Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses.

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.

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen.

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.

Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

Generation in yeast and antigenic characterization of hepatitis E virus capsid protein virus-like particles.

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.

Yeast-generated virus-like particles as antigens for detection of human bocavirus 1-4 specific antibodies in human serum.

Human bocaviruses (HBoV) are non-enveloped, single-stranded DNA viruses, classified into the genus Bocavirus in the family Parvoviridae. Self-assembled virus-like particles (VLPs) composed of the major capsid protein VP2 of HBoV1-4 and mosaic VLPs composed of both VP2 and VP1 capsid proteins of HBoV1 were generated in yeast Saccharomyces cerevisiae and used to detect HBoV-specific IgG in human serum. Recombinant HBoV VLPs were similar to native HBoV particles in size and morphology. The prevalence of HBoV infection in a group of Lithuanian patients with clinical symptoms of respiratory tract infection was studied using purified yeast-generated VLPs as antigens in a competitive enzyme immunoassay (EIA). After depletion of cross-reactive antibodies, the seroprevalence of HBoV1 was 44.2 % and the seroprevalence of HBoV2-4 was 35.7 %. Mosaic VLPs consisting of HBoV1 VP1 and VP2 proteins showed a stronger reactivity with HBoV1 IgG-positive human serum specimens, and two equivocal serum specimens were reinterpreted as positive. Thus, mosaic VLPs offer a more sensitive tool for HBoV1 serology than currently available serodiagnostics tests based on VP2 VLPs. In conclusion, yeast S. cerevisiae represents an efficient expression system for generating recombinant HBoV1-4 VLPs of diagnostic relevance.

Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines.

BACKGROUND: Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. Enzyme-linked immunosorbent assays (ELISA) are commercially available for detection of SBV-specific antibodies in bovine sera and milk. Here we describe the development and evaluation of an indirect ELISA based on a yeast derived recombinant SBV nucleocapsid protein (N) for the detection of SBV-specific antibodies in bovine saliva. Development of a non-invasive test to detect antibodies in individual bovine saliva samples could potentially provide a test suitable for calves and adult cattle. The aim of this study was to investigate the agreement between the levels of antibodies (IgG) measured in milk and sera, and the level of antibodies (IgG and IgA) in saliva, in comparison with the antibody levels detected in sera and milk with commercially available test. RESULTS: Serum, milk and saliva samples from 58 cows were collected from three dairy herds in Lithuania and tested for the presence of SBV-specific antibodies. The presence of IgG antibodies was tested in parallel serum and milk samples, while the presence of IgA and IgG antibodies was tested in saliva samples. The presence of SBV-specific IgG and IgA in saliva was tested using an indirect ELISA based on a yeast-derived recombinant N protein. The presence of SBV-specific IgG in milk and sera was tested in parallel using a commercial recombinant protein based test. The sensitivities of the newly developed tests were as follows: 96 % for the IgG serum assay and 94 % for the IgG milk assay and 85 % and 98 % for IgG and IgA in saliva tests, when compared with data generated by a commercial IgG assay. CONCLUSIONS: Data from testing the saliva IgG and IgA and also the milk and serum IgG with indirect SBV-specific ELISAs showed close agreement with the commercial serum and milk IgG assay data. The level of IgG in saliva was notably lower in comparison to IgA. The newly developed method exhibits the potential to serve as an easily transferable tool for epidemiological studies.

Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

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.

Immunogenicity of novel vB_EcoS_NBD2 bacteriophage-originated nanotubes as a carrier for peptide-based vaccines.

Non-infectious virus-like nanoparticles mimic native virus structures and can be modified by inserting foreign protein fragments, making them immunogenic tools for antigen presentation. This study investigated, for the first time, the immunogenicity of long and flexible polytubes formed by yeast-expressed tail tube protein gp39 of bacteriophage vB_EcoS_NBD2 and evaluated their ability to elicit an immune response against the inserted protein fragments. Protein gp39-based polytubes induced humoral immune response in mice, even without the use of adjuvant. Bioinformatics analysis guided the selection of protein fragments from Acinetobacter baumannii for insertion into the C-terminus of gp39. Chimeric polytubes, displaying 28-amino acid long OmpA protein fragment, induced IgG response against OmpA protein fragment in immunized mice. These polytubes demonstrated their effectiveness both as antigen carrier and an adjuvant, when the OmpA fragments were either displayed on chimeric polytubes or used alongside with the unmodified polytubes. Our findings expand the potential applications of long and flexible polytubes, contributing to the development of novel antigen carriers with improved immunogenicity and antigen presentation capabilities.

Immunological comparison of recombinant shrimp allergen Pen m 4, produced in Pichia pastoris and Escherichia coli.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3-2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.

Characterization of a Panel of Cross-Reactive Hantavirus Nucleocapsid Protein-Specific Monoclonal Antibodies.

Hantaviruses are emerging pathogens with a worldwide distribution that can cause life-threatening diseases in humans. Monoclonal antibodies (MAbs) against hantavirus nucleocapsid (N) proteins are important tools in virus diagnostics, epidemiological studies and basic research studies on virus replication and pathogenesis. Here, we extend the collection of previously generated MAbs raised against a segment of Puumala orthohantavirus (PUUV) N protein harbored on virus-like particles (VLPs) and MAbs against N proteins of Sin Nombre orthohantavirus/Andes orthohantavirus by generating nine novel MAbs against N proteins of Dobrava-Belgrade orthohantavirus (DOBV), Tula orthohantavirus (TULV), Thottapalayam thottimvirus (TPMV) and PUUV. In order to have a wide collection of well-described hantavirus-specific MAbs, the cross-reactivity of novel and previously generated MAbs was determined against N proteins of 15 rodent- and shrew-borne hantaviruses by different immunological methods. We found that all MAbs, excluding TPMV-specific MAbs, demonstrated different cross-reactivity patterns with N proteins of hantaviruses and recognized native viral antigens in infected mammalian cells. This well-characterized collection of cross-reactive hantavirus-specific MAbs has a potential application in various fields of hantavirus research, diagnostics and therapy.

Pet Rats as the Likely Reservoir for Human Seoul Orthohantavirus Infection.

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient's rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007-2020) as well as 594 wild rats from the Netherlands (2013-2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

Seroprevalence study in forestry workers of a non-endemic region in eastern Germany reveals infections by Tula and Dobrava-Belgrade hantaviruses.

Highly endemic and outbreak regions for human hantavirus infections are located in the southern, southeastern, and western parts of Germany. The dominant hantavirus is the bank vole transmitted Puumala virus (PUUV). In the eastern part of Germany, previous investigations revealed Tula virus (TULV) and Dobrava-Belgrade virus (DOBV) infections in the respective rodent reservoirs. Here, we describe a seroprevalence study in forestry workers from Brandenburg, eastern Germany, using IgG ELISA and immunoblot tests based on recombinant TULV, DOBV, and PUUV antigens. Out of the 563 sera tested, 499 from male and 64 from female workers, we found 41 out of the 499 (8.2%) sera from men (mean age 47 years) and 10 out of 64 (15.6%) from the women (mean age 48 years) anti-hantavirus-positive. The majority of the 51 seropositive samples reacted exclusively in the TULV (n=22) and DOBV tests (n=17). Focus reduction neutralization assay investigations on selected sera confirmed the presence of TULV- and DOBV-specific antibodies in the forestry workers. These investigations demonstrated a potential health threat for forestry workers and also the average population in non-endemic geographical regions where TULV and DOBV are circulating in the corresponding reservoir hosts. The infections in this region might be frequently overlooked due to their unspecific and mild symptoms.

Characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples.

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.

Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein.

Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.

Antigenicity study of the yeast-generated human parvovirus 4 (PARV4) virus-like particles.

Human parvovirus 4 (PARV4) is a novel tetraparvovirus that was isolated from intravenous drug users in 2005. Recombinant PARV4 capsid protein VP2 can form stable virus-like particles (VLPs) in yeast. These VLPs could act as antigen carriers during vaccine development. Therefore, the information about PARV4 VP2 VLP antigenic sites could advance further research in this area. In this work, human parvovirus 4 VLPs obtained from yeast were used to generate monoclonal antibodies (mAbs) in mice. Epitope mapping of the obtained mAbs showed at least three distinct antigenic sites of the VP2 protein. On top of that, molecular cloning was used to replace PARV4 VP2 antigenic sites with heterologous peptides. The chimeric PARV4 VLPs bearing polyhistidine inserts obtained from yeast were observed using electron microscopy while polyhistidine-specific antibodies detected heterologous peptides of the chimeric VP2 proteins.

Identification of a novel hantavirus strain in the root vole (Microtus oeconomus) in Lithuania, Eastern Europe.

Hantaviruses are zoonotic pathogens that can cause subclinical to lethal infections in humans. In Europe, five orthohantaviruses are present in rodents: Myodes-associated Puumala orthohantavirus (PUUV), Microtus-associated Tula orthohantavirus, Traemmersee hantavirus (TRAV)/ Tatenale hantavirus (TATV)/ Kielder hantavirus, rat-borne Seoul orthohantavirus, and Apodemus-associated Dobrava-Belgrade orthohantavirus (DOBV). Human PUUV and DOBV infections were detected previously in Lithuania, but the presence of Microtus-associated hantaviruses is not known. For this study we screened 234 Microtus voles, including root voles (Microtus oeconomus), field voles (Microtus agrestis) and common voles (Microtus arvalis) from Lithuania for hantavirus infections. This initial screening was based on reverse transcription-polymerase chain reaction (RT-PCR) targeting the S segment and serological analysis. A novel hantavirus was detected in eight of 79 root voles tentatively named "Rusne virus" according to the capture location and complete genome sequences were determined. In the coding regions of all three genome segments, Rusne virus showed high sequence similarity to TRAV and TATV and clustered with Kielder hantavirus in phylogenetic analyses of partial S and L segment sequences. Pairwise evolutionary distance analysis confirmed Rusne virus as a strain of the species TRAV/TATV. Moreover, we synthesized the entire nucleocapsid (N) protein of Rusne virus in Saccharomyces cerevisiae. We observed cross-reactivity of antibodies raised against other hantaviruses, including PUUV, with this new N protein. ELISA investigation of all 234 voles detected Rusne virus-reactive antibodies exclusively in four of 79 root voles, all being also RNA positive, but not in any other vole species. In conclusion, the detection of Rusne virus RNA in multiple root voles at the same trapping site during three years and its absence in sympatric field voles suggests root voles as the reservoir host of this novel virus. Future investigations should evaluate host association of TRAV, TATV, Kielder virus and the novel Rusne virus and their evolutionary relationships.

vB_EcoS_NBD2 bacteriophage-originated polytubes as a carrier for the presentation of foreign sequences.

Virus-based nanoparticles constitute a promising platform for the creation of efficient vaccines and nanomaterials. Previously we demonstrated, that the recombinant tail tube protein gp39 of vB_EcoS_NBD2 bacteriophage self-assembles into extremely long (from 0.1 to >3.95 µm), flexible, and stable polytubes when produced in Saccharomyces cerevisiae. To develop a tubular platform for multivalent display of foreign antigens, yeast-derived recombinant tail tube protein gp39 was chosen as a scaffold. The carboxy-terminal fusions of gp39 with various antigens up to 238 amino acids in length resulted in different synthesis efficiency and self-assembly capacity. Recombinant gp39 fused with green fluorescent protein (eGFP) comprising 238 amino acid residues was capable to self-assemble into short fluorescent polytubes with retained eGFP functional activity. By demonstrating the display of active foreign antigens on the exterior surface of polytubes, these structures may provide a promising tool for diverse applications in nanotechnology.

Antibodies to Epstein-Barr virus and neurotropic viruses in multiple sclerosis and optic neuritis.

Antibody indices to Measles, Mumps, Varicella Zoster (MRZ) are of diagnostic value in multiple sclerosis (MS). Here, we have investigated, if this panel could be extended to increase diagnostic value. Samples from relapsing-remitting (RR) MS and optic neuritis (ON) patients were tested for reactivity to antigens from Epstein-Barr, Varicella Zoster, Measles, Mumps and Rubella (EMMRZ) viruses. Increased IgG levels in serum and cerebrospinal fluid (CSF) were found in RRMS patients, along with a significant correlation between serum and CSF. The sensitivity of the EMMRZ panel was increased approximately 40% compared to the MRZ panel, suggesting that the EMMRZ panel may be useful in MS and ON diagnostics.

Human Parvoviruses May Affect the Development and Clinical Course of Meningitis and Meningoencephalitis.

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 1-4 (HBoV1-4), 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 HBoV1-4 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 HBoV1-4 and B19V infection should be included in the diagnostics to reduce the number of meningitis/meningoencephalitis with unknown/unexplained etiology.

Self-Assembly of Tail Tube Protein of Bacteriophage vB_EcoS_NBD2 into Extremely Long Polytubes in E. coli and S. cerevisiae.

Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%⁻90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and 0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.