, Alabama; Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas (R.E.L.); Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (K.V.H.); Department of Pathology, National Zoological Park, Smithsonian Institution, Washington, D.C. (R.J.M.) An ELISA was developed for measuring serum antibodies against the arenavirus lymphocytic choriomeningitis virus (LCMV) and a closely related isolate termed callitrichid hepatitis virus (CHV). The ELISA was used to test sera from healthy adults and from hepatitis patients. In Birmingham, Alabama, the seropositivity rate for healthy black women was 5.1% (7/138), and the rate for patients with all types of hepatitis or cirrhosis was 4.3% (2/46). In San Antonio, Texas, the seropositivity rate among a clinical series of patients with non-A, non-B hepatitis was 0 (0/20), and the rate among persons rejected from blood donation because of high serum alanine aminotransferase levels was 2.4% (2/82). These results indicate that infection with LCMV or CHV is common in Birmingham but that infection is not associated with hepatitis. 0 1992 Witey-Liss, inc.
antigenic relative of LCMV [Montali et al., 1989; Stephensen et al., 19901. The clinical course and pathologic changes seen in CH are strikingly similar to those of other arenavirus infections, particularly Lassa fever [Stephensen et al., 19911. Since some zoo workers exposed to primates with CH were found to have serum antibodies against CHV when tested with a dot immunoblot assay [Montali et al., 19891, we developed a simpler ELISA to test human and nonhuman primate sera for antibodies against CHV. We tested sera from healthy humans and, in view of the hepatotropic nature of the CHV isolate, sera from individuals with clinical and/or biochemical evidence of hepatitis. I n addition to validating the ELISA by comparing it to previously developed dot and western immunoblot assays [Stephensen et al., 19911, we also tested this assay against the standard immunofluorescent antibody (IFA) test for detecting serum antibodies against LCMV [Lehman-Grube et al., 19791.
KEY WORDS arenavirus, callitrichid, hepatitis
MATERIALS AND METHODS ELISA for Serum Antibody Against CHV Immulon 1plates (Dynatech Laboratories, Chantilly, VA) were coated for 1 h r at room temperature (while shaking) with 100 p.1 of a liver homogenate (diluted in 0.06 M Na,C03, pH 9.6) prepared both from a common marmoset (Cullithrix jucchus) experimentally inoculated with CHV and from a healthy control. Five micrograms of total protein was used to coat each well. The preparation of this antigen has been described [Stephensen et al., 19901. Wells were then blocked in dilution buffer (50 mM Tris, 150 mM NaC1, 1 mM
INTRODUCTION Arenaviruses, which persist in rodent reservoirs, cause several serious zoonotic diseases of humans, most notably Lassa fever and Bolivian and Argentine hemorrhagic fevers [Walker and Murphy, 19871. The viruses which cause these diseases are not found in the United States but lymphocytic choriomeningitis virus (LCMV), which is found in this country, can also cause debilitating infections, ranging in severity from a protracted, flu-like illness to meningitis and encephalitis, to a fatal, hemorrhagic syndrome [Vanzee et al., 1975; Meyer et al., 1960; Smadel et al., 19421. Callitrichid hepatitis (CH) is a recently described disease that occurs in zoo collections of New World primates in the United States [Ramsay et al., 19891. The etiologic agent, callitrichid hepatitis virus (CHV), is a close 8 1992 WILEY-LISS, INC.
Accepted for publication February 12,1992. Address reprint requests to C.B. Stephensen, Division of International Health, Dept. of Public Health Sciences, School of Public Health, Univ. of Alabama at Birmingham, Birmingham, AL 35294.
28
EDTA, 0.05% Tween 20, 2% bovine serum albumin [BSA], 2% normal goat serum [NGS], pH 7.4). The wells were then washed 3 times in wash buffer (dilution buffer without NGS and with only 0.1% BSA) before incubation with sera (diluted in dilution buffer) for 1h r at 24°C with shaking. Wells were then washed 5 times before incubation with goat anti-mouse IgG conjugated with alkaline phosphatase (Kirkegaard and Perry Laboratories, Gaithersburg, MD) a t a final dilution of 0.5 pg/ml. After a final 5 washes, p-nitrophenyl phosphate substrate was added at a concentration of 1 p.g/ml in diethanolamine buffer (0.25 M MgCl,, 10% diethanolamine, pH 9.8). Absorbance was read at 405 nm a t 1h r and 18 hr. Mean absorbance values were calculated from 2 wells coated with CH liver extract (S)and from 2 negative control wells coated with liver extract from a n uninfected animal (N), and results were expressed a s a signal-to-noise ratio (S/N) or as the difference between the 2 values (S-N).
Western Immunoblot and Dot Immunoblot Assays Western and dot immunoblots were carried out exactly a s described [Montali et al., 1989; Stephensen et al., 19901. Antibody binding was visualized using '251-staphylococcal protein A. Sera were preabsorbed against acetone-extracted liver powder from a marmoset without CH to remove reactivity against normal liver antigens. Immunofluorescence Test for Serum Antibody Against LCMV The immunofluorescent antibody (IFA) test reagents (LCMV-infected and uninfected Vero-E6 cell suspensions and human anti-LCMV-positive control serum 700871) were kindly provided by Bertha Farrar, Special Pathogens Branch, Centers for Disease Control (Atlanta, GA). Cell suspensions were spotted onto glass slides, air-dried, and stored a t -70°C until use. Antisera were diluted 1:4, 1:16, and 1:64 in PBS and slides were incubated a t 24°C €or 1 hr. After rinsing twice in PBS and soaking for 10 min in PBS the slides were dried with a hair drier and stained with 1%Evans blue in PBS. They were then incubated for 30 min at room temperature with a 1:20 dilution of goat anti-human IgG conjugated with FITC at a final concentration of 25 pg/ml (Kirkegaard and Perry Laboratories, Gaithersburg, MD). After washing, as above, coverslips were seated with buffered glycerol and the cells were examined with a Zeiss immunofluorescence microscope. Hepatitis C Serology All sera from hepatitis patients were screened for serum antibody against HCV using both the HCV ELISA Antibody Test System (Ortho) and a n ELISA using peptides from the NS4 protein of HCV. Sera and MAbs The reaction of arenavirus-specific MAbs 33.6 and 1-1.3 with CHV have been described [Montali et al.,
Stephensen et al. 19891. These MAbs were kindly provided by Michael Buchmeier, Research Institute of Scripps Clinic (La Jolla, CA). Sera from marmosets and tamarins with CH and from 2 veterinarians (E.E. and L.L.) who cared for marmosets with CH and who were found to be seropositive by dot immunoblot and western immunoblot assays were used as positive controls in developing the ELISA. Sera from uninfected callitrichids [Stephensen et al., 19901 and from several humans not exposed to callitrichids with CH were used as negative controls. A mouse polyclonal anti-LCMV ascitic fluid was purchased from Microbiological Associates (Bethesda,MD) and a human anti-LCMV serum was provided with the IFA reagents, a s described above. Four sets of human sera were tested; 1) 138 sera from healthy, black women recruited from among UAB employees and local community groups for use in a study concerning zinc nutriture and hypertension (mean age, 32 years); 2) 24 sera collected from patients with non-A, non-B hepatitis during 1988-1989 by the Jefferson County Department of Health a s part of their hepatitis surveillance program; 3) 82 serum samples from units of blood rejected in San Antonio, Texas due to high alanine aminotransferase (ALT) levels; and 4) 20 sera from a series of patients seen in San Antonio with acute non-A, non-B hepatitis.
RESULTS Liver extracts from the experimental CH case were examined by western immunoblot analysis using MAbs specific for the arenavirus nucleocapsid protein and the GP2 surface glycoprotein. Both antigens were found in abundance (data not shown). This extract was used a s the CHV antigen precoat in both the dot immunoblot assay and the ELISA. No arenaviral antigens were detected with these MAbs in the extract of uninfected marmoset liver, which was used a s a negative control to detect binding of antibodies to normal liver antigens. Seventeen human sera were screened by dot immunoblot analysis to determine if they reacted with CHV antigens found in the livers of infected marmosets (Fig. 1).Two individuals who cared for callitrichids with CH were found to be seropositive, while the remaining 15 individuals without such exposure were found to be seronegative. The two positive sera also reacted with CHV antigens (principally the nucleocapsid protein) by western immunoblot analysis (data not shown). Sera from the 2 seropositive individuals and from 9 seronegative individuals (the 6 remaining sera screened by dot immunoblot were not available), and from 6 callitrichids previously shown to be seropositive and 5 shown to be seronegative by the same methods I Stephensen et al., 19901 were tested in the ELISA. Positive sera typically gave a high absorbance in the wells precoated with CH liver and much lower levels in the negative control wells (Fig. 2). Positive reactions were typically seen a t dilutions in excess of 1:1,600. ELISA results from all 8 positive and 14 negative sera reveal that all of the negative samples had SIN ratios s 1 . 5 and S-N values of
Prevalence of Serum Antibodies Against Lymphocytic Choriomeningitis Virus in Selected Populations From Two U.S. Cities Charles B. Stephensen, Sharon R. Blount, Robert E. Lanford, Kathryn V. Holmes, Richard J. Montali, Michael E. Fleenor, and Joseph F.E. Shaw Division of International Health, Department of Public Health Sciences, School of Public Health, University of Alabama at Birmingham (C.B.S., S.R.B.) and Bureau of Disease Control, Jefferson County Department of Health (M.E.F., J.F.E.S.), Birmingham, Alabama; Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas (R.E.L.); Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (K.V.H.); Department of Pathology, National Zoological Park, Smithsonian Institution, Washington, D.C. (R.J.M.) An ELISA was developed for measuring serum antibodies against the arenavirus lymphocytic choriomeningitis virus (LCMV) and a closely related isolate termed callitrichid hepatitis virus (CHV). The ELISA was used to test sera from healthy adults and from hepatitis patients. In Birmingham, Alabama, the seropositivity rate for healthy black women was 5.1% (7/138), and the rate for patients with all types of hepatitis or cirrhosis was 4.3% (2/46). In San Antonio, Texas, the seropositivity rate among a clinical series of patients with non-A, non-B hepatitis was 0 (0/20), and the rate among persons rejected from blood donation because of high serum alanine aminotransferase levels was 2.4% (2/82). These results indicate that infection with LCMV or CHV is common in Birmingham but that infection is not associated with hepatitis. 0 1992 Witey-Liss, inc.
antigenic relative of LCMV [Montali et al., 1989; Stephensen et al., 19901. The clinical course and pathologic changes seen in CH are strikingly similar to those of other arenavirus infections, particularly Lassa fever [Stephensen et al., 19911. Since some zoo workers exposed to primates with CH were found to have serum antibodies against CHV when tested with a dot immunoblot assay [Montali et al., 19891, we developed a simpler ELISA to test human and nonhuman primate sera for antibodies against CHV. We tested sera from healthy humans and, in view of the hepatotropic nature of the CHV isolate, sera from individuals with clinical and/or biochemical evidence of hepatitis. I n addition to validating the ELISA by comparing it to previously developed dot and western immunoblot assays [Stephensen et al., 19911, we also tested this assay against the standard immunofluorescent antibody (IFA) test for detecting serum antibodies against LCMV [Lehman-Grube et al., 19791.
KEY WORDS arenavirus, callitrichid, hepatitis
MATERIALS AND METHODS ELISA for Serum Antibody Against CHV Immulon 1plates (Dynatech Laboratories, Chantilly, VA) were coated for 1 h r at room temperature (while shaking) with 100 p.1 of a liver homogenate (diluted in 0.06 M Na,C03, pH 9.6) prepared both from a common marmoset (Cullithrix jucchus) experimentally inoculated with CHV and from a healthy control. Five micrograms of total protein was used to coat each well. The preparation of this antigen has been described [Stephensen et al., 19901. Wells were then blocked in dilution buffer (50 mM Tris, 150 mM NaC1, 1 mM
INTRODUCTION Arenaviruses, which persist in rodent reservoirs, cause several serious zoonotic diseases of humans, most notably Lassa fever and Bolivian and Argentine hemorrhagic fevers [Walker and Murphy, 19871. The viruses which cause these diseases are not found in the United States but lymphocytic choriomeningitis virus (LCMV), which is found in this country, can also cause debilitating infections, ranging in severity from a protracted, flu-like illness to meningitis and encephalitis, to a fatal, hemorrhagic syndrome [Vanzee et al., 1975; Meyer et al., 1960; Smadel et al., 19421. Callitrichid hepatitis (CH) is a recently described disease that occurs in zoo collections of New World primates in the United States [Ramsay et al., 19891. The etiologic agent, callitrichid hepatitis virus (CHV), is a close 8 1992 WILEY-LISS, INC.
Accepted for publication February 12,1992. Address reprint requests to C.B. Stephensen, Division of International Health, Dept. of Public Health Sciences, School of Public Health, Univ. of Alabama at Birmingham, Birmingham, AL 35294.
28
EDTA, 0.05% Tween 20, 2% bovine serum albumin [BSA], 2% normal goat serum [NGS], pH 7.4). The wells were then washed 3 times in wash buffer (dilution buffer without NGS and with only 0.1% BSA) before incubation with sera (diluted in dilution buffer) for 1h r at 24°C with shaking. Wells were then washed 5 times before incubation with goat anti-mouse IgG conjugated with alkaline phosphatase (Kirkegaard and Perry Laboratories, Gaithersburg, MD) a t a final dilution of 0.5 pg/ml. After a final 5 washes, p-nitrophenyl phosphate substrate was added at a concentration of 1 p.g/ml in diethanolamine buffer (0.25 M MgCl,, 10% diethanolamine, pH 9.8). Absorbance was read at 405 nm a t 1h r and 18 hr. Mean absorbance values were calculated from 2 wells coated with CH liver extract (S)and from 2 negative control wells coated with liver extract from a n uninfected animal (N), and results were expressed a s a signal-to-noise ratio (S/N) or as the difference between the 2 values (S-N).
Western Immunoblot and Dot Immunoblot Assays Western and dot immunoblots were carried out exactly a s described [Montali et al., 1989; Stephensen et al., 19901. Antibody binding was visualized using '251-staphylococcal protein A. Sera were preabsorbed against acetone-extracted liver powder from a marmoset without CH to remove reactivity against normal liver antigens. Immunofluorescence Test for Serum Antibody Against LCMV The immunofluorescent antibody (IFA) test reagents (LCMV-infected and uninfected Vero-E6 cell suspensions and human anti-LCMV-positive control serum 700871) were kindly provided by Bertha Farrar, Special Pathogens Branch, Centers for Disease Control (Atlanta, GA). Cell suspensions were spotted onto glass slides, air-dried, and stored a t -70°C until use. Antisera were diluted 1:4, 1:16, and 1:64 in PBS and slides were incubated a t 24°C €or 1 hr. After rinsing twice in PBS and soaking for 10 min in PBS the slides were dried with a hair drier and stained with 1%Evans blue in PBS. They were then incubated for 30 min at room temperature with a 1:20 dilution of goat anti-human IgG conjugated with FITC at a final concentration of 25 pg/ml (Kirkegaard and Perry Laboratories, Gaithersburg, MD). After washing, as above, coverslips were seated with buffered glycerol and the cells were examined with a Zeiss immunofluorescence microscope. Hepatitis C Serology All sera from hepatitis patients were screened for serum antibody against HCV using both the HCV ELISA Antibody Test System (Ortho) and a n ELISA using peptides from the NS4 protein of HCV. Sera and MAbs The reaction of arenavirus-specific MAbs 33.6 and 1-1.3 with CHV have been described [Montali et al.,
Stephensen et al. 19891. These MAbs were kindly provided by Michael Buchmeier, Research Institute of Scripps Clinic (La Jolla, CA). Sera from marmosets and tamarins with CH and from 2 veterinarians (E.E. and L.L.) who cared for marmosets with CH and who were found to be seropositive by dot immunoblot and western immunoblot assays were used as positive controls in developing the ELISA. Sera from uninfected callitrichids [Stephensen et al., 19901 and from several humans not exposed to callitrichids with CH were used as negative controls. A mouse polyclonal anti-LCMV ascitic fluid was purchased from Microbiological Associates (Bethesda,MD) and a human anti-LCMV serum was provided with the IFA reagents, a s described above. Four sets of human sera were tested; 1) 138 sera from healthy, black women recruited from among UAB employees and local community groups for use in a study concerning zinc nutriture and hypertension (mean age, 32 years); 2) 24 sera collected from patients with non-A, non-B hepatitis during 1988-1989 by the Jefferson County Department of Health a s part of their hepatitis surveillance program; 3) 82 serum samples from units of blood rejected in San Antonio, Texas due to high alanine aminotransferase (ALT) levels; and 4) 20 sera from a series of patients seen in San Antonio with acute non-A, non-B hepatitis.
RESULTS Liver extracts from the experimental CH case were examined by western immunoblot analysis using MAbs specific for the arenavirus nucleocapsid protein and the GP2 surface glycoprotein. Both antigens were found in abundance (data not shown). This extract was used a s the CHV antigen precoat in both the dot immunoblot assay and the ELISA. No arenaviral antigens were detected with these MAbs in the extract of uninfected marmoset liver, which was used a s a negative control to detect binding of antibodies to normal liver antigens. Seventeen human sera were screened by dot immunoblot analysis to determine if they reacted with CHV antigens found in the livers of infected marmosets (Fig. 1).Two individuals who cared for callitrichids with CH were found to be seropositive, while the remaining 15 individuals without such exposure were found to be seronegative. The two positive sera also reacted with CHV antigens (principally the nucleocapsid protein) by western immunoblot analysis (data not shown). Sera from the 2 seropositive individuals and from 9 seronegative individuals (the 6 remaining sera screened by dot immunoblot were not available), and from 6 callitrichids previously shown to be seropositive and 5 shown to be seronegative by the same methods I Stephensen et al., 19901 were tested in the ELISA. Positive sera typically gave a high absorbance in the wells precoated with CH liver and much lower levels in the negative control wells (Fig. 2). Positive reactions were typically seen a t dilutions in excess of 1:1,600. ELISA results from all 8 positive and 14 negative sera reveal that all of the negative samples had SIN ratios s 1 . 5 and S-N values of
