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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.

Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic “a” determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV genotypes. Recombinant scFv consisting of immunoglobulin VH and VL regions joined by a 20 aa-long linker was generated by cloning the respective cDNA sequences from hybridoma HB1. The recombinant scFv generated in Escherichia coli recognized the same epitope as the parental MAb HB1. Cloning of HB1 VH and VL regions allowed determination of their primary structure and subsequent computer modeling of antibody–epitope interaction. The generated molecular models of HB1 variable region with its target peptides were in accordance with experimental data showing the importance of certain aa residues in antibody binding. In conclusion, the current study describes new HBsAg-specific antibodies with HBV-neutralizing potency and a broad cross-reactivity against different HBV strains. The generated MAb HB1 will be of great value in diagnostic and research settings, while the recombinant HB1-derived scFv represents a promising “building block” for producing anti-HBV tools with a potential biopharmaceutical application.

Epigenetic alterations of human papillomavirus (HPV) genome play an important role in virus life cycle and carcinogenic progression. The aim of the current study was to investigate the correlation between the grade of cervical pathology and DNA methylation status within the L1 gene and the long control region (LCR) of HPV16, HPV18 and HPV51. HPV genomes were analyzed using bisulfite DNA modification procedure with the subsequent amplification of target DNA regions and sequencing. A collection of 202 cervical specimens was analyzed: 157 HPV16-positive specimens, 21 HPV18-positive specimens and 24 HPV51-positive specimens. This study revealed that methylation of CpG was significantly more prevalent in L1 gene as compared to LCR region of all three studied HPV types and the degree of DNA methylation level correlated with the severity of cervical neoplasia. An increased DNA methylation level of HPV16 promoter region in case of cervical cancer was determined.

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400–519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein. Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter. High levels of rMVN (20 mg/litre of yeast culture) were generated. Electron microscopy showed that the purified rMVN assembled into typical herring-bone structures. Monoclonal antibodies produced to the rMVN also reacted with native measles virus N in immunofluorescence tests. The purified rMVN and a monoclonal antibody to the rMVN conjugated to horseradish peroxidase were used to develop a measles specific IgM capture EIA (MACEIA) in both serum and oral fluid specimens. Evaluations of the MACEIA were performed by testing a) serum samples (n=80) and b) paired oral fluid/serum samples from measles cases (n=50, representing 16 cases) and oral fluids from controls with non-measles rash (n=59, representing 48 cases). The samples were also tested for measles IgM, using a reference radioimmunoassay (MACRIA). The sensitivity and specificity of the MACEIA compared with MACRIA for a) the serum samples were 100 and 96.6% respectively and b) for paired serum/oral fluids samples 100 and 100%, respectively.

The expression of mumps virus nucleocapsid protein in yeast Pichia pastoris was investigated. Viral nucleocapsid proteins usually elicit a strong long-term humoral immune response in patients and experimental animals. Therefore, the detection of antibodies specific to mumps virus nucleoprotein can play an important role in immunoassays for mumps diagnosis. For producing a high-level of recombinant mumps virus nucleoprotein the expression system of yeast P. pastoris was employed. The recombinant nucleocapsid protein was purified by cesium chloride ultracentrifugation of yeast lysates. Electron microscopy of the purified recombinant nucleocapsid protein revealed a herring-bone structure similar to the one discovered in mammalian cells infected with mumps virus. The yield of purified nucleocapsid-like particles from P. pastoris constituted 2.1 mg per 1 g of wet biomass and was considerably higher in comparison to the other expression systems.