Puumala Hantavirus Nucleocapsid Research Articles

Human Puumala hantavirus infection in northern Sweden; increased seroprevalence and association to risk and health factors

BackgroundThe rodent borne Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome in central and northern Europe. The number of cases has increased and northern Sweden has experienced large outbreaks in 1998 and 2006–2007 which raised questions regarding the level of immunity in the human population.MethodsA randomly selected population aged between 25 and 74 years from northern Sweden were invited during 2009 to participate in a WHO project for monitoring of trends and determinants in cardiovascular disease. Health and risk factors were evaluated and sera from 1,600 participants were available for analysis for specific PUUV IgG antibodies using a recombinant PUUV nucleocapsid protein ELISA.ResultsThe overall seroprevalence in the investigated population was 13.4 %, which is a 50 % increase compared to a similar study only two decades previously. The prevalence of PUUV IgG increased with age, and among 65–75 years it was 22 %. More men (15.3 %) than women (11.4 %) were seropositive (p < 0.05). The identified risk factors were smoking (OR = 1.67), living in rural areas (OR = 1.92), and owning farmland or forest (OR = 2.44). No associations were found between previous PUUV exposure and chronic lung disease, diabetes, hypertension, renal dysfunction, stroke or myocardial infarction.ConclusionsPUUV is a common infection in northern Sweden and there is a high life time risk to acquire PUUV infection in endemic areas. Certain risk factors as living in rural areas and smoking were identified. Groups with increased risk should be targeted for future vaccination when available, and should also be informed about appropriate protection from rodent secreta.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1879-2) contains supplementary material, which is available to authorized users.

Surface-activated microtiter-plate microarray for simultaneous CRP quantification and viral antibody detection

Microarrays are widely used in high-throughput DNA and RNA hybridization tests and recently adopted to protein and small molecule interaction studies in basic research and diagnostics. Parallel detection of serum antibodies and antigens has several potential applications in epidemiologic research, vaccine development, and in the diagnosis of allergies, autoimmunity, and infectious diseases. This study demonstrates an immobilization method for immunoassay-based microarray in conventional 96-well polystyrene plates for a serologic diagnostic method combined with quantitative C-reactive protein (CRP) assay. A synthetic peptide (HIV-1), a recombinant protein (Puumala hantavirus nucleocapsid), and purified virus preparations (Sindbis and adenoviruses) were used as antigens for virus-specific antibody detection and monoclonal anti-CRP antibody for antigen detection. The microarray was based on conventional enzyme immunoassays and densitometry from photographed results. Peptide and recombinant antigens functioned well, while whole virus antigens gave discrepant results in 1 out of 23 samples from the reference method, tested with human sera with various antibody responses. The CRP results were in concordance in the concentration range 0.5–150 mg/L with 2 commercially available CRP assays: ReaScan rapid test (R2 = 0.9975) and Cobas 6000 analyzer (R2 =0.9595). The results indicate that microtiter plates provide a promising platform for further development of microarrays for parallel antibody and antigen detection.

Hantavirus‐specific IgA in saliva and viral antigen in the parotid gland in patients with hemorrhagic fever with renal syndrome

The Hantavirus genus comprises rodent borne, zoonotic viruses of the Bunyaviridae family that cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Rodent saliva contains infectious hantavirus and evidence suggests that hantavirus is also shed in human saliva, but person-to-person transmission is rare. In saliva, immunoglobulin (Ig) A is the predominant immunoglobulin class. Secretory IgA serves as an important first line of defence on epithelial surfaces and the binding of secretory IgA to pathogens can inhibit adherence of microorganisms to mucosal cells and neutralize viruses. This study investigated the presence and importance of salivary IgA in relation to viral antigen in the saliva by testing Puumala hantavirus (PUUV) specific IgA, and RNA in saliva in acutely ill patients with HFRS. In saliva samples, PUUV specific IgA was detected in 12 of 33 (36%) patients with HFRS and 20 (61%) were PUUV RNA positive. There was a statistically significant inverse association between the presence of salivary IgA antibodies and PUUV RNA in the saliva. PUUV-specific IgA in saliva was not found in a long-term follow-up, while PUUV IgA in serum was detected in three patients, 28-32 months after the initial study. Notably, both PUUV RNA and PUUV nucleocapsid antigen were detected in endothelial cells within the parotid gland of a deceased patient with HFRS.

Detection of Puumala hantavirus antibody with ELISA using a recombinant truncated nucleocapsid protein expressed in Escherichia coli.

A truncated recombinant nucleocapsid protein (rNp118) consisting of the first 118 amino-terminal amino acids (AA) of the Puumala hantavirus (PUUV) nucleocapsid protein expressed in Escherichia coli, was evaluated for its antigenicity and reliability as serodiagnostic antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of PUUV antibodies in human sera. The PUUV nucleocapsid protein has been shown to contain several B-cell epitopes, mapped within the first 118 amino-terminal AA. This finding makes the rNp118 an interesting recombinant protein to use as serodiagnostic antigen. The sensitivity of this new PUUV-rNp118 ELISA, was compared with those of a commercially available PUUV ELISA assay and an home-made ELISA based on a recombinant whole nucleocapsid protein of PUUV. Eighty-six human serum samples clinically suspected for PUUV-induced nephropathia epidemica, and previously screened with the reference assays, were tested. The sensitivity of the new assay was compared with that of the reference assays and an excellent correlation between the assays was found. Sera found to be negative by other methods were also negative in our assay. The ELISA based on rNp118 represents an alternative and valid test for detection of antibodies to PUUV in human sera.

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice.

Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles. Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles. In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced. Immunization of BALB/c mice with chimeric virus-like particles induced a high-titered antibody response against the hantavirus nucleocapsid protein, even in the absence of any adjuvant. The strongest response was observed in mice immunized with virus-like particles harboring 120 amino acids of hantavirus nucleocapsid protein. According to the immunoglobulin subclass distribution of nucleocapsid protein-specific antibodies a mixed Th1/Th2 response was detected. The VP1 carrier itself also induced a mixed Th1/Th2 response, which was found to be reduced in mice immunized with virus-like particles harboring 120 amino acid-long inserts. In conclusion, hamster polyomavirus VP1 represents a promising carrier moiety for future vaccine development.

PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine

The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g. the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated. In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead. Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved. This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein. Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo.

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/Hällnäs into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 μg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAgΔ) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV ‘mosaic particles’ in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAgΔ and the HBcAgΔ/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1–45 and aa 75–119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.

HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein.

Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.

Chimaeric HBV core particles carrying a defined segment of Puumala hantavirus nucleocapsid protein evoke protective immunity in an animal model

Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carries of foreign protective epitopes.