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Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.

Abstract To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

Meningitis and meningoencephalitis are neurological inflammatory diseases, and although routine diagnostics include testing of a wide range of pathogens, still in many cases, no causative agent is detected. Human parvovirus B19 (B19V), human bocaviruses 1&ndash, 4 (HBoV1&ndash, 4), and human parvovirus 4 (hPARV4) are members of the Parvoviridae family and are associated with a wide range of clinical manifestations including neurological disorders. The main aim of this study was to determine whether human parvoviruses infection markers are present among patients with meningitis/meningoencephalitis in Latvia as well as to clarify the role of these viruses on the clinical course of the mentioned diseases. Our study revealed HBoV1&ndash, 4 and B19V genomic sequences in 52.38% and 16.67% of patients, respectively. Furthermore, symptoms such as the presence of a headache and its severity, fatigue, disorientation, and difficulties to concentrate were significantly frequently present in patients with active parvovirus infection in comparison with parvoviruses negative patients, therefore we suggest that HBoV1&ndash, 4 and B19V infection should be included in the diagnostics to reduce the number of meningitis/meningoencephalitis with unknown/unexplained etiology.

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 E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products and with blood components through transfusions. There are no approved or golden standard serologic systems for HEV diagnostics. Commercially available HEV tests often provide inconsistent results which may differ among the assays. In this study, we describe generation in yeast and characterization of HEV genotype 3 (HEV-3) and rat HEV capsid proteins self-assembled into virus-like particles (VLPs) and the development of HEV-specific monoclonal antibodies (MAbs). Full-length HEV-3 and rat HEV capsid proteins and their truncated variants comprising amino acids (aa) 112-608 were produced in yeast S. cerevisiae. The yeast-expressed rat HEV capsid protein was found to be glycosylated. The full-length HEV-3 capsid protein and both full-length and truncated rat HEV capsid proteins were capable to self-assemble into VLPs. All recombinant proteins contained HEV genotype-specific linear epitopes and cross-reactive conformational epitopes recognized by serum antibodies from HEV-infected reservoir animals. Two panels of MAbs against HEV-3 and rat HEV capsid proteins were generated. Their cross-reactivity pattern was investigated by Western blot, ELISA, and immunofluorescence assay on HEV-3-infected cell cultures. The analysis revealed cross-reactive, genotype-specific, and virus-reactive MAbs. MAb epitopes were localized within S, M, and P domains of HEV-3 and rat HEV capsid proteins. Yeast-generated recombinant VLPs of HEV-3 and rat HEV capsid proteins and HEV-specific MAbs might be employed to develop novel HEV detection systems.

Monoclonal antibodies are important tools for various applications in hantavirus diagnostics. Recently, we generated Puumala virus (PUUV)-reactive monoclonal antibodies (mAbs) by immunisation of mice with chimeric polyomavirus-derived virus-like particles (VLPs) harbouring the 120-amino-acid-long amino-terminal region of the PUUV nucleocapsid (N) protein. Here, we describe the generation of two mAbs by co-immunisation of mice with hexahistidine-tagged full-length N proteins of Sin Nombre virus (SNV) and Andes virus (ANDV), their characterization by different immunoassays and comparison with the previously generated mAbs raised against a segment of PUUV N protein inserted into VLPs. All of the mAbs reacted strongly in ELISA and western blot tests with the antigens used for immunization and cross-reacted to varying extents with N proteins of other hantaviruses. All mAbs raised against a segment of the PUUV N protein presented on chimeric VLPs and both mAbs raised against the full-length AND/SNV N protein reacted with Vero cells infected with different hantaviruses. The reactivity of mAbs with native viral nucleocapsids was also confirmed by their reactivity in immunohistochemistry assays with kidney tissue specimens from experimentally SNV-infected rodents and human heart tissue specimens from hantavirus cardiopulmonary syndrome patients. Therefore, the described mAbs represent useful tools for the immunodetection of hantavirus infection. Fil: Kucinskaite-Kodze, Indre. Vilnius University. Institute of Biotechnology; Lituania. Fil: Petraityte-Burneikiene, Rasa. Vilnius University. Institute of Biotechnology; Lituania. Fil: Zvirbliene, Aurelija. Vilnius University. Institute of Biotechnology; Lituania. Fil: Hjelle, Brian. University of New Mexico School of Medicine. Department of Pathology; Estados Unidos. Fil: Medina, Rafael A. University of New Mexico School of Medicine. Department of Pathology; Estados Unidos. Fil: Gedvilaite, Alma. Vilnius University. Institute of Biotechnology; Lituania. Fil: Razanskiene, Ausra. Vilnius University. Institute of Biotechnology; Lituania. Fil: Schmidt-Chanasit, Jonas. Federal Research Institute for Animal Health. Institute of Epidemiology; Alemania. Fil: Mertens, Marc. Federal Research Institute for Animal Health. Institute of Epidemiology; Alemania. Fil: Padula, Paula. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología; Argentina. Fil: Sasnauskas, Kestutis. Vilnius University. Institute of Biotechnology; Lituania. Fil: Ulrich, Rainer G. Federal Research Institute for Animal Health. Institute of Epidemiology; Alemania.

Background Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production. Results In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test. Conclusions We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.

Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.