Andrea Normann

Acute hepatitis A virus infection in Turkey.

Anti‐HAV IgM positive serum samples from acute phase hepatitis A patients from various areas in Turkey were tested for viral RNA by RT‐PCR (reverse transcriptase polymerase chain reaction), using primer pairs from two different regions of the HAV genome. The PCR products amplified from both genomic regions underwent phylogenetic analyses. A comparison of the regions showed the same genotyping results, and the RT‐PCR‐2 in the 5′NCR demonstrated greater sensitivity compared to RT‐PCR‐1 in the VP1‐P2A region. The majority of the isolates belonged to genotype IB and are related closely to each other; however, two isolates related even more strongly to the HAV HM175 strain. Two (n = 37) RT‐PCR positive sera were classified under genotype IA. A surprising finding emerged for the mean levels of serum transaminases AST and ALT: higher levels were found in patients under 10 years of age compared to older patients. Anti‐HAV IgM levels were determined quantitatively and, in addition, the HAV‐RNA genome equivalents were ascertained by real time RT‐PCR. No evidence was found for an association between viral load and the higher transaminase levels in the younger group. J. Med. Virol. 80:785–790, 2008.

Sequence Variability of Hepatitis A Virus and Factor VIII Associated Hepatitis A Infections in Hemophilia Patients in Europe: An Update

Outbreaks and sporadic cases of hepatitis A have been observed in 4 European countries in hemophilia patients receiving factor VIII preparations. PCR amplification of potential hepatitis A virus (HAV) nucleic acid present in plasma pools, purified factor VIII and acute‐phase sera from infected individuals has been performed and the nucleic acid sequence determined for those samples that resulted in a positive PCR product. HAV sequences were detected in the serum of 2 German patients, but not in the factor VIII lots administered to these individuals. Screening of plasma pools and the corresponding 5 lots of factor VIII associated with the outbreak in Ireland did not reveal any HAV sequences. In contrast, a study of samples from Italy detected HAV sequences in 5 of 12 lots and in 2 hemophilia patients who developed hepatitis A. These data suggest that implicated factor VIII preparations might have been involved in the outbreaks of HAV infection among Italian hemophiliacs. However, no molecular evidence was obtained for a similar association in Germany or Ireland. The preliminary data from these two investigations must be verified by animal inoculation studies and supported by epidemiologic analysis.

Detection of Hepatitis A Virus in a Factor VIII Preparation by Antigen Capture/PCR

The antigen capture/PCR (AC/PCR) has been applied in the analysis of various factor VIII preparations, which were suspected to be contaminated with hepatitis A virus (HAV). AC/PCR involves capturing the antigen, i.e. the intact virus particles, by binding to the HAV monoclonal antibody mAb 7e7, reverse transcription of the viral RNA and amplification of the cDNA with HAV‐specific primer pairs. The PCR analysis of one factor VIII concentrate yielded an HAV‐specific DNA product, which could be confirmed by Southern blot analysis. The HAV strain recovered by AC/PCR from this factor VIII concentrate could be classified into genotype III after solid‐phase sequencing of the product and comparison with the consensus sequences for the known HAV genotypes. Analysis of the sera from 3 haemophiliacs treated with this batch has not resulted in a reliable product. However, the results obtained indicate that HAV can be detected in purified factor VIII preparations by AC/PCR.

Transmission of Hepatitis A Virus Infection despite Vaccination

To the Editor: In 1989, a killed hepatitis A virus (HAV) vaccine produced from cell culture was described.1 Today, several vaccines are available commercially in countries all over the world. The W...

A new monoclonal antibody (Cox mAB 31A2) detects VP1 protein of coxsackievirus B3 with high sensitivity and specificity.

Human enteroviruses, e.g. coxsackieviruses, induce a variety of severe acute and chronic forms of disease, including myocarditis, meningitis and diabetes mellitus type 1. To visualize enterovirus infection with a diagnostic intent, many studies have applied a commercially available antibody (anti-CVB5 VP1, clone 5-D8/1, Dako, Hamburg, Germany) that identifies VP1 of different enteroviral serotypes. Many antibodies, however, have been found to bind non-specifically to proteins of cardiomyocytes and in the interstitial space, resulting in non-specific staining in immunohistochemistry. In this paper we show that the anti-CVB5 VP1 antibody, recognizing VP1 of coxsackieviruses and widely used in diagnostics and research, shows strong cross-reactivity with cellular proteins in the heart (and pancreas) of humans and mice, which calls for a more specific antibody to be used for diagnostic purposes. We observed by Western blot analyses of lysates from human heart tissue samples and HeLa cells two cross-reactive bands when using clone 5-D8/1. Peptide mass fingerprinting (MALDI-TOF) identified these proteins as creatine kinase (B-type) and tubulin, confirming that this mAb detects cellular proteins in addition to viral VP1. In order to overcome the problems of false positive VP1 staining we generated a new highly specific and sensitive monoclonal antibody (Cox mAB 31A2) that recognizes VP1 from CVB3. The new antibody was characterized and was found to function well in immunohistochemistry, immunofluorescence staining, Western blotting, ELISA and FACS analyses.