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The burden of seasonal human coronavirus (HCoV) infections in early childhood and adults has not been well analyzed in longitudinal serological studies. Here we analyzed the changes in HCoV (229E, HKU1, NL63, OC43, MERS, and SARS-CoV-2) spike-specific antibody levels in follow-up serum specimens of 140 children at the age of 1, 2, and 3 years, and of 113 healthcare workers vaccinated for Covid-19 with BNT162b2-vaccine. IgG antibody levels against six recombinant HCoV spike subunit 1 (S1) proteins were measured by enzyme immunoassay. We show that by the age of three years the cumulative seropositivity for seasonal HCoVs increased to 38-81% depending on virus type. BNT162b2 vaccinations increased anti-SARS-CoV-2 S1 antibodies, but no increase in seasonal coronavirus antibodies associated with vaccinations. In healthcare workers, during a one year follow-up, diagnostic antibody rises were seen in 5, 4 and 14% of the cases against 229E, NL63 and OC43 viruses, respectively, correlating well with the circulating HCoVs. In 6% of the HCWs, a diagnostic antibody rise was seen against S1 of HKU1, however, these rises coincided with anti-OC43 S1 antibody rises. Rabbit and guinea pig immune sera against HCoV S1 proteins indicated immunological cross-reactivity within alpha-CoV (229E and NL63) and beta-CoV (HKU1 and OC43) genera.

Background Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. Methods We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. Results The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. Conclusions The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.

Vaccination shows efficacy in protecting from COVID-19, but regime and dosing optimization is still ongoing. Here the authors show that BNT162b2, mRNA-1273, or their combination with ChAdOx1 induces similar antibody responses, and those receiving three doses of BNT162b2 induce neutralizing antibodies against the Omicron variant. Two COVID-19 mRNA (of BNT162b2, mRNA-1273) and two adenovirus vector vaccines (ChAdOx1 and Janssen) are licensed in Europe, but optimization of regime and dosing is still ongoing. Here we show in health care workers (n = 328) that two doses of BNT162b2, mRNA-1273, or a combination of ChAdOx1 adenovirus vector and mRNA vaccines administrated with a long 12-week dose interval induce equally high levels of anti-SARS-CoV-2 spike antibodies and neutralizing antibodies against D614 and Delta variant. By contrast, two doses of BNT162b2 with a short 3-week interval induce 2-3-fold lower titers of neutralizing antibodies than those from the 12-week interval, yet a third BNT162b2 or mRNA-1273 booster dose increases the antibody levels 4-fold compared to the levels after the second dose, as well as induces neutralizing antibody against Omicron BA.1 variant. Our data thus indicates that a third COVID-19 mRNA vaccine may induce cross-protective neutralizing antibodies against multiple variants.

Two COVID-19 mRNA and two adenovirus vector vaccines have been licensed in Europe and various vaccine combinations and dosing strategies have been exploited to maximize the immunity against COVID-19. Here, we show that among health care workers (n=328) two doses of BNT162b2, mRNA-1273, or ChAdOx1 as also a combination of an adenovirus vector and mRNA vaccines induces equally high levels of anti-SARS-CoV-2 spike antibodies and neutralizing antibodies against B.1 and B.1.617.2 when administrated with a long 12-week dose interval. Two doses of BNT162b2 with a short 3-week interval induce 2-3-fold lower titers of neutralizing antibodies compared to the long interval. Third mRNA vaccine dose for the short dose interval group increased the antibody levels 4-fold compared to the levels after the second dose. Importantly, sera from all three-times vaccinated neutralized B.1.1.529 (Omicron). The data indicates that a third COVID-19 mRNA vaccine dose efficiently induces cross-protective neutralizing antibodies against multiple variants.

Here, we describe a scalable and automated, high-content microscopy -based mini-immunofluorescence assay (mini-IFA) for serological testing i.e., detection of antibodies. Unlike conventional IFA, which often relies on the use of cells infected with the target pathogen, our assay employs transfected cells expressing individual viral antigens. The assay builds on a custom neural network-based image analysis pipeline for the automated and multiplexed detection of immunoglobulins (IgG, IgA, and IgM) in patient samples. As a proof-of-concept, we employed high-throughput equipment to set up the assay for measuring antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with spike (S), membrane (M), and nucleo (N) proteins, and the receptor-binding domain (R) as the antigens. We compared the automated mini-IFA results from hundreds of patient samples to the visual observations of human experts and to the results obtained with conventional ELISA. The comparisons demonstrated a high correlation to both, suggesting high sensitivity and specificity of the mini-IFA. By testing pre-pandemic samples and those collected from patients with RT-PCR confirmed SARS-CoV-2 infection, we found mini-IFA to be most suitable for IgG and IgA detection. The results demonstrated N and S proteins as the ideal antigens, and the use of these antigens can serve to distinguish between vaccinated and infected individuals. The assay principle described enables detection of antibodies against practically any pathogen, and none of the assay steps require high biosafety level environment. The simultaneous detection of multiple Ig classes allows for distinguishing between recent and past infection.Public abstractThe manuscript describes a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response in patient blood samples. The automated method builds on machine-learning -guided image analysis with SARS-CoV-2 as the model pathogen. The method enables simultaneous measurement of IgM, IgA, and IgG responses against different virus antigens in a high throughput manner. The assay relies on antigens expressed through transfection and allows for differentiation between vaccine-induced and infection-induced antibody responses. The transfection-based antigen expression enables performing the assay at a low biosafety level laboratory and allows fast adaptation of the assay to emerging pathogens. Our results provide proof-of-concept for the approach, demonstrating fast and accurate measurement of antibody responses in a clinical and research set-up.

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n = 180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees’ neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides strong evidence that the second dose of the BNT162b2 vaccine induces efficient cross-neutralization of SARS-CoV-2 variants currently circulating in the world.

Background Human parechoviruses (HPeVs) (family Picornaviridae), are common pathogens in young children. Despite their high prevalence, research on their genetic identity, diversity and evolution have remained scarce. Objectives Complete coding regions of three previously reported HPeV-4 isolates from Finnish children with sepsis-like disease were sequenced in order to elucidate the phylogenetic relationships and potential recombination events during the evolution of these isolates. Study design The isolated viruses were sequenced and aligned with all HPeV complete genome sequences available in GenBank. Phylogenetic trees were constructed and similarity plot and bootscanning methods were used for recombination analysis. Results The three HPeV-4 isolates had 99.8% nucleotide sequence similarity. The phylogenetic analysis indicated that capsid-encoding sequences of these HPeV-4 isolates were closely related to other HPeV-4 strains (80.7-94.7% nucleotide similarity), whereas their non-structural region genes 2A to 3C clustered together with several HPeV-1 and HPeV-3 strains, in addition to the HPeV-4 strain K251176-02 (isolated 2002 in the Netherlands), but not with other HPeV-4 strains. However, in 3D-encoding sequence the Finnish HPeV-4 isolates did not cluster with the strain HPeV-4/K251176-02, but instead, formed a distinct group together with several HPeV-1 and HPeV-3 strains. Similarity plot and Bootscan analyses further confirmed intertypic recombination events in the evolution of the Finnish HPeV-4 isolates. Conclusion Intertypic recombination event(s) have occurred during the evolution of HPeV-4 isolates from children with sepsis-like disease. However, due to the low number of parechovirus complete genomes available, the precise recombination partners could not be detected. The results suggest frequent intratypic recombination among parechoviruses.

Picornaviruses (family Picornaviridae) are small, nonenveloped, positive-sense, single-stranded RNA viruses. The members of this family are currently classified into 47 genera and 110 species. Of picornaviruses, entero-and parechoviruses are associated with aseptic meningitis. They are transmitted via fecal-oral and respiratory routes, and occasionally, these viruses may cause a brief viremia and gain access to central nervous system (CNS). During the diagnostic screening of entero-and parechovirus types in Finland in year 2013-14, we detected a cluster of echovirus 4 (E4) infections in young adults and adolescents. As E4 is infrequently detected in Finland, we contacted several Northern and Central European laboratories that conduct routine surveillance for enteroviruses and, for those who have had E4 cases, we send a query for E4 sequences and data. Here we report CNS infections caused by E4 in Finland, Sweden, Norway, Denmark, Iceland and Germany in 2013 and 2014, and show that the E4 detected in these countries form a single lineage. In contrast, E4 strains circulating in these countries preceding the year 2013, and those circulating elsewhere in Europe during 2013-2014, formed several independent clusters.

Background: Ljungan virus (LV) has not confirmed to associate with any human disease, but a possible connection with type 1 diabetes has been suggested. LV is a rodent-borne picornavirus that induces a diabetes-like condition in rodents. Approximately 30% of adults and 60% of children are seropositive in Finland. The Finnish Type 1 Diabetes Prediction and Prevention study enabled the use of very well characterized sample panels from children seroconverted to positivity for multiple islet autoantibodies during their prospective observation from birth; in addition, samples from age, sex, human leukocyte antigen (HLA), and residence area matched control children. Methods: We analyzed LV IgG seroprevalence in 102 case children (65 had also developed type 1 diabetes), in addition to nondiabetic control children. LV and human parechovirus (HPeV) immunofluorescence assays were used to analyze LV and HPeV-specific IgG from 102 plasma samples taken at the time of islet autoantibody appearance and from 204 samples from the matched control children. Results: Altogether 46.1% of the case and 50.7% of the control children were positive for LV IgG (odds ratio 0.8; 95% confidence interval, 0.47–1.36; P = 0.416) and 67.6% versus 79.8% were positive for HPeV IgG, respectively (odds ratio 0.49, 0.27–0.9, P = 0.023). Conclusions: Thus, no risk associations between LV or HPeV-specific IgG and islet autoimmunity were observed. However, a trend for significantly higher prevalence of HPeV antibodies in control children (P = 0.023) suggests a possible protective association of this virus with islet autoimmunity.

Human parechoviruses (HPeVs) are RNA viruses associated mainly with mild gastrointestinal and respiratory infections in children and also cause neonatal sepsis and CNS infections. Human enteroviruses, close relatives of HPeVs, associate with the development of type 1 diabetes. In this study, the potential role of HPeV infections in promoting beta cell autoimmunity was investigated by analyzing stool samples of 54 prediabetic case and 134 healthy control children for the presence of HPeV RNA and comparing the derived infection frequencies. All 188 children were participants of the Finnish prospective Diabetes Prediction and Prevention study. Viral RNA was screened for using an HPeV-specific RT-PCR method coupled to liquid hybridization of the PCR product. The overall HPeV infection frequency did not differ between prediabetic case and control children. However, case boys had more HPeV positive samples in the 6-month period before becoming autoantibody positive, when compared to the matching time-period in controls (P

Ljungan virus (LV, genus Parechovirus, family Picornaviridae) is considered currently to be a rodent-borne virus. Despite suggested human disease associations, its zoonotic potential remains unclear. To date, LV antibody prevalence in both humans and rodents has not been studied. In this study, two different LV immunofluorescence assays (LV IFAs) were developed with LV genotypes 1 (LV strain 87-012G) and 2 (LV strain 145SLG), and cross-neutralization and -reaction studies were carried out with LV strain 145SLG. Finally, a panel of 37 Finnish sera was screened for anti-LV antibodies using two different LV IFAs (LV 145SLG and LV 87-012G) and a neutralization (NT) assay (LV 145SLG), and 50 samples from Myodes glareolus by LV IFA (LV 145SLG). The LV seroprevalence study showed 38% and 18% positivity in humans and M. glareolus, respectively. LV IFAs and NT assays were compared, and the results were in good agreement. The data are the first evidence of humans and rodents coming into contact with LV in Finland. Additional studies are required in order to acquire a better understanding of the prevalence, epidemiological patterns and possible disease association of LV infections.

Background Human parechoviruses (HPeVs) are common viruses mainly infecting young children. Most infections are mild, but HPeV3 causes severe CNS infections in new-born infants. Objectives The aim was to study the epidemiology of HPeVs in Finnish general population in the period 1996–2007, with special emphasis on the different types circulating in Finland. Study design A total of 2236 stool specimens were collected from 200 healthy Finnish children in a prospective birth cohort study, most before the age of 2 years. Samples were tested for the presence of HPeV RNA using a specific RT-PCR. The genotype of HPeV was determined by sequencing the VP1 genomic region. Results HPeV RNA was detected in 144 (6.4%) specimens from 78 (39%) children. The dominant type was HPeV1 (93% of the type-identified 105 samples), although types 3 and 6 were also identified. HPeV was found sequentially in more than one sample in 43 infections lasting up to 93 days. The positive findings were distributed equally in young ages and declined towards the age of 2 years. Infections clustered around the autumn months with no obvious change between years. No significant differences were seen between boys and girls. Conclusions HPeV is a common virus infecting Finnish children under 2 years of age. HPeVs circulate throughout the year with clear accumulation on autumn, also seen in individual years over the 11-year study period. The virus deserves increased attention and should be included in the test panel of clinical virus laboratories.

Fig. 1. T1 weighted pre- (a) and post-contrast (b) images. Multiple lesions of white matter show contrast enhancement.∗ Corresponding author at: Haartman institute, Department of Virology, Haartmaninkatu 3, POB 21 FIN-00014 University of Helsinki, Finland. Tel.: +358 9 19126579;fax: +358 9 19126491.E-mail address: jenna.pietilainen@helsinki.fi (J. Pietilainen-Nicklen).

Human parechoviruses are a family of viruses closely related to enteroviruses, and associated with neonatal sepsis-like syndrome, respiratory symptoms and gastrointestinal infection. Here we present clinical details of two neonatal sepsis cases suspected to be caused by HPeV4 infection. The patients were hospitalized in October, 2012. No other causative agents were detected.

The symptoms observed in children with human parechovirus (HPeV) infection vary widely from asymptomatic or mild gastrointestinal infections to more severe central nervous system infections and sepsis-like disease. Many of the disease associations are, however, only suggestive. In this study, we examined the connection between HPeV and acute otitis media, lower respiratory infections and suspected central nervous system infections.An HPeV specific real-time reverese transcriptase polymerase chain reaction was used to detect HPeV RNA. We analyzed altogether 200 middle-ear fluid samples, 192 nasopharyngeal aspirates, 79 cerebrospinal fluid specimens and 50 serum and 5 fecal or fecal culture samples. Positive samples were typed by sequencing the VP1 region.Seven (8%) of 85 children with suspected central nervous system infections were positive for HPeV. Of these, 4 (all in autumn 2012 and from children3 months of age) were typed to be HPeV4, whereas 1 child had HPeV3. HPeV4 was detected from stool, serum and cerebrospinal fluid. The children with acute otitis media tested HPeV positive in 2.5% episodes. In the lower respiratory cases, HPeV was absent.The findings reported in this study suggest that HPeV4 can cause sepsis-like disease in young infants and be present in cerebrospinal fluid. Furthermore, this report shows that HPeV findings in children with more severe symptoms occur also in Finland.

Ljungan virus (LV, genus Parechovirus, family Picornaviridae) is considered currently to be a rodent-borne virus. Despite suggested human disease associations, its zoonotic potential remains unclear. To date, LV antibody prevalence in both humans and rodents has not been studied. In this study, two different LV immunofluorescence assays (LV IFAs) were developed with LV genotypes 1 (LV strain 87-012G) and 2 (LV strain 145SLG), and cross-neutralization and -reaction studies were carried out with LV strain 145SLG. Finally, a panel of 37 Finnish sera was screened for anti-LV antibodies using two different LV IFAs (LV 145SLG and LV 87-012G) and a neutralization (NT) assay (LV 145SLG), and 50 samples from Myodes glareolus by LV IFA (LV 145SLG). The LV seroprevalence study showed 38% and 18% positivity in humans and M. glareolus, respectively. LV IFAs and NT assays were compared, and the results were in good agreement. The data are the first evidence of humans and rodents coming into contact with LV in Finland. Additional studies are required in order to acquire a better understanding of the prevalence, epidemiological patterns and possible disease association of LV infections.

Background Human parechoviruses (HPeVs) are RNA viruses associated with mild gastrointestinal and respiratory infections in children, but may also cause neonatal sepsis and CNS infections in infants. While the prevalence of HPeVs is known mostly among hospitalized populations, the knowledge of HPeV seroprevalence in the general population is poor. Objectives The aim of this study was to identify and compare the HPeV1-6 seroprevalence in Finnish and Dutch populations. Study design : A type specific microneutralization assay was set up for detecting neutralizing antibodies (nABs) against HPeV types 1–6. Altogether 616 serum samples from Finnish and Dutch population were analyzed for antibodies against HPeVs. The samples were collected from Finnish children aged 1, 5 or 10 years, Finnish adults, 0- to 5-year-old Dutch children, Dutch women of childbearing age and Dutch HIV-positive men. Results In both adult populations, seropositivity was high against HPeV1 (99% in Finnish and 92% in Dutch samples) and HPeV2 (86% and 95%). Against HPeV4, the seropositivity was similar (62% and 60%). In Dutch adults, nABs against HPeV5 and 6 (75% and 74%) were detected more often than in Finnish adults (35% and 57%, respectively). In contrast, seropositivity against HPeV3 was as low as 13% in the Finnish and 10% in the Dutch adults. The seroprevalence of all HPeV types increased with age. Conclusions The seroprevalence of HPeVs is high in Finnish and Dutch populations and HPeV type 2 and types 4–6 are significantly more prevalent compared to earlier reports. The seroprevalence of antibodies observed against HPeV3 was low.