Characterization of anti-idiotypic antibodies mimicking antibody- and receptor-binding sites on hepatitis A virus.

Two anti-idiotypic monoclonal antibodies (mAb2s; named 94-2 and 94-7), were generated from a BALB/c mouse immunized with human monoclonal anti-hepatitis A virus (HAV) neutralizing antibody KF94. We characterized the properties of the mAb2s and determined interactions between mAb2s, KF94 and HAV using enzyme-linked immunosorbent assay, immunofluorescence assay and HAV infectivity assay. Inactivated HAV inhibited mAb2 binding to KF94, indicating that the mAb2s mimicked the HAV neutralization site that was complementary to the paratope of KF94. MAb2 94-7 competed with an anti-HAV cellular receptor antibody for binding to HAV-susceptible cells and partially blocked virus infection. We speculated that mAb2 94-7 mimicked a portion of the HAV receptor-binding site. The ability to generate mAb2 implies that HAV receptor-binding sites are exposed on the surface of HAV, permitting antibody access.

An in-house-anti-hepatitis A virus (HAV)-specific immunoglobulin M capture enzyme-linked immunosorbent assay: evaluation and application to an HAV outbreak.

An anti-hepatitis A virus (HAV)-specific immunoglobulin M capture enzyme-linked immunosorbent assay (anti-HAV IgM ELISA) kit was re-designed for laboratory use and compared with a commercial anti-HAV IgM detection system using 58 serum samples collected from patients, vaccines, and healthy individuals. Because concordance between the two systems was high (r = 0.93, P < 0.05), 19 sets of serum and fecal samples obtained from individuals exposed to an HAV outbreak were also examined. Serum levels of anti-HAV IgM were determined using the in-house ELISA kit and the HAV genome in fecal samples was detected using the polymerase chain reaction (PCR). Among the 19 sets of sample, 14 were positive for both anti-HAV IgM and the HAV genome. All of those whose serum samples were anti-HAV IgM negative were also negative for the HAV genome in fecal samples. The results of the in-house IgM ELISA were consistent with those of the HAV genome detected by PCR and with the commercial IgM ELISA. The in-house anti-HAV IgM ELISA kit was therefore proven suitable for laboratory use and applicable to epidemiological studies of HAV infection.

Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay.

A one-step, single tube, real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting sequences of the untranslated region in the genome of hepatitis A virus (HAV). The RT-LAMP assay reported in this study was very simple and rapid; the HAV-specific amplification was obtained in 50 min under isothermal conditions at 62.5 degrees C by employing a set of seven primers. The RNAs of three cell-adapted HAV strains belonging to different subgenotypes (IA, IB and IIIB) were equally well amplified. The detection limits of the RT-LAMP assay for these HAV strains were 0.4-0.8 focus forming units (FFU)/reaction. The results of the calibration using the WHO international standard indicated that the RT-LAMP assay had similar sensitivity to the conventional RT-PCR method. A comparison of the results from the RT-LAMP and the LightCycler PCR assay using clinical samples in feces revealed that the findings were similar between the two methods. Although several genotypes remain to be tested, it is concluded that the new real-time RT-LAMP assay is very suitable for detection and quantitation of most prevalent genotypes of HAV in diagnostic laboratories.

Shifting seroepidemiology of hepatitis A in Japan, 1973-2003.

BACKGROUND: Hepatitis A infection is caused by hepatitis A virus (HAV) contracted through fecal-oral transmission. Life-long immunity is conferred after infection. Improved sanitary conditions have generally resulted in a significant decline in the incidence of hepatitis A. However, a low incidence of infection results in increased HAV susceptibility. The present study investigates the prevalence of anti-HAV antibody and clarifies the current HAV status and HAV susceptibility in Japan at 2003. METHODS: A total of 2,430 serum specimens collected during 2003 from Japanese individuals ranging in age from 0-92 years, were tested for anti-HAV antibody using an inhibition enzyme linked immunosorbent assay. All specimens were obtained from the WHO and the National Serum Reference Bank/National Institute of Infectious Diseases, Tokyo, Japan. RESULTS: The overall seroprevalence was 12.2%. Anti-HAV antibodies were rarely detected in individuals between 0-44 years of age. Starting from the age of 45-49 years, seropositivity gradually increased through age 65 years and above. Seroprevalence was not affected by gender, and geographic distribution did not affect age-specific seroprevalence until the age of 60 years. CONCLUSIONS: HAV susceptibility in Japan is increasing annually. Particularly, the prevalence of anti-HAV antibody in individuals older than 50 years in 2003 was 50.3%, which is significantly lower than that of corresponding studies in 1994 (74.3%), 1984 (96.9%) and 1973 (96.9%). The growing susceptible population of advanced age results in more frequent HAV infection among them. The surveillance of anti-HAV antibody prevalence is useful for implementing preventive measures and for controlling the spread of HAV.

A Sabin 3-derived poliovirus recombinant contained a sequence homologous with indigenous human enterovirus species C in the viral polymerase coding region.

Outbreaks of poliomyelitis caused by circulating vaccine-derived polioviruses (cVDPVs) have been reported in areas where indigenous wild polioviruses (PVs) were eliminated by vaccination. Most of these cVDPVs contained unidentified sequences in the nonstructural protein coding region which were considered to be derived from human enterovirus species C (HEV-C) by recombination. In this study, we report isolation of a Sabin 3-derived PV recombinant (Cambodia-02) from an acute flaccid paralysis (AFP) case in Cambodia in 2002. We attempted to identify the putative recombination counterpart of Cambodia-02 by sequence analysis of nonpolio enterovirus isolates from AFP cases in Cambodia from 1999 to 2003. Based on the previously estimated evolution rates of PVs, the recombination event resulting in Cambodia-02 was estimated to have occurred within 6 months after the administration of oral PV vaccine (99.3% nucleotide identity in VP1 region). The 2BC and the 3D(pol) coding regions of Cambodia-02 were grouped into the genetic cluster of indigenous coxsackie A virus type 17 (CAV17) (the highest [87.1%] nucleotide identity) and the cluster of indigenous CAV13-CAV18 (the highest [94.9%] nucleotide identity) by the phylogenic analysis of the HEV-C isolates in 2002, respectively. CAV13-CAV18 and CAV17 were the dominant HEV-C serotypes in 2002 but not in 2001 and in 2003. We found a putative recombination between CAV13-CAV18 and CAV17 in the 3CD(pro) coding region of a CAV17 isolate. These results suggested that a part of the 3D(pol) coding region of PV3(Cambodia-02) was derived from a HEV-C strain genetically related to indigenous CAV13-CAV18 strains in 2002 in Cambodia.

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001.

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.

Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993.

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.

Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus.

An outbreak of paralytic poliomyelitis occurred in the Dominican Republic (13 confirmed cases) and Haiti (8 confirmed cases, including 2 fatal cases) during 2000-2001. All but one of the patients were either unvaccinated or incompletely vaccinated children, and cases occurred in communities with very low (7 to 40%) rates of coverage with oral poliovirus vaccine (OPV). The outbreak was associated with the circulation of a derivative of the type 1 OPV strain, probably originating from a single OPV dose given in 1998-1999. The vaccine-derived poliovirus associated with the outbreak had biological properties indistinguishable from those of wild poliovirus.