Cross-reactive monoclonal antibodies against fish parvalbumins as a tool for studying antigenic similarity of different parvalbumins and analysis of fish extracts.

Fish parvalbumins are heat-stable calcium-binding proteins that are highly cross-reactive in causing allergy symptoms in fish-sensitized patients. The reactivities of parvalbumin-specific monoclonal or polyclonal antibodies with parvalbumins of different fish species allowed their application for development of various immunoassays for allergen identification in fish samples. In this study, monoclonal antibodies (MAbs) were generated against two parvalbumins - natural Atlantic cod parvalbumin and recombinant common carp ß-parvalbumin expressed in E. coli. Large collections of recombinant parvalbumins and natural allergen extracts of different fish species and other animals were used to identify the specificities of these MAbs using ELISA, Western blot, and dot blot. MAbs demonstrated different patterns of cross-reactivities with recombinant parvalbumins. Their binding affinities were affected by the addition and removal of Ca2+ ions. Moreover, all MAbs showed a broad reactivity with the target antigens in natural fish, chicken, and pork extracts. The ability of two MAbs (clones 7B2 and 3F6) to identify and isolate native parvalbumins from allergen extracts was confirmed by Western blot. Epitope mapping using recombinant fragments of Atlantic cod parvalbumin (Gad m 1) and common carp parvalbumin (Cyp c 1) revealed that 4 out of 5 MAbs recognize parvalbumin regions that contain calcium binding sites. In conclusion, the generated broadly reactive well-characterized MAbs against fish ß-parvalbumins could be applied for investigation of parvalbumins of fish and other animals and their detection in allergen extracts.

Detection of rat hepatitis E virus, but not human pathogenic hepatitis E virus genotype 1-4 infections in wild rats from Lithuania.

Rat hepatitis E virus (HEV) is an orthohepevirus which is related to other HEV found in humans and other mammals. It was first identified in Norway rats (Rattus norvegicus) from Germany in 2010, and later it has been detected in Black rats (Rattus rattus) and Norway rats from USA, China, Indonesia, Vietnam and many European countries. In this study, we describe molecular and serological investigations of Black and Norway rats trapped in Lithuania, Eastern Europe, for infections with rat HEV and human HEV genotypes 1-4. Rat HEV-specific real-time reverse transcription-PCR (RT-qPCR) analysis of rat liver samples revealed the presence of rat HEV in 9 of 109 (8.3%) samples. In contrast, a RT-qPCR specific for HEV genotypes 1-4 did not reveal any positive samples. A nested broad spectrum RT-PCR was used for a confirmation of rat HEV infection with a subsequent sequencing of the amplified rat HEV genome fragment. Phylogenetic analysis revealed a clustering of all newly identified rat HEV sequences with Norway rat-derived rat HEV sequences from Germany within the species Orthohepevirus C. An indirect ELISA using a yeast-expressed truncated rat HEV capsid protein variant revealed 31.2% seropositive samples indicating a high rate of rat HEV circulation in the rat population examined. In conclusion, the current investigation confirms rat HEV infections in Norway and Black rats in Lithuania, Eastern Europe, and the non-persistent nature of HEV infection.