Detection of Antibodies to the Nonstructural 3C Proteinase of Hepatitis A Virus

Stewart, Deneen R.; Morris, Tina S.; Purcell, Robert H.; Emerson, Suzanne U.

doi:10.1086/514079

Cited by 23 publications

(10 citation statements)

References 15 publications

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“…Antibodies to nonstructural proteins have been detected in humans and experimentally infected chimpanzees but are absent in vaccinated individuals (126,202). However, because of what appears to be a variable host antibody response during HAV replication, a diagnostic test for these antibodies, which could be used to complement current anti-HAV testing and differentiate previously infected from vaccinated persons, has not been developed (201,233).…”

Section: Pathogenesis and Natural History Of Hav Infectionmentioning

confidence: 99%

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

et al. 2006

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SUMMARY Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

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Section: Pathogenesis and Natural History Of Hav Infectionmentioning

confidence: 99%

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

et al. 2006

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“…Furthermore, detection of an immune response mediated against nonstructural proteins that are not present in the virion is suggestive of viral replication in vivo. Previous studies of hepatitis A virus (HAV) infection also suggested the severity of infection is correlated with the development of anti-3C antibodies (5,9). However, these studies also suggest that there is heterogeneity in the development of anti-3C and antistructural protein antibodies.…”

Section: Discussionmentioning

confidence: 98%

“…Klassevirus 3C protease shares only 38% amino acid identity and only one potential 7-mer epitope with its closest relative, the 3C protease of Aichi virus. Previous studies of hepatitis A virus (HAV), a prototypic picornavirus, indicate that antibodies are made against the picornaviral 3C protease during bona fide picornavirus replication and thus can distinguish between vaccinated and actively infected chimpanzees, humans, and tamarins (5,9).…”

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confidence: 99%

Serological Evidence of Human Klassevirus Infection

Greninger

Holtz

Kang

et al. 2010

Clin Vaccine Immunol

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Klassevirus is a proposed new genus of picornavirus that has been associated with pediatric diarrhea. In this study, we used recombinant klassevirus 3C protease as the capture antigen for an indirect serological enzymelinked immunosorbent assay (ELISA). Four of six klassevirus reverse transcription (RT)-PCR-positive individuals demonstrated seroconversion against the 3C protease, suggesting that klassevirus infection and replication occur in humans. Additional screening of 353 samples from an age-banded serological cohort from two St. Louis hospitals indicated a seroprevalence of 6.8%.Klassevirus is the prototype member of a new picornavirus genus that has recently been discovered in human stool (3,4,7). Phylogenetic analysis shows klassevirus is most closely related to Aichi virus, a known cause of oyster-associated gastroenteritis in humans. Molecular detection studies have found klassevirus in pediatric stool and municipal sewage from the United States, Spain, Australia, Tunisia, and Nigeria (3, 4, 7). A small case control study found a statistically significant association between klassevirus and pediatric diarrhea (7). Furthermore, klassevirus RNA has been detected in high titer in pediatric stool (3). However, klassevirus has not yet been detected in any sterile sites in the human body, and thus it is still not clear whether the presence of the virus in stool represents a bona fide human infection.In this study, we established a serological assay for klassevirus infection using recombinant klassevirus 3C protease to demonstrate seroconversion and human infection and to test for seroprevalence in an age-banded pediatric cohort from hospitals in the St. Louis area. Klassevirus 3C protease shares only 38% amino acid identity and only one potential 7-mer epitope with its closest relative, the 3C protease of Aichi virus. Previous studies of hepatitis A virus (HAV), a prototypic picornavirus, indicate that antibodies are made against the picornaviral 3C protease during bona fide picornavirus replication and thus can distinguish between vaccinated and actively infected chimpanzees, humans, and tamarins (5, 9). MATERIALS AND METHODSExpression and purification of 3C protease. The klassevirus 3C protease gene (nucleotides [nt] 5825 to 6409 from strain 2394-01; NC_012986.1) flanked with NdeI and XhoI restriction sites was amplified from stool total RNA by reverse transcription (RT)-PCR with the following primers: klasse3C-NdeI (5Ј-CATAT GGGTTTCGACCCTGCCGTCATGAAG-3Ј and klasse3C-XhoI (5Ј-CTCGAG TCATCACTGAGGTGTGGCCAGGTTAGAGA-3Ј) (restriction sites italicized and stop codons underlined). The resulting product was sequence confirmed, digested with NdeI and XhoI, and subcloned into NdeI/XhoI-digested pET15b (Novagen), which contains a 6ϫHis tag on the N terminus. The sequenceconfirmed pET15b vector containing the klassevirus 3C gene was transformed into Escherichia coli BL21(DE3)LysS/pRIL and recombinant 6ϫHis-klassevirus 3C protein expression was induced with 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at 37°C for 5...

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“…Other studies have found that children and adults naturally infected with hepatitis A virus (HAV) make antibodies to the viral protease and these are detected at least up to 15 months after infection (10,17). This has been useful in distinguishing antibody acquired in response to HAV infection and antibody induced by immunization with an inactivated vaccine.…”

mentioning

confidence: 99%