key: cord-0010031-98258wy6
authors: Baer, G. M.; Yager, P. A.; Hierholzer, J. C.; Walker, J. A.; Almeida, J. D.
title: Studies of an outbreak of acute hepatitis A: II. Antibody changes to cytomegalovirus and herpesvirus
date: 2005-12-09
journal: J Med Virol
DOI: 10.1002/jmv.1890010103
sha: a53c321e34b1ef94aa87a67febdf94783a2e4c71
doc_id: 10031
cord_uid: 98258wy6

The acute and convalescent sera from 14 schoolchildren with acute hepatitis A were tested for antibody changes to 70 viral antigens. Marked decreases were noted in the levels of antibody to cytomegalovirus in 5 of the 14 children and in the levels of antibody to herpesvirus type 1 in 3. No such changes were noted in 9 sex‐ and age‐matched healthy control children from the same classes.

Compared with the relative paucity of virus-related antibody studies in chronic hepatitis, many studies have shown an association between various respiratory and enetric viruses and outbreaks of acute hepatitis A (Davis, 1961; Liebhaber et al., 1965; Hatch and Siem, 1966; Hartwell et al., 1968; Zuckerman et al., 1968; Hatch and Swanson: 1969; Shulman and Barker, 1969; Berquist et al., 1972; Alwen, 1973) . Moreover, recently Almeida reported seroconversions to several unidentified faecal antigens with virus morphology in patients with infectious hepatitis (Almeida et al., 1974) . The early reporting of an outbreak of hepatitis A in a rural elementary school in Alabama in October and November 1972 permitted us to obtain acute and convalescent blood samples from 50 sick cluldren and 24 healthy matched controls. Briefly, the children affected were between 6 and 12 years old, and the sharp "epidemic curve" suggested a common-source outbreak, one eventually traced to a contaminated water source. A more complete description of the details surrounding the outbreak is given in a companion paper (Baer et al., 1977) . We noted a significant drop in the C'3 and C'4 components of complement in the acute specimens. Selected sera from this same outbreak were tested for possible antibody rises against a variety of viruses and mycoplasma, and the results of that testing are reported here.

We tested paired sera from 14 randomly selected schoolchildren age 6-12 with acute hepatitis A (the definition of a case is given in the companion paper) and from 9 age-and sex-matched healthy children from the same classes. The acute sera were collected approximately one week after the peak of the outbreak (1 1 /9/72), while the convalescent sera were drawn one month later (1 2/12/72).

The sera were tested for antibodies to the prototype strains of a large number of respiratory and enteric viruses by complement-fixation (CF), hemagglutination-inhibition (HI), indirect hemagglutination (IHA), and/or serum neutralization (SN) tests, as appropriate for each particular virus. The sera were heat-inactivated (56"C, 30 min) for all serologic tests. Antibodies to influenza A and B, parainfluenza 1,2, and 3, the "soluble" and "viral" antigens of mumps virus, the group-specific hexon antigen of adenovirus, respiratory syncytial virus (RSV), Mycoplasma pneumoniae, herpes simplex 1, rubella, and cytomegalovirus (CMV) were assayed by the standardized CF test with overnight fixation of 5 units of complement (Casey, 1965) . Antibodies to adenovirus types 1 to 33, influenza C, parainfluenza 4A and 4B, Newcastle disease virus (NDV), Coxsackievirus A-24, and coronavirus OC 43 were assayed by the standardized HI test with 0.01 M phosphate-buffered saline diluent and spectrophotometrically standardized 0.4% mammalian or 0.5% avian red blood cells (Hierholzer and Suggs, 1969; Hierholzer et al., 1969) . test as described by Killgore and Dowdle (1970) . Antibodies to herpes simplex 1 and CMV were determined by the IHA procedures of Bernstein and Stewart (1971a, b) , and coronavirus 229 E antibodies were measured by a similar IHA test (Kaye et al., 1972) . Neutrahzing antibodies t o the six Coxsackie B viruses, echovirus types 4 and 9, and Coxsackievirus A-10 were assayed by SN tests in monkey kidney (MK) tissue culture (Hierholzer et al., 1972) . Neutralizing antibody titers to Coxsackievirus A-9, A-16, and A-21 and t o four strains of enterovirus 70 were similarly assayed by SN tests in human diploid fibroblast (RU-1) tissue Hemagglutinating antigens of parainfluenza 4A and 4B were prepared for use in the HI culture, and to Coxsackievirus A-4 in suckling mice brain (Melnick and Wenner, 1969) . Antibodies to the agent of infectious mononucleosis (Epstein-Barr virus) were measured by the ox cell hemolysin test with 2% erythrocytes (Mason, 1951) .

surface antigen (HBsAg) and antibody (anti-HBs) (Gocke and Howe, 1970) . Sera were tested by counterimmunoelectrophoresis for the presence of hepatitis B

All sera were negative for HBsAg and anti-HBs. Table I shows the variety of serologic tests performed on the selected sera, and the agents or antigens used. No significant increases in antibody titers were noted between the acute and convalescent specimens. Occasionally, low but stable complement-fixing antibody titers were noted for parainfluenza, adenoviruses, mumps, rubella and Coxsackie-viruses, but nothng beyond a twofold rise or fall was observed. It was seen, however, that marked falls (fourfold or greater) against cytomegalovirus and herpesvirus 1 occurred in sera of 5 (35%) and 3 (21%) of the sick children, respectively, whereas no such changes occurred in the matched healthy controls (Table 11 ). There also appeared to be a relationship between the degree of iUness and the antibody changes: Antibody changes were noted in 4 (67%) of the 6 icteric children whose sera we tested. 

The fall in antibody titers to cytomegalovirus and herpesvirus that occurred in some of the hepatitis-A-infected schoolchildren was so dramatic as to demand a plausible explanation. The onset of illness in hepatitis A occurs after the beginning of virus excretion (Dienstag et al., 1975; Ward et al., 1958) . In view of the usual preponderance of active CMV and herpesvirus infections in the young age groups represented here, natural infections probably developed years before this hepatitis A outbreak. It appears, therefore, that the stimulation of the already existing CMV and herpesvirus antibody titers in the affected children occurred a few weeks before the hepatitis symptoms appeared and that these levels were either at their peak or already beginning to fall when our acute samples were drawn. Such an occurrence could account for the titer decreases seen in t h s outbreak of acute hepatitis A in confrast to the titer increases reported in chronic hepatitis (Triger, Kurtz, MacCallum, and Wright, 1972) . diagnosed in the last few years and often involves the liver (Hanshaw et al., 1965) . If temporary liver damage by acute hepatitis A infection stimulates Kupffer cells and liver macrophages, and this causes antibody rises to childhood viruses, CMV would likely be involved. Although the antibody titers to two other primarily childhood viruses, measles and rubella, were reported to be increased in chronic active hepatitis (Triger, Kurtz, MacCallum, and Wright, 1972) no examination for CMV antibody was made in that study; nor are we aware of acute and convalescent sera from acute hepatitis A cases being examined for antibody changes to such a variety of viral antigens as in the present study.

occurrence of CMV antibody in hepatitis B: "A relatively high proportion of patients with type-B transfusion hepatitis, or no hepatitis at all, have also developed antibody to CMV, (Prince et al., 1971; Purcell et al., 1971; Stevens et al., 1970) , and it is difficult at present to evaluate the etiologic significance, if any, of this virus posttransfusion hepatitis." It also should be noted that we found no antibodies against Epstein-Barr virus, an agent recently incriminated in numerous cases of HBsAg-negative hepatitis in a dialysis unit (Corey et al., 1975) .

Out data add more evidence that the critical liver changes that occur during acute hepatitis attacks may alter antibody levels to key antigens. Walker (1972) has reviewed two mechanisms that might affect antibody production: an increased processing of antigen in the proliferated Kupffer cells, which thus triggers antibody synthesis in immunocytes attracted to the liver (Paronetto et al., 1962; Hadziyannis et al., 1969) ; and, as mentioned earlier, a failure of Kupffer cells to sequester antigens absorbed from the gut. In addition, Popper et al. (1965) has shown that liver injury is associated with activation of the various components of the hepatic mesenchyma and some parts of the lymphoid system, and has suggested that this might explain the frequently encountered erratic false-positive Wasserman tests. The antibody rises to enteric bacteria noted by other workers (Triger, Alp, and Wright, 1972; Bjdrneboe et al., 1972; Protell et al., 1971) during chronic hepatitis also suggest a failure in the sequestering ability of the liver.

Sera from other acute hepatitis A outbreaks should be examined to determine whether infectious childhood viruses can actually be recovered. It is again apparent, however, from the findings reported here that various antibody changes not due to the inciting virus may occur in infectious hepatitis A, and that such changes must be placed in proper perspective during the search for the precise etiologic agent of the disease.

Cytomegalovirus infection, common in childhood (Welier, 1970) , has been increasingly Of great interest are the comments recently made by Purcell et al. (1974) on the

Standardized diagnostic complement fixation method and adapatation to micro test

Applied Microbiology 16: 1859. Hatch MH, Siem RA

Proceedings of the Society for Experimental Biology and Medicine

Lennette EH, Schmidt NF (cds): "Diagnostic Procedures for Viral

Transmissible Disease and Blood Transfusion

Eighth Symposium on Advanced Medicine

British Journalof Experimental Pathology 49: 235. Publich Health Monograph #74

Rickettsia1 Infections

The authors wish to acknowledge the assistance of Dr. &chard Levine, Dr. John Bryan, Mr. Bruce Wood, Dr. Harold C. Woodworth, and Mr. Roger Norris in these studies.