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Background Previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primes the immune system; thus individuals who have recovered from infection have enhanced immune responses to subsequent vaccination (hybrid immunity). However, it remains unclear how well hybrid immunity induced by severe or mild infection can cross-neutralize emerging variants. We aimed to compare the strength and breadth of antibody responses in vaccinated recovered and uninfected subjects. Methods We measured spike-specific immunoglobulin (Ig)G and neutralizing antibodies (NAbs) from vaccinated subjects including 320 with hybrid immunity and 20 without previous infection. From 29 subjects with a previous severe or mild infection, we also measured NAb responses against Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529/BA.1) variants following vaccination. Results A single vaccine dose induced 2-fold higher anti-spike IgG concentrations and up to 4-fold higher neutralizing potency of antibodies in subjects with a previous infection compared with vaccinated subjects without a previous infection. Hybrid immunity was more enhanced after a severe than a mild infection, with sequentially decreasing NAb titers against Alpha, Beta, Delta, and Omicron variants. We found similar IgG concentrations in subjects with a previous infection after 1 or 2 vaccine doses. Conclusions Hybrid immunity induced strong IgG responses, particularly after severe infection. However, the NAb titers were low against heterologous variants, especially against Omicron.

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.

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that minks remain virus RNA positive for days, develop clinical signs and histopathological changes, and transmit the virus to uninfected recipients warranting further studies and preparedness.

The emergence of SARS-CoV-2 Omicron variant (B.1.1.529) with major spike protein mutations has raised concern over potential neutralization escape and breakthrough infections among vaccinated and previously SARS-CoV-2-infected subjects. We measured cross-protective antibodies against variants in health care workers (HCW, n = 20) and nursing home residents (n = 9) from samples collected at 1-2 months, following the booster (3rd) dose. We also assessed the antibody responses in subjects infected before the Omicron era (n = 38) with subsequent administration of a single mRNA vaccine dose. Following booster vaccination, HCWs had high IgG antibody concentrations to the spike protein and neutralizing antibodies (NAb) were detectable against all variants. IgG concentrations among the elderly remained lower, and some lacked NAbs against the Beta and Omicron variants. NAb titers were significantly reduced against Delta, Beta, and Omicron compared to WT virus regardless of age. Vaccination induced high IgG concentrations and variable titers of cross-reactive NAbs in previously infected subjects, whereas NAb titers against Omicron were barely detectable 1 month postinfection. High IgG concentrations with cross-protective neutralizing activity were detected after three Coronavirus Disease 2019 (COVID-19) vaccine doses in HCWs. However, lower NAb titers seen in the frail elderly suggest inadequate protection against Omicron breakthrough infections, yet protection against severe COVID-19 is expected.

Zika virus (ZIKV) infections in humans are considered to be mild or subclinical. However, during the recent epidemics in the Pacific Islands and the Americas, the infection was associated with Quillain-Barré syndrome and congenital infections with fetal brain abnormalities, including microcephaly. Thus, more detailed understanding of ZIKV-host cell interactions and regulation of innate immune responses by strains of differential evolutionary origin is required. Here, we characterized the infection and immune responses triggered by two epidemic Asian/American lineage viruses, including an isolate from fetal brains, and a historical, low passage 1947 African lineage virus in human monocyte-derived dendritic cells (DCs) and macrophages. The epidemic Asian/American ZIKV replicated well and induced relatively good antiviral responses in human DCs whereas the African strain replicated less efficiently and induced weaker immune responses. In macrophages both the African and Asian strains showed limited replication and relatively weak cytokine gene expression. Interestingly, in macrophages we observed host protein degradation, especially IRF3 and STAT2, at early phases of infection with both lineage viruses, suggesting an early proteasomal activation in phagocytic cells. Our data indicates that ZIKV evolution has led to significant phenotypic differences in the replication characteristics leading to differential regulation of host innate immune responses.

The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

The emergence of SARS-CoV-2 Omicron variant (B.1.1.529) with major spike protein mutations has raised concern over potential neutralization escape and breakthrough infections among vaccinated and previously SARS-CoV-2 infected subjects. We measured cross-protective antibodies against variants in health care workers (HCW, n=20) and nursing home residents (n=9) from samples collected 1-2 months following the booster (3rd) dose. We also assessed the antibody responses in prior to Omicron era infected subjects (n=38) with subsequent administration of a single mRNA vaccine dose. Following booster vaccination HCWs had high IgG antibody concentrations to the spike protein and neutralizing antibodies (NAb) were detectable against all variants. IgG concentrations among the elderly remained lower, and some lacked NAbs against the Beta and Omicron variants. NAb titers were significantly reduced against Delta, Beta and Omicron compared to wild-type virus regardless of age. Vaccination induced high IgG concentrations and variable titers of cross-reactive NAbs in previously infected subjects, whereas NAb titers against Omicron were barely detectable 1-month post-infection. High IgG concentrations with cross-protective neutralizing activity were detected after three COVID-19 vaccine doses in HCWs. However, lower NAb titers seen in the frail elderly suggest inadequate protection against Omicron breakthrough infections, yet protection against severe COVID-19 is expected.Clinical trial registrationEudraCT 2021-004788-29

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21–42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). The relative quantity of SARS-CoV-2 RNA fragments in the 24h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and RT-qPCR assay targeted with N2-assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of new and confirmed COVID-19 cases in the sewer network area.Over the 10-month surveillance period, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results), while only 24% of all samples exhibited gene copy (GC) numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7, 18 and 36 cases within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 GC required significantly more COVID-19 cases: the quantification rate was 50%, 75% and 95% when the 14-day incidence was 110, 152 and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 GC quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the GC of SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Most subjects develop antibodies to SARS‐CoV‐2 following infection. In order to estimate the duration of immunity induced by SARS‐CoV‐2 it is important to understand for how long antibodies persist after infection in humans. Here, we assessed the persistence of serum antibodies following WT SARS‐CoV‐2 infection at 8 and 13 months after diagnosis in 367 individuals. The SARS‐CoV‐2 spike IgG (S‐IgG) and nucleoprotein IgG (N‐IgG) concentrations and the proportion of subjects with neutralizing antibodies (NAb) were assessed. Moreover, the NAb titers among a smaller subset of participants (n = 78) against a WT virus (B) and variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) were determined. We found that NAb against the WT virus persisted in 89% and S‐IgG in 97% of subjects for at least 13 months after infection. Only 36% had N‐IgG by 13 months. The mean S‐IgG concentrations declined from 8 to 13 months by less than one third; N‐IgG concentrations declined by two‐thirds. Subjects with severe infection had markedly higher IgG and NAb levels and are expected to remain seropositive for longer. Significantly lower NAb titers against the variants compared to the WT virus, especially after a mild disease, suggests reduced protection against VOCs., A year after WT SARS‐CoV‐2 infection high seropositivity rate was observed: 89% of subjects had persisting neutralizing antibodies and up to 97% had anti‐spike IgG antibodies. Compared to the WT virus, neutralizing antibody titers were reduced for variants of concern Alpha, Beta, and Delta.

The primary target organ of coronavirus disease 2019 (COVID-19) infection is the respiratory tract. Currently, there is limited information on the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect and regulate innate immunity in human immune cells and lung epithelial cells. Here, we compared the ability of four Finnish isolates of SARS-CoV-2 from COVID-19 patients to replicate and induce interferons (IFNs) and other cytokines in different human cells. All isolates failed to replicate in dendritic cells, macrophages, monocytes, and lymphocytes, and no induction of cytokine gene expression was seen. However, most of the isolates replicated in Calu-3 cells, and they readily induced type I and type III IFN gene expression. The hCoV-19/Finland/FIN-25/2020 isolate, originating from a traveler from Milan in March 2020, showed better ability to replicate and induce IFN and inflammatory responses in Calu-3 cells than other isolates of SARS-CoV-2. Our data increase the knowledge on the pathogenesis and antiviral mechanisms of SARS-CoV-2 infection in human cell systems. IMPORTANCE With the rapid spread of the coronavirus disease 2019 (COVID-19) pandemic, information on the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and regulation of innate immunity in human immune cells and lung epithelial cells is needed. In the present study, we show that SARS-CoV-2 failed to productively infect human immune cells, but different isolates of SARS-CoV-2 showed differential ability to replicate and regulate innate interferon responses in human lung epithelial Calu-3 cells. These findings will open up the way for further studies on the mechanisms of pathogenesis of SARS-CoV-2 in human cells.

SummaryBackgroundHousehold transmission studies offer the opportunity to assess both secondary attack rate and persistence of SARS-CoV-2 antibodies over time.MethodsWe invited confirmed COVID-19 cases and their household members to attend up to four household visits with collection of nasopharyngeal and serum samples over 28 days after index case onset. We calculated secondary attack rates (SAR) based on the presence of SARS-CoV-2 nucleoprotein IgG antibodies (IgG Ab) and/or neutralizing antibodies (NAb) overall and per households. Three and six months later, we assessed the persistence of SARS-CoV-2 antibodies.FindingsWe recruited 39 index cases and 90 household members. Among 87 household members evaluated, SAR was 48% (n=42), including 37 symptomatic secondary cases. In total, 80/129 (62%) participants developed both IgG Ab and NAb, while three participants only developed IgG Ab. Among participants who had both IgG Ab and NAb during the initial follow-up, 68/69 (99%) and 63/70 (90%) had IgG Ab and NAb at 3 months, while at 6 months, 59/75 (79%) and 63/75 (84%) had IgG Ab and NAb, respectively. Participants who required hospital care had initially 5-fold IgG Ab concentrations compared to cases with mild symptoms and 8-fold compared to asymptomatic cases.InterpretationFollowing detection of a COVID-19 case in a household, other members had a high risk of becoming infected. Follow-up of participants showed strong persistence of antibodies in most cases.FundingThis study was supported by THL coordinated funding for COVID-19 research (Finnish Government’s supplementary budget) and by the Academy of Finland (Decision number 336431).Research in contextEvidence before this studyHousehold transmission studies are pivotal to the characterization of transmission dynamics of emerging infectious diseases in a closed setting with homogenous exposure, including proportion of asymptomatic cases using serologic assessment of infection. Additionally, data on long-term persistence of immune response, including neutralizing antibodies following COVID-19 remains scarce. Our search on PubMed for articles published between January 1st 2020, and June 1st, 2021 using the search terms “household” AND “transmission” AND (“COVID-19” OR “SARS-CoV-2”) retrieved 381 results including 35 relevant articles: 21 original household transmission studies, 5 reviews and 9 statistical transmission, modelling or register linkage studies. Depending on the diagnosis method and the duration of follow-up, secondary attack rates (SAR) ranged from 4.6% when household contacts were followed for 14 days and tested only in case of symptoms to close to 90%. None of the household transmission studies involved long-term convalescent follow-up.Added value of this studyThis extensive (one month) active follow-up, using RT-PCR diagnosis and serological testing for SARS-CoV-2 nucleoprotein IgG antibodies (IgG Ab) and neutralizing antibodies (NAb) showed that household transmission was high, with a 48% (42/87) SAR overall and 50% [IQR: 0-100%] at the level of the household. All but one out of 64 RT-PCR confirmed participants had developed both IgG Ab and NAb after immediate convalescence. Six months after inclusion, majority of previously seropositive (IgG and/or NAb) participants still had IgG Ab (59/75) or NAb (63/75) showing long-term persistence of humoral immunity to SARS-CoV-2.Implications of all the available evidenceThe risk of transmission of SARS-CoV-2 infections within households is considerable. Isolation of the primary case, especially from household contacts with a high risk of severe disease, e.g. due to age or comorbidities, should be considered even though viral shedding might occur before confirmed diagnosis in household contacts. Long-term persistence of antibodies following infection, even in asymptomatic and mild cases, suggests enduring natural immunity and possibly protection from severe COVID-19.

Understanding for how long antibodies persist following Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection provides important insight into estimating the duration of immunity induced by infection.We assessed the persistence of serum antibodies following wild-type SARS-CoV-2 infection six and twelve months after diagnosis in 367 individuals of whom 13% had severe disease requiring hospitalization. We determined the SARS-CoV-2 spike (S-IgG) and nucleoprotein IgG concentrations and the proportion of subjects with neutralizing antibodies (NAb). We also measured the NAb titers among a smaller subset of participants (n=78) against a wild-type virus (B.1) and three variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2).We found that NAb against the wild-type virus and S-IgG persisted in 89% and 97% of subjects for at least twelve months after infection, respectively. IgG and NAb levels were higher after severe infection. NAb titers were significantly lower against variants compared to the wild-type virus.

The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility. We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, -20 °C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus.

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (~ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area.& nbsp;Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARSCoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.& nbsp;Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

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.

BackgroundSensitive and highly specific antibody tests are critical for detection of SARS-CoV-2 antibodies especially in populations where seroprevalence is low.AimTo set up, optimize and evaluate the analytical and clinical performance of a new in-house microsphere immunoassay for measurement of IgG antibodies to SARS-CoV-2 nucleoprotein for assessment of population seroprevalence in Finland.MethodsWe set up a new in-house microsphere immunoassay (FMIA) with SARS-CoV-2 nucleoprotein and optimized its analytical performance. For evaluation of clinical performance, we tested sera collected in a well-characterized cohort of PCR positive-confirmed SARS-CoV-2 patients (n=89) with mostly mild symptoms, and before the COVID-19 pandemic (n=402), for nucleoprotein specific IgG concentrations by FMIA and a commercial chemiluminescent immunoassay and for neutralizing antibodies by the microneutralization test.ResultsThe analytical performance of FMIA was established in terms of sensitivity, linearity and precision. FMIA discriminated between COVID-19 patient and control samples with high specificity (100%) and sensitivity (100%). We generated FMIA seropositivity cut-offs, 0.46 and 1.71 U/ml, for low- and high-seroprevalence settings, respectively. In addition, we obtained high level of agreement between FMIA results and results by the microneutralization test.ConclusionThe fluorescent microsphere immunoassay showed excellent analytical and clinical performance and is well suited for serosurveillance studies of SARS-CoV-2. However, to optimize analytical sensitivity and clinical specificity of the assay, different seropositivity thresholds depending on the intended use of the assay and the target population, may be needed.

SummaryBackgroundThe role of children in SARS-CoV-2 transmission is unclear. We investigated two COVID-19 school exposure incidents in the Helsinki area.MethodsWe conducted two retrospective cohort studies after schools exposures, with a household transmission extension. We defined a case as an exposed person with either a positive RT-PCR, or positive microneutralisation testing (MNT) as confirmation of SARS-CoV-2 nucleoprotein IgG antibodies detection via fluorescent microsphere immunoassay (FMIA). We recruited close school contacts and families of school cases, calculated attack rates (AR) on school level and families, and identified transmission chains.FindingsIn incident A, the index was a pupil. Participation rate was 74% (89/121), and no cases were identified. In incident B, the index was a member of school personnel. Participation rate was 81% (51/63). AR was 16% (8/51): 6 pupils and 1 member of school personnel were MNT and FMIA positive; 1 pupil had a positive RT-PCR, but negative serology samples. We visited all school cases’ families (n=8). The AR among close household contacts was 42% (9/20 in 3/8 families) but other plausible sources were always reported. At three months post-exposure, 6/8 school cases were re-sampled and still MNT positive.InterpretationWhen the index was a child, no school transmission was identified, while the occurrence of an adult case led to a 16% AR. Further cases were evidenced in 3 families, but other transmission chains were plausible. It is likely that transmission from children to adults is limited.FundingThe Finnish Institute for Health and Welfare funded this study.Research in contextEvidence before the studyThe first autochthonous case of COVID-19 in Finland was identified on February 29th. Transmission of the virus has led to more than 7250 cases and over 300 deaths (As of July 12th 2020). On March 16th, assuming that children might have a role in transmission, the Finnish government ordered school closures, to the exclusion of pre-school and grades 1-3. Schools were closed from March 18 and reopened on May 14th. At the stage of closure, a very limited number of reports of school related COVID-19 clusters or exposure incidents had been published, and the potential extent of transmission in a school setting was unknown.Added value of this studyWe investigated two exposure incidents in two different schools from the Helsinki area to assess transmission among pupils, school personnel and household contacts of identified cases. In school A, contact with a COVID-19 pupil did not lead to further transmission, while in school B, out of 51 recruited contacts, eight (16%) were proved to have had COVID-19 infection, including one member of staff. Among the close household contacts of pupils who were tested positive, COVID-19 attack rate was 31% (5/16). However, in all investigated households, other sources of infections were plausible; hence household transmission following a pediatric COVID-19 case appears to be limited.Implications of all of the available evidenceIncidence of COVID-19 infections in children following school related exposure was limited, as well as secondary transmission within their household. We hope our findings will help prioritize mitigation measures as well as reduce worry among parents of school aged children as most EU countries are preparing for the start of a new school year in autumn.

The first case of coronavirus disease (COVID-19) in Finland was confirmed on 29 January 2020. No secondary cases were detected. We describe the clinical picture and laboratory findings 3–23 days since the first symptoms. The SARS-CoV-2/Finland/1/2020 virus strain was isolated, the genome showing a single nucleotide substitution to the reference strain from Wuhan. Neutralising antibody response appeared within 9 days along with specific IgM and IgG response, targeting particularly nucleocapsid and spike proteins.

Another host factor for SARS-CoV-2 Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science, this issue p. 856, p. 861; see also p. 765, NRP1 serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19., The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

The Zika virus (ZIKV) is a member of the Flaviviridae family and an important human pathogen. Most pathogenic viruses encode proteins that interfere with the activation of host innate immune responses. Like other flaviviruses, ZIKV interferes with the expression of interferon (IFN) genes and inhibits IFN-induced antiviral responses. ZIKV infects through epithelial barriers where IFN-&lambda, 1 is an important antiviral molecule. In this study, we analyzed the effects of ZIKV proteins on the activation of IFN-&lambda, 1 promoter. All ZIKV proteins were cloned and transiently expressed. ZIKV NS5, but no other ZIKV protein, was able to interfere with the RIG-I signaling pathway. This inhibition took place upstream of interferon regulatory factor 3 (IRF3) resulting in reduced phosphorylation of IRF3 and reduced activation of IFN-&lambda, 1 promoter. Furthermore, we showed that ZIKV NS5 interacts with the protein kinase IKK&epsilon, which is likely critical to the observed inhibition of phosphorylation of IRF3.

Ebolaviruses (EBOV) cause severe disease with a recent outbreak in West Africa in 2014-2015 leading to more than 28 000 cases and 11 300 fatalities. This emphasizes the urgent need for better knowledge on these highly pathogenic RNA viruses. Host innate immune responses play a key role in restricting the spread of a viral disease. In this study we systematically analyzed the effects of cloned EBOV genes on the main host immune response to RNA viruses: the activation of RIG-I pathway and type I and III interferon (IFN) gene expression. EBOV VP24, in addition of inhibiting IFN-induced antiviral responses, was found to efficiently inhibit type III IFN-λ1 gene expression. This inhibition was found to occur downstream of IRF3 activation and to be dependent on VP24 importin binding residues. These results emphasize the importance of VP24 in EBOV infection cycle, making VP24 as an excellent target for drug development.

Mammalian cells express different types of RNA molecules that can be classified as protein coding RNAs (mRNA) and non-coding RNAs (ncRNAs) the latter of which have housekeeping and regulatory functions in cells. Cellular RNAs are not recognized by cellular pattern recognition receptors (PRRs) and innate immunity is not activated. RNA viruses encode and express RNA molecules that usually differ from cell-specific RNAs and they include for instance 5'capped and 5'mono- and triphosphorylated RNAs, small viral RNAs and viral RNA-protein complexes called vRNPs. These molecules are recognized by certain members of Toll-like receptor (TLR) and RIG-I-like receptor (RLR) families leading to activation of innate immune responses and the production of antiviral cytokines, such as type I and type III interferons (IFNs). Virus-specific ssRNA and dsRNA molecules that mimic the viral genomic RNAs or their replication intermediates can efficiently be produced by bacteriophage T7 DNA-dependent RNA polymerase and bacteriophage phi6 RNA-dependent RNA polymerase, respectively. These molecules can then be delivered into mammalian cells and the mechanisms of activation of innate immune responses can be studied. In addition, synthetic viral dsRNAs can be processed to small interfering RNAs (siRNAs) by a Dicer enzyme to produce a swarm of antiviral siRNAs. Here we describe the biology of RNAs, their in vitro production and delivery into mammalian cells as well as how these molecules can be used to inhibit virus replication and to study the mechanisms of activation of the innate immune system.

In this study we assessed the ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to replicate and induce innate immunity in human monocyte-derived macrophages and dendritic cells (MDDCs), and compared it with severe acute respiratory syndrome coronavirus (SARS-CoV). Assessments of viral protein and RNA levels in infected cells showed that both viruses were impaired in their ability to replicate in these cells. Some induction of IFN-λ1, CXCL10 and MxA mRNAs in both macrophages and MDDCs was seen in response to MERS-CoV infection, but almost no such induction was observed in response to SARS-CoV infection. ELISA and Western blot assays showed clear production of CXCL10 and MxA in MERS-CoV-infected macrophages and MDDCs. Our data suggest that SARS-CoV and MERS-CoV replicate poorly in human macrophages and MDDCs, but MERS-CoV is nonetheless capable of inducing a readily detectable host innate immune response. Our results highlight a clear difference between the viruses in activating host innate immune responses in macrophages and MDDCs, which may contribute to the pathogenesis of infection.

Influenza A viruses cause recurrent epidemics and occasional global pandemics. Wild birds are the natural reservoir of influenza A virus from where the virus can be transmitted to poultry or to mammals including humans. Mortality among humans in the highly pathogenic avian influenza H5N1 virus infection is even 60%. Despite intense research, there are still open questions in the pathogenicity of the H5N1 virus in humans. To characterize the H5N1 virus infection in human monocyte-derived macrophages (Mɸs) and dendritic cells (DCs), we used human isolates of highly pathogenic H5N1/2004 and H5N1/1997 and low pathogenic H7N9/2013 avian influenza viruses in comparison with a seasonal H3N2/1989 virus. We noticed that the H5N1 viruses have an overwhelming ability to replicate and spread in primary human immune cell cultures, and even the addition of trypsin did not equalize the infectivity of H7N9 or H3N2 viruses to the level seen with H5N1 virus. H5N1 virus stocks contained more often propagation-competent viruses than the H7N9 or H3N2 viruses. The data also showed that human DCs and Mɸs maintain 1,000- and 10,000-fold increase in the production of infectious H5N1 virus, respectively. Both analyzed highly pathogenic H5N1 viruses showed multi-cycle infection in primary human DCs and Mɸs, whereas the H3N2 and H7N9 viruses were incapable of spreading in immune cells. Interestingly, H5N1 virus was able to spread extremely efficiently despite the strong induction of antiviral interferon gene expression, which may in part explain the high pathogenicity of H5N1 virus infection in humans.

Recognition of viral nucleic acids leads to type I and type III IFN gene expression and activation of host antiviral responses. At present, type III IFN genes are the least well-characterized IFN types. Here, we demonstrate that the p38 MAPK signaling pathway is involved in regulating IFN-λ1 gene expression in response to various types of RNA molecules in human moDCs. Inhibition of p38 MAPK strongly reduced IFN gene expression, and overexpression of p38α MAPK enhanced IFN-λ1 gene expression in RNA-stimulated moDCs. The regulation of IFN gene expression by p38 MAPK signaling was independent of protein synthesis and thus, a direct result of RNA stimulation. Moreover, the RIG-I/MDA5-MAVS-IRF3 pathway was required for p38α MAPK to up-regulate IFN-λ1 promoter activation, whereas the MyD88-IRF7 pathway was not needed, and the regulation was not involved directly in IRF7-dependent IFN-α1 gene expression. The stimulatory effect of p38α MAPK on IFN-λ1 mRNA expression in human moDCs did not take place directly via the activating TBK1/IKKɛ complex, but rather, it occurred through some other parallel pathways. Furthermore, mutations in ISRE and NF-κB binding sites in the promoter region of the IFN-λ1 gene led to a significant reduction in p38α MAPK-mediated IFN responses after RNA stimulation. Altogether, our data suggest that the p38α MAPK pathway is linked with RLR signaling pathways and regulates the expression of early IFN genes after RNA stimulation cooperatively with IRF3 and NF-κB to induce antiviral responses further.

The activation of the interferon (IFN) system, which is triggered largely by the recognition of viral nucleic acids, is one of the most important host defense reactions against viral infections. Although influenza A and B viruses, which both have segmented negative-strand RNA genomes, share major structural similarities, they have evolutionarily diverged, with total genetic incompatibility. Here we compare antiviral-inducing mechanisms during infections with type A and B influenza viruses in human dendritic cells. We observed that IFN responses are induced significantly faster in cells infected with influenza B virus than in cells infected with type A influenza virus and that the early induction of antiviral gene expression is mediated by the activation of the transcription factor IFN regulatory factor 3 (IRF3). We further demonstrate that influenza A virus infection activates IFN responses only after viral RNA (vRNA) synthesis, whereas influenza B virus induces IFN responses even if its infectivity is destroyed by UV treatment. Thus, initial viral transcription, replication, and viral protein synthesis are dispensable for influenza B virus-induced antiviral responses. Moreover, vRNA molecules from both type A and B viruses are equally potent activators of IFN induction, but incoming influenza B virus structures are recognized directly in the cytosol, while influenza A virus is able to evade early recognition. Collectively, our data provide new evidence of a novel antiviral evasion strategy for influenza A virus without a contribution of the viral NS1 protein, and this opens up new insights into different influenza virus pathogenicities.

Induction of type III IFNs in bacteria-infected human moDCs requires bacterial phagocytosis in conjunction with signaling. Type I IFNs (IFN-α/βs) and type III IFNs (IFN-λ1–3) play an important role in host defense against viral infections. The induction of type I IFNs has recently been found to take place also in bacterial infections, and therefore, this study focuses on analyzing the regulation of type III IFNs in response to bacterial stimulation. We found by quantitative RT-PCR that the expression of IFN-λ1 and IFN-λ2/3 mRNAs, as well as that of IFN-β, was similarly up-regulated in response to stimulation with live Salmonella typhimurium or TLR4 agonist LPS in human moDCs. The induction of IFN-λ mRNAs did not require ongoing protein synthesis, and only IFN-λ1 was detected at the protein level. The induction of IFN-λ mRNAs was sensitive to SB202190, Ly294002, and PDTC, which inhibit p38 MAPK, PI3K, and NF-κB activation, respectively. Furthermore, we observed that blocking dynamin-dependent endocytosis pathways with dynasore led to decreased cell surface expression of CD86 and HLA class II molecules and reduced production of IFN-λ1, CXCL10, and IL-6 when the cells were infected with S. typhimurium. Cytokine production was also impaired in dynasore-treated, Streptococcus thermophilus-stimulated cells. Further, inhibition of dynamin prevented S. typhimurium-induced phosphorylation of IRF3 and the internalization of the bacteria. In summary, induction of type III IFNs in bacteria-infected human moDCs requires multiple signaling pathways and involves bacterial phagocytosis.

Virus replication induces the expression of antiviral type I (IFN-αβ) and type III (IFN-λ1–3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-λ1 and IFN-λ3 gene promoters revealed them to be similar to IFN-β and IFN-α genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-κB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-λ1 promoter, whereas the IFN-λ3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-λ1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-β gene, whereas IFN-λ2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-α genes.

Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IFN regulatory factor 3 (IRF3) activation and IFN-λ1 gene expression with faster kinetics than does influenza A virus, without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes, and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I-, MDA5-, and RIG-I/MDA5-deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids early IRF3 activation and IFN gene induction. IMPORTANCE Recently, a great deal of interest has been paid to identifying the ligands for RIG-I under conditions of natural infection, as many previous studies have been based on transfection of cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of influenza B virus by human macrophages. We show that influenza B virus induces IRF3 activation, leading to IFN gene expression after viral RNPs (vRNPs) are released into the cytosol and are recognized by RIG-I receptor, meaning that the incoming influenza B virus is already able to activate IFN gene expression. In contrast, influenza A (H3N2) virus failed to activate IRF3 at very early times of infection, suggesting that there are differences in innate immune recognition between influenza A and B viruses.

Lung epithelial cells are the primary cellular targets for respiratory virus pathogens such as influenza and parainfluenza viruses. Here, we have analyzed influenza A, influenza B and Sendai virus-induced chemokine response in human A549 lung epithelial cells. Influenza virus infection resulted in low CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8 and CXCL10/IP-10 production at late times of infection. However, when cells were pretreated with TNF-alpha or IFN-alpha, influenza-A-virus-induced chemokine production was greatly enhanced. Cytokine pretreatment resulted in enhanced expression of RIG-I, IKKepsilon, interferon regulatory factor (IRF)1, IRF7 and p50 proteins. Most importantly, influenza-A-virus-induced DNA binding of IRF1, IRF3, IRF7 and NF-kappaB onto CXCL10 ISRE and NF-kappaB elements, respectively, was markedly enhanced in cytokine-pretreated cells. Our results suggest that IFN-alpha and TNF-alpha have a significant role in priming epithelial cells for higher cytokine and chemokine production in influenza A virus infection.

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-α], IFN-β, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-α/β-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.

The precise role of leukocytes and mediators in human milk is still unresolved. Eosinophils are uncommonly detected in human milk and their presence has previously been associated with maternal atopy and development of cow's milk allergy (CMA) in the breast-fed infant. The purpose of this study was to examine the levels of eosinophil cationic protein (ECP) in human milk and to compare the levels with development of allergic diseases in breast-fed infants. Altogether 94 breast-feeding mothers (58 atopic, 36 nonatopic) with their babies were prospectively followed from birth for development of CMA or atopic dermatitis. Colostrum and mature milk samples (at 3 mo of lactation), together with mother's peripheral blood samples, were collected. Milk and blood leukocyte content was evaluated with a light microscope. ECP concentration in human milk was measured by commercial UniCAP method. By the end of a 2-y follow-up, 51 mothers had an infant with CMA, 24 had an infant with atopic dermatitis, and 19 had a healthy infant. ECP concentration in milk was under the detection limit (2 microg/L) in all the mothers with a healthy infant, whereas detectable levels were found in 27% of mothers with a CMA infant and in 42% of those with a baby with atopic dermatitis. Measurable ECP in milk was detected in 26% of the atopic and 25% of the nonatopic mothers. Presence of ECP in human milk is associated with development of CMA and atopic dermatitis in the breast-fed infant, but has no direct association with the maternal atopy.