Laboratory-based surveillance of COVID-19 in the Greater Helsinki area, Finland, February-June 2020.

OBJECTIVES: The aim was to characterise age- and sex-specific severe acute respiratory syndrome coronavirus disease-2 (SARS-CoV-2) RT-PCR sampling frequency and positivity rate in Greater Helsinki area in Finland during February-June 2020. We also describe the laboratory capacity building for these diagnostics. METHODS: Laboratory registry data for altogether 80,791 specimens from 70,517 individuals was analysed. The data included the date of sampling, sex, age and the SARS-CoV-2 RT-PCR test result on specimens collected between 1 February and 15 June 2020. RESULTS: Altogether, 4057/80,791 (5.0%) of the specimens were positive and 3915/70,517 (5.6%) of the individuals were found positive. In all, 37% of specimens were from male and 67% from female subjects. While the number of positive cases was similar in male and female subjects, the positivity rate was significantly higher in male subjects: 7.5% of male and 4.4% of female subjects tested positive. The highest incidence/100,000 was observed in those aged ≥80 years. The proportion of young adults in positive cases increased in late May 2020. Large dips in testing frequency were observed during every weekend and also during public holidays. CONCLUSIONS: Our data suggest that men pursue SARS-CoV-2 testing less frequently than women. Consequently, a subset of coronavirus disease-2019 infections in men may have gone undetected. People sought testing less frequently on weekends and public holidays, and this may also lead to missing of positive cases. The proportion of young adults in positive cases increased towards the end of the study period, which may suggest their returning back to social behaviour with an increased risk of infection.

Chikungunya virus infections in Finnish travellers 2009-2019.

The mosquito-borne chikungunya virus (CHIKV) causes an acute febrile illness with rash, joint and muscle pain.A realtime RT-PCR assay for CHIKV detecting non-structural protein (nsP2; CHIKV nsP2-RT-qPCR) was set up. All the serodiagnosed CHIKV cases detected during 2009-2019 in Finland were screened with the assay, followed by isolations attempts and sequencing using Sanger and next generation sequencing (NGS). To validate the assay external and in-house quality control samples were used and all were correctly identified. Specificity of the assay was 100%. Assay was sensitive to detect CHIKV RNA in dilution of 10-8.During years 2009-2019 34 patients were diagnosed for acute CHIKV infection. Twelve out of 34 cases were positive by CHIKV nsP2-RT-qPCR.Two CHIKV isolations succeeded from two individuals infected originally in Thailand, 2019. From 12 CHIKV nsP2-RT-qPCR positive samples, five (42%) CHIKVs were successfully sequenced. In this study, CHIKVs from year 2019 clustered with CHIKV ECSA-lineage forming sub-cluster with strains from ones detected in Bangladesh 2017, and the ones from Jamaica (2014) within Asian lineage showing highest similarity to strains detected in Caribbean outbreak 2013-15.  Majority of the CHIKV infections detected in Finland originates from Asia and virus lineages reflect the global circulation of the pathogen.

Performance of six SARS-CoV-2 immunoassays in comparison with microneutralisation.

There is an urgent need for reliable high-throughput serological assays for the management of the ongoing COVID-19 pandemic. Preferably, the performance of serological tests for a novel virus should be determined with clinical specimens against a gold standard, i.e. virus neutralisation. We compared the performance of six commercial immunoassays for the detection of SARS-COV-2 IgG, IgA and IgM antibodies, including four automated assays [Abbott SARS-COV-2 IgG (CE marked), Diasorin Liaison® SARS-COV-2 S1/S2 IgG (research use only, RUO), and Euroimmun SARS-COV-2 IgG and IgA (CE marked)], and two rapid lateral flow (immunocromatographic) tests [Acro Biotech 2019-nCoV IgG/IgM (CE marked) and Xiamen Biotime Biotechnology SARS-COV-2 IgG/IgM (CE marked)] with a microneutralisation test (MNT). Two specimen panels from serum samples sent to Helsinki University Hospital Laboratory (HUSLAB) were compiled: the patient panel (N=70) included sera from PCR confirmed COVID-19 patients, and the negative panel (N=81) included sera sent for screening of autoimmune diseases and respiratory virus antibodies in 2018 and 2019. The MNT was carried out for all COVID-19 samples (70 serum samples, 62 individuals) and for 53 samples from the negative panel. Forty-one out of 62 COVID-19 patients showed neutralising antibodies.The specificity and sensitivity values of the commercial tests against MNT, respectively, were as follows: 95.1 %/80.5 % (Abbott Architect SARS-CoV-2 IgG), 94.9 %/43.8 % (Diasorin Liaison SARS-CoV-2 IgG; RUO), 68.3 %/87.8 % (Euroimmun SARS-CoV-2 IgA), 86.6 %/70.7 % (Euroimmun SARS-CoV-2 IgG), 74.4 %/56.1 % (Acro 2019-nCoV IgG), 69.5 %/46.3 % (Acro 2019-nCoV IgM), 97.5 %/71.9 % (Xiamen Biotime SARS-CoV-2 IgG), and 88.8 %/81.3 % (Xiamen Biotime SARS-CoV-2 IgM). This study shows variable performance values. Laboratories should carefully consider their testing process, such as a two-tier approach, in order to optimize the overall performance of SARS- CoV-2 serodiagnostics.

Serological survey in the Finnish human population implies human-to-human transmission of Ljungan virus or antigenically related viruses.

Ljungan virus (LV) is a picornavirus related to human parechoviruses (HPeV). The virus has been found in bank voles (Myodes glareolus) and several other rodent species, and suggested to have zoonotic potential. Thus far, seroepidemiological data on LV infections in humans are scarce. In this study, we aimed to characterize the demographic and geographical distribution of LV-reactive antibodies in Finland, and to investigate its occurrence in patients suspected of having a rodent-borne disease, nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV). Using an immunofluorescence assay (LV strain 145SLG), we screened human sera (n = 1378) and found LV-reactive antibodies in 36% of samples. The probability of possessing LV-reactive antibodies peaked at age of 14 years, suggesting that most infections occur in childhood. The prevalence of LV-reactive antibodies was significantly higher in the urbanized area surrounding Helsinki than in more rural Central Finland. These findings are uncharacteristic of a rodent-borne pathogen, and therefore we consider human-to-human transmission of one or several Ljungan-like viruses as a likely cause for most of the observed antibody responses.

Serological survey for viral pathogens in Turkish rodents.

Wild rodents (n = 330) were trapped around the villages of Altindere and Cosandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihemsin, Rize Province), and Bozdag (Odemis, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.

Is there an association of Pneumocystis infection with the presence of arena-, hanta-, and poxvirus antibodies in wild mice and shrews in Finland?

As part of studies on the nature of the endemic virus infections in natural rodent hosts, the possible association of cyst forms of Pneumocystis spp. with the presence of hanta-, cowpox-, and arenavirus antibodies in wild mice (Apodemus flavicollis, N=105; Apodemus agrarius, N=63; Micromys minutus, N=50) and the common shrew (Sorex araneus, N=101) was studied in south-central Finland. One hantavirus (Saaremaa virus, SAAV) seropositive A. agrarius, and 2 cowpoxvirus (CPXV) seropositive S. araneus were detected, and antibodies against an arenavirus (Lymphocytic choriomeningitis virus, LCMV) were found in all 3 mouse species but not in shrews. Cyst forms of Pneumocystis spp. were detected in all species except A. agrarius. There was no significant association between virus antibodies (LCMV in mice, and CPXV in shrews) and cyst forms of Pneumocystis in any of the species. Concurrent presence of virus antibodies (LCMV) and cyst forms of Pneumocystis were detected only in 1 M. minutus. In conclusion, we found no evidence of any association between Pneumocystis and antibodies to any of the viruses tested.

Corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by Puumala virus infection.

Reported here are two cases of hantavirus pulmonary syndrome caused by Puumala virus infection, which rapidly resolved after initiation of corticosteroid treatment combined with continuous veno-venous hemodiafiltration. These cases emphasize the role of the inflammatory response in the pathogenesis of hantavirus pulmonary syndrome.

Hantavirus and arenavirus antibody prevalence in rodents and humans in Trentino, Northern Italy.

The spatial and temporal distribution of hantavirus and arenavirus antibody-positive wild rodents in Trentino, Italy, was studied using immunofluorescence assays (IFA) in two long-term sites trapped in 2000-2003, and six other sites trapped in 2002. The overall hantavirus seroprevalence in the bank voles, Clethrionomys glareolus (n=229) screened for Puumala virus (PUUV) antibodies was 0.4%, and that for Apodemus flavicollis mice (n=1416) screened for Dobrava virus (DOBV) antibodies was 0.2%. Antibodies against lymphocytic choriomeningitis virus (LCMV) were found in 82 (5.6%) of the 1472 tested rodents; the seroprevalence being 6.1% in A. flavicollis (n=1181), 3.3% in C. glareolus (n=276), and 14.3% in Microtus arvalis (n=7). Of the serum samples of 488 forestry workers studied by IFA, 12 were LCMV-IgG positive (2.5%) and one DOBV-IgG positive (0.2%), however, the latter could not be confirmed DOBV-specific with a neutralization assay. Our results show a widespread distribution but low prevalence of DOBV in Trentino, and demonstrate that the arenavirus antibodies are a common finding in several other rodent species besides the house mouse.

Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells.

Puumala hantavirus (PUUV) glycoproteins G1 and G2 and nucleocapsid protein (N) were expressed in BHK-21 cells by transfection of a plasmid producing a recombinant alphavirus replicon. Coexpression of G1 and G2 from separate constructs seemed to be important for the optimal folding of the glycoproteins, as evaluated by a panel of MAbs detecting conformational epitopes. To evaluate the human antibody response against recombinant G1, G2 and N, several panels of sera were tested by immunofluorescence assay (IFA). Also human sera showed the best reactivity towards G1 and G2 coexpressed from separate transcripts (G1 + G2). Notably, only 2% of the acute sera (total number = 133) contained IgG antibodies against G1 + G2, whereas of old-immunity sera (total number = 100) 87% were G1 + G2 positive. Analysis of a panel of serial patient sera showed that as the immunity matured, IgG antibodies against the recombinant glycoproteins appeared and the titers increased in the course of time, while antibodies against the recombinant N were present already in the acute phase in high titers. The granular fluorescence pattern in PUUV IgG-IFA, associated with the acute phase of immunity, was linked to the presence of antibodies against N, whereas the diffuse fluorescence pattern associated with old-immunity, was linked to the development of antibodies against G1 + G2. The granular fluorescence pattern in PUUV IgG-IFA had a predictive value of 100% for acute PUUV infection. Weak cross-reaction with PUUV glycoproteins was observed in 36% of old-immunity DOBV-specific human sera.

Antigenic properties and diagnostic potential of recombinant dobrava virus nucleocapsid protein.

Dobrava hantavirus (DOBV) causes severe hemorrhagic fever with renal syndrome in the Balkan region and has been detected recently also in Russia, Estonia, and Germany. DOBV nucleocapsid protein (N) was produced in insect cells, using the baculovirus expression system (bac-DOBV-N), and in E. coli as a truncated (aa 1-165) glutathione-S transferase fusion protein (DOBV-dN-GST). The antigenic properties of bac-DOBV-N were found identical to native DOBV-N when examined by a panel of hantavirus-specific monoclonal antibodies. Enzyme immunoassays for detection of IgM and IgG antibodies were set up using DOBV recombinant N proteins and compared with those based on recombinant Hantaan and Puumala virus N, using panels of sera collected from DOBV, Hantaan and Puumala virus-infected patients. Full-length N protein (bac-DOBV-N) was found to be a more sensitive antigen than DOBV-dN-GST. The sensitivity values for sera from DOBV-infected patients were 100% for bac-DOBV-N and 86% for DOBV-dN-GST by IgM assays, and 98% for bac-DOBV-N and 88% for DOBV-dN-GST by IgG assays. The specificity values were 100% for bac-DOBV-N and 99% for DOBV-dN-GST by IgM assays, and 100% for both antigens by IgG assays.

Evaluation of Puumala virus IgG and IgM enzyme immunoassays based on recombinant baculovirus-expressed nucleocapsid protein for early nephropathia epidemica diagnosis.

BACKGROUND: Puumala virus (PUU), a member of Hantavirus genus, is the causative agent of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome (HFRS). Rapid diagnosis is essential for clinical management of NE. OBJECTIVES: To evaluate the usefulness of recombinant protein-based IgM (direct- and mu-capture) and IgG (direct-and antigen (Ag)-capture) enzyme immunoassays (EIA) in early diagnosis of NE in comparison to IgG immunofluorescence assay (IF), and to find out the time limit for PUU-specific antibody seroconversion. STUDY DESIGN: The specific IgM and IgG antibody responses in serum were analyzed in 109 patients (235 serial sera) and 114 patients (233 serial sera), respectively. The serum panel used was selected from a larger material according to the availability of information concerning the date after onset of symptoms, the panel also containing NE patients who had been IgG-IF negative in their first (early) samples to find out the possible differences between sensitivities of the EIAs and IF. RESULTS: All NE patients tested became IgM-positive at the latest on the 6th (mu-capture EIA) or 7th (direct-IgM EIA) day after onset of symptoms. Out of a panel of very early NE-patient sera (n = 38) that could not be detected by IgG-IF, 66% were already positive with both direct-IgM EIA and mu-capture EIA. When comparing IgG EIAs and IgG-IF, 98% of IF-positive sera from NE patients were also positive with direct-IgG EIA, and 99% with Ag-capture IgG EIA. Out of a panel of very early NE-patient sera (n = 37) that could not be detected by IgG-IF, 57% were positive with direct-IgG EIA, and 27% with Ag-capture IgG EIA. CONCLUSIONS: The baculovirus-expressed PUU-N-based IgG and IgM EIAs were found most suitable for NE diagnosis, giving the opportunity in some cases for earlier diagnosis as compared with PUU-IgG IF.

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

A panel of seven human monoclonal Fabs against Puumala virus (PUU) nucleocapsid protein (N) was obtained by panning an antibody phage-display library prepared from the spleen of a PUU-immune individual. Three antibodies reacted in immunoblotting and cross-reacted strongly with Tula and Sin Nombre virus recombinant N proteins. These antibodies mapped to the amino terminus of the N protein. One PUU glycoprotein 2 (G2)-specific Fab obtained against a novel epitope (G2c) cross-reacted with Khabarovsk virus but not with the other hantavirus serotypes. An N protein-specific Fab was successfully used as capture antibody to detect PUU-specific serum IgG and IgM antibodies in an enzyme immunoassay. The result demonstrates the usefulness of recombinant human Fabs as potential diagnostic tools.

Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica).

Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.

Characterization of Puumala virus nucleocapsid protein: identification of B-cell epitopes and domains involved in protective immunity.

B-cell epitopes in the nucleocapsid protein (N) of Puumala (PUU) virus were investigated by use of truncated recombinant proteins and overlapping peptides. Six of seven epitopes, recognized by bank vole monoclonal antibodies, were localized within the amino-terminal region of the protein (aa 1-79). Polyclonal antibodies from wild-trapped or experimentally infected bank voles identified epitopes located over the entire protein. Antibody end-point titers to different N fragments indicated that the amino-terminal region is the major antigenic target in PUU virus-infected bank voles. To investigate the role of PUU virus N in protective immunity, we analyzed the immunogenicity of truncated recombinant N and developed an animal model based on colonized bank voles. No PUU virus N antigen, nor any glycoprotein-specific antibodies, could be detected after virus challenge in animals immunized with an amino-terminal fragment (aa 1-118), a fragment covering two thirds of the animals immunized with shorter N fragments displayed either N antigen, or glycoprotein-specific antibodies, suggestive of partial protection. Prechallenge sera from all groups of immunized animals were found negative or only weakly positive for neutralizing antibodies when assayed by focus reduction neutralization test, which indicated an important role for cell-mediated immunity in protection.

Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells.

Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.

Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied.

Tula virus: a newly detected hantavirus carried by European common voles.

A novel hantavirus has been discovered in European common voles, Microtus arvalis and Microtus rossiaemeridionalis. According to sequencing data for the genomic RNA S segment and nucleocapsid protein and data obtained by immunoblotting with a panel of monoclonal antibodies, the virus, designated Tula virus, is a distinct novel member of the genus Hantavirus. Phylogenetic analyses of Tula virus indicate that it is most closely related to Prospect Hill, Puumala, and Muerto Canyon viruses. The results support the view that the evolution of hantaviruses follows that of their primary carriers. Comparison of strains circulating within a local rodent population revealed a genetic drift via accumulation of base substitutions and deletions or insertions. The Tula virus population from individual animals is represented by quasispecies, indicating the potential for rapid evolution of the agent.

Puumala virus antibody and immunoglobulin G avidity assays based on a recombinant nucleocapsid antigen.

Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.

Cloning and sequencing of Puumala virus Sotkamo strain S and M RNA segments: evidence for strain variation in hantaviruses and expression of the nucleocapsid protein.

The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV Hällnäs strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the Hällnäs strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a beta-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.