The antibody titres to P. falciparum and Epstein-Barr Virus-associated antigens were assayed in 22 patients with NPC and 43 controls. All, but one patient had antimalarial titres; 14 had titres greater than 80 and 4 patients greater than 640. Compared to controls the mean anti-malarial titre for most age groups were higher in the patients. Those patients with high anti-malarial titres also had high IgA anti-VCA titre, an antibody which has been demonstrated to be diagnostic for NPC. The peak anti-VCA (IgG) and anti-EA (IgG) antibody titres were associated with anti-falciparum titres of 320-640 and 80-160, respectively. The results are discussed in relation to the possible association between malarial infection and etiology of NPC.
The pattern of seroconversion to Epstein-Barr virus (EBV) was determined in 98 Malaysian children aged 2 weeks to 12 years. Maternal IgG antibodies to EBV viral capsid antigen, ranging between 1:10 to 1:160 titer, were found in 70.6 percent of infants less than three months old, and dropped to 26 percent by seven to nine months. Primary infection, as denoted by emergence of EBV-IgM antibody, occurred at 4 to 6 months, and by eight years all children were seropositive. Maternal antibody titers to EBV nuclear antigen were detected in 52.9 percent of infants less than 3 months old, declined to undetectable levels by 4 to 12 months, and then increased to 40 percent by the age of 12 years. The IgA antibody to viral capsid antigen was absent in all but one infant aged one year; the child also had IgG anti-early antigen, The IgG antibody to EBV early antigen were present in 17.7 percent of the infants aged 3 months or less. This seroconversion to EBV in early life explains the absence of infectious mononucleosis in the Malaysian population. The data suggest that a subunit vaccine to protect against EBV-associated diseases, most notably nasopharyngeal carcinoma, commonly observed in Malaysians would have to be administered to infants 6-12 months of age.
Studies were carried out into the immunopathogenesis and laboratory diagnosis of dengue virus infections. Using an experimental system it was shown that cell-mediated immunity (CMI), as measured by delayed-type hypersensitivity (DTH) was induced in mice infected with dengue virus. The nature of the DTH response satisfies most criteria for a classical DTH reaction. In addition, it was also shown that infection with dengue virus causes a transient immunosuppression as measured by the immune response to other, unrelated antigens. With regard to the laboratory diagnosis of dengue infections, it was found that mosquito cells were a sensitive system for the isolation of dengue viruses and that the success of isolation was related to the antibody content of the serum. A new method for the rapid isolation of dengue viruses was also developed involving the intracerebral inoculation of mosquito larvae. By the use of this method viral antigens can be detected as early as 2-3 days after specimen inoculation. The significance of these findings in relation to the immunopathogenesis, prevention and control of disease syndromes due to dengue viruses is discussed.
Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.
The titres of IgA against Epstein-Barr virus, viral capsid antigens and the titres of IgG against early antigen were found to be useful markers for the diagnosis of different histopathological types of nasopharyngeal carcinoma.
Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.
Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared. They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus. A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin. All the MAbs were also tested against representative strains of BVDV and border disease virus. Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses. Among the other HCV MAbs geographical variation in reaction patterns was observed. There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.
The ability of passage in HeLa cells to attenuate flaviviruses was investigated for three different strains of the mosquito-borne West Nile (WN) virus and two tick-borne viruses, louping-ill and Langat. One strain of WN virus, Sarawak, was attenuated 4000-fold for adult mice by intraperitoneal or intranasal challenge after six HeLa passages. The HeLa-passaged virus was also found to be antigenically different and temperature-sensitive in its growth characteristics compared with the parent. After six HeLa cell passages the Egypt 101 and Smithburn strains of WN virus lost their ability to infect monkey kidney cells and no longer killed adult mice, although inoculated animals became sick for several days. In contrast, two tick-borne flaviviruses remained as virulent for mice after six HeLa passages as the parent non-HeLa-passaged virus. Neither of the tick-borne viruses exhibited characteristics associated with temperature sensitivity. The results, therefore, indicate that the mosquito-borne, but not tick-borne, flaviviruses can be attenuated by very few passages in HeLa cells. This observation may provide a model system with which to analyse the molecular basis of attenuation and/or virulence of mosquito-borne flaviviruses.
150 specimens from suspected herpes simplex genital and skin lesions were received in virus transport medium. They were inoculated into Hep-2 cell monolayers, examined for the presence of virus by cytopathic effect (CPE), direct immunoperoxidase (DIP) and direct immunofluorescence (DIF). Of 39 (26.0%) virus-positive specimens by CPE, 37 (24.7%) were HSV-positive by DIP and 36 (24.0%) by DIF staining. DIP staining had a sensitivity of 100%, specificity of 99.1%, positive predictive value of 97.3% and negative predictive value of 100% in relation to DIF as a standard test. Of 39 specimens positive by CPE, only 25.6% were HSV-positive within 24 h post-inoculation compared to 94% HSV-positive by DIP and DIF staining at the same time.
The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.
A total of 234 sera from healthy Malaysians of diverse ethnic origins were tested for antibody to the Z29 and prototype GS strains of HHV-6. The prevalence in the races ranged from 58 to 80% for the GS strain and 49 to 76% for the Z29 strain. The highest prevalence was in Malays with semi-urban cultural lifestyles and lowest was in the indigenous rural tribes (Ibans, Kadazans, Bidayuhs, and Orang Asli). The antibody titres to GS and Z29 virus capsid antigens differed in 11 (4.7%) samples by more than 2 dilutions. In 9 of the 11 sera the titres to GS strain were higher than to the Z29 strain. The differences in the antibody titres between strains of HHV-6 may reflect subtle changes in antigen structure of the virus recognised by some individuals.
A total of 44 patients suspected of streptococcal infections were studied in outpatient clinics in Tokyo during the one year from December 1988 to December 1989. Employing bacteriological culturing and serodiagnosis, the following results were obtained. 1) There were 9 cases of impetigo and 15 cases of erysipelas with typical clinical manifestations and age distributions. 2) It seemed that some of the skin infections were caused by group A streptococci whose M-types were different from those of upper respiratory infections typically occurring in Japan. 3) The type distribution of group A streptococci found were quite similar to those isolated in Thailand or Malaysia. 4) There were found group A streptococci exhibiting unique combinations of T- and M-types, such as T11 and M9, T11 and M62 or T13-49 and MOD8 (Provisional type). 5) As for serodiagnostic method, ADNB (anti-deoxyribonuclease B) titer reflected infection by group A streptococcus only, while ASK (anti-streptokinase) and ASO (anti-streptolysin O) reflected not only group A streptococcal infections but group G infections as well.
The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.
An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.
The distribution of rotavirus G (VP7) serotypes circulating in four locations in Malaysia, representing three geographical areas, was evaluated in 341 RNA-positive stool specimens obtained discontinuously between 1977 and 1988 from infants and young children under the age of five years who were hospitalized with acute gastroenteritis. A total of 306 specimens (256 stool suspensions and 50 that were adapted to growth in tissue culture) that were rotavirus positive by the confirmatory enzyme-linked immunosorbent assay (ELISA) were examined for serotype by ELISA utilizing monoclonal antibodies to rotavirus G serotype 1, 2, 3, 4, or 9. One hundred eighty (59%) of the 306 specimens could be serotyped; of these 180 specimens, 71% were serotype 4, 15% were serotype 1, 4% were serotype 2, and 4% were serotype 3. Serotype 9 rotavirus was not detected. Most (71%) of the specimens tested were obtained in 1988, when serotype 4 predominated in three locations in West Malaysia; no single serotype was predominant in a limited number of specimens from East Malaysia.
Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.
The BamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzyme-linked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.
A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) was developed for the fast-growing and cytopathic cell culture-adapted hepatitis A virus (HAV) strain HM175A.2. Infectivity titration by EIA correlated well with titration by cytopathic effects. The reliability of this assay was demonstrated by close agreement in virus infectivity titers among different assays of the same virus aliquot and between assays of different virus aliquots. HAV infected cell cultures after fixation could be stored for up to 1 week before testing without decline in virus titer.