A species-independent lateral flow microarray immunoassay to detect WNV and USUV NS1-specific antibodies in serum.

Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws (Corvus monedula), and common blackbirds (Turdus merula). Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to "gold standard" WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions.

The increasing complexity of arbovirus serology: An in-depth systematic review on cross-reactivity.

Diagnosis of arbovirus infection or exposure by antibody testing is becoming increasingly difficult due to global expansion of arboviruses, which induce antibodies that may (cross-)react in serological assays. We provide a systematic review of the current knowledge and knowledge gaps in differential arbovirus serology. The search included Medline, Embase and Web of Science databases and identified 911 publications which were reduced to 102 after exclusion of studies not providing data on possible cross-reactivity or studies that did not meet the inclusion criteria regarding confirmation of virus exposure of reference population sets. Using a scoring system to further assess quality of studies, we show that the majority of the selected papers (N = 102) provides insufficient detail to support conclusions on specificity of serological outcomes with regards to elucidating antibody cross-reactivity. Along with the lack of standardization of assays, metadata such as time of illness onset, vaccination, infection and travel history, age and specificity of serological methods were most frequently missing. Given the critical role of serology for diagnosis and surveillance of arbovirus infections, better standards for reporting, as well as the development of more (standardized) specific serological assays that allow discrimination between exposures to multiple different arboviruses, are a large global unmet need.

The pathology of co-infection with Usutu virus and Plasmodium spp. in naturally infected Eurasian blackbirds (Turdus merula).

Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus causing mortality in Eurasian blackbirds (Turdus merula) in Europe. In dead blackbirds, avian malaria co-infection due to mosquito-borne hemosporidians (e.g., Plasmodium spp.) has been reported. In humans, a similar co-infection of a flavivirus, Dengue virus, and Plasmodium spp. is causing increased severity of clinical disease. Currently, the effects of co-infection of arboviruses and hemosporidians in blackbirds remain unclear. This study investigates the rate of USUV and Plasmodium spp. co-infection in found-dead blackbirds (n = 203) from 2016 to 2020 in the Netherlands. Presence of Plasmodium spp. was evaluated by cytology (43/203; 21,2%), histopathology (94/186; 50,5%) and qPCR (179/203; 88,1%). The severity of histological lesions in USUV and Plasmodium spp. co-infected dead blackbirds (121/203; 59,6%) were compared with those in Plasmodium spp. single-infected cases. Additionally, since no knowledge is present on the infection rate on live birds and mosquitoes in the Netherlands, a small group of live blackbirds (n = 12) and selected in the field-collected mosquito pools (n = 96) in 2020 were tested for the presence of Plasmodium spp. The latter was detected in the tested live blackbirds by qPCR (8/10; 80%), and cytology (3/11; 27,3%) and in the mosquito pools by qPCR (18/96; 18,7%). For this study, co-infection between USUV and Plasmodium spp. was observed only in the dead blackbirds. The high Plasmodium spp. presence, associated with lower lesions score, in single infected found dead birds suggest a predominantly smaller pathogenic role as single agent. On the other hand, the higher histological lesion scores observed in USUV and Plasmodium spp. co-infected birds suggests a major pathogenic role for the virus or an increased severity of the lesions due to a possible interplay of the two agents.

Assessing West Nile virus (WNV) and Usutu virus (USUV) exposure in bird ringers in the Netherlands: a high-risk group for WNV and USUV infection?

Introduction: In 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained neurological disease revealed human WNV infections in July and August 2020. Bird ringers are highly exposed to mosquito bites and possibly avian excrements during ringing activities. This study therefore investigates whether bird ringers are at higher risk of exposure to WNV and Usutu virus (USUV). Methods: Dutch bird ringers were asked to provide a single serum sample (May - September 2021) and to fill out a survey. Sera were screened by protein microarray for presence of specific IgG against WNV and USUV non-structural protein 1 (NS1), followed by focus reduction virus neutralization tests (FRNT). Healthcare workers (2009-2010), the national immunity cohort (2016-2017) and blood donors (2021) were used as control groups without this occupational exposure. Results: The majority of the 157 participating bird ringers was male (132/157, 84%) and the median age was 62 years. Thirty-seven participants (37/157, 23.6%) showed WNV and USUV IgG microarray signals above background, compared to 6.4% (6/94) in the community cohort and 2.1% (2/96) in blood donors (p < 0.01). Two seroreactive bird ringers were confirmed WNV or USUV positive by FRNT. The majority of seroreactive bird ringers travelled to EU countries with reported WNV human cases (30/37, 81%) (p = 0.07). No difference was observed between bird ringers with and without previous yellow fever vaccination. Discussion: The higher frequency of WNV and/or USUV IgG reactive bird ringers indicates increased flavivirus exposure compared to the general population, suggesting that individuals with high-exposure professions may be considered to complement existing surveillance systems. However, the complexity of serological interpretation in relation to location-specific exposure (including travel), and antibody cross-reactivity, remain a challenge when performing surveillance of emerging flaviviruses in low-prevalence settings.

Monkeypox Virus Cross-Neutralizing Antibodies in Clinical Trial Participants Vaccinated With Modified Vaccinia Virus Ankara Encoding Middle East Respiratory Syndrome-Coronavirus Spike Protein.

Modified vaccinia virus Ankara (MVA) is used as a vaccine against monkeypox virus and as a viral vaccine vector. MVA-MERS-S is a vaccine candidate against Middle East respiratory syndrome (MERS)-associated coronavirus. Here, we report that cross-reactive monkeypox virus neutralizing antibodies were detectable in only a single study participant after the first dose of MVA-MERS-S vaccine, in 3 of 10 after the second dose, and in 10 of 10 after the third dose.

Evaluation of the use of alternative sample types for mosquito-borne flavivirus surveillance: Using Usutu virus as a model.

Wild birds are reservoirs of several zoonotic arboviruses including West Nile virus (WNV) and Usutu virus (USUV), and are often monitored as indicators for virus introduction and spread. To optimize the bird surveillance for arboviruses in the Netherlands and to explore the possibilities for citizen science in surveillance, we investigated the suitability of using alternative sample types from live and dead birds. The sensitivity of molecular detection via RT-PCR of viral RNA in feather, heart, lung, throat and cloaca swabs from dead birds, and serum, dried blood spots (DBS) and throat and cloaca swabs from live birds were compared. IgY antibody detection was also assessed from DBS relative to serum on protein-microarray and virus neutralization test. Feathers showed a high detection sensitivity for USUV RNA in both live and dead birds, and no significant decrease was observed in the RNA loads in the feathers after being stored dry at room temperature for 43 days. Additionally, viral RNAs extracted from feathers of day 0 and 43 were successfully sequenced. The results indicated no statistical significant difference in sensitivity and viral loads detection in heart, spleen, and lung relative to corresponding brain samples in dead birds. In live birds, viral RNA loads did not differ between throat and cloaca swabs. This study identified less-invasive sample types that allows involvement of citizens in collecting samples from wild birds for arbovirus surveillance. Sensitivity and specificity of DBS-based antibody detections were significantly lower and therefore need optimization.

Risks of SARS-CoV-2 transmission between free-ranging animals and captive mink in the Netherlands.

In the Netherlands, 69 of the 126 (55%) mink farms in total became infected with SARS-CoV-2 in 2020. Despite strict biosecurity measures and extensive epidemiological investigations, the main transmission route remained unclear. A better understanding of SARS-CoV-2 transmission between mink farms is of relevance for countries where mink farming is still common practice and can be used as a case study to improve future emerging disease preparedness. We assessed whether SARS-CoV-2 spilled over from mink to free-ranging animals, and whether free-ranging animals may have played a role in farm-to-farm transmission in the Netherlands. The study encompassed farm visits, farm questionnaires, expert workshops and SARS-CoV-2 RNA and antibody testing of samples from target animal species (bats, birds and free-ranging carnivores). In this study, we show that the open housing system of mink allowed access to birds, bats and most free-ranging carnivores, and that direct and indirect contact with mink was likely after entry, especially for free-ranging carnivores and birds. This allowed SARS-CoV-2 exposure to animals entering the mink farm, and subsequent infection or mechanical carriage by the target animal species. Moreover, mink can escape farms in some cases, and two SARS-CoV-2-positive mink were found outside farm premises. No other SARS-CoV-2-RNA-positive free-ranging animals were detected, suggesting there was no abundant circulation in the species tested during the study period. To investigate previous SARS-CoV-2 infections, SARS-CoV-2 antibody detection using lung extracts of carcasses was set up and validated. One tested beech marten did have SARS-CoV-2 antibodies, but the closest SARS-CoV-2-infected mink farm was outside of its home range, making infection at a mink farm unlikely. Knowing that virus exchange between different species and the formation of animal reservoirs affects SARS-CoV-2 evolution, continued vigilance and monitoring of mink farms and surrounding wildlife remains vital.

Profiling of humoral immune responses to norovirus in children across Europe.

Norovirus is a leading cause of epidemic acute gastroenteritis. More than 30 genotypes circulate in humans, some are common, and others are only sporadically detected. Here, we investigated whether serology can be used to determine which genotypes infect children. We established a multiplex protein microarray with structural and non-structural norovirus antigens that allowed simultaneous antibody testing against 30 human GI and GII genotypes. Antibody responses of sera obtained from 287 children aged < 1 month to 5.5 years were profiled. Most specific IgG and IgA responses were directed against the GII.2, GII.3, GII.4, and GII.6 capsid genotypes. While we detected antibody responses against rare genotypes, we found no evidence for wide circulation. We also detected genotype-specific antibodies against the non-structural proteins p48 and p22 in sera of older children. In this study, we show the age-dependent antibody responses to a broad range of norovirus capsid and polymerase genotypes, which will aid in the development of vaccines.

Transmission of SARS-CoV-2 within households: a remote prospective cohort study in European countries.

Household transmission studies are useful to quantify SARS-CoV-2 transmission dynamics. We conducted a remote prospective household study to quantify transmission, and the effects of subject characteristics, household characteristics, and implemented infection control measures on transmission. Households with a laboratory-confirmed SARS-CoV-2 index case were enrolled < 48 h following test result. Follow-up included digitally daily symptom recording, regular nose-throat self-sampling and paired dried blood spots from all household members. Samples were tested for virus detection and SARS-CoV-2 antibodies. Secondary attack rates (SARs) and associated factors were estimated using logistic regression. In 276 households with 920 participants (276 index cases and 644 household members) daily symptom diaries and questionnaires were completed by 95%, and > 85% completed sample collection. 200 secondary SARS-CoV-2 infections were detected, yielding a household SAR of 45.7% (95% CI 39.7-51.7%) and per-person SAR of 32.6% (95%CI: 28.1-37.4%). 126 (63%) secondary cases were detected at enrollment. Mild (aRR = 0.57) and asymptomatic index cases (aRR = 0.29) were less likely to transmit SARS-CoV-2, compared to index cases with an acute respiratory illness (p = 0.03 for trend), and child index cases (< 12 years aRR = 0.60 and 12-18 years aRR = 0.85) compared to adults (p = 0.03 for trend). Infection control interventions in households had no significant effect on transmission. We found high SARs with the majority of transmissions occuring early after SARS-CoV-2 introduction into the household. This may explain the futile effect of implemented household measures. Age and symptom status of the index case influence secondary transmission. Remote, digitally-supported study designs with self-sampling are feasible for studying transmission under pandemic restrictions.

Poxvirus MVA Expressing SARS-CoV-2 S Protein Induces Robust Immunity and Protects Rhesus Macaques From SARS-CoV-2.

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials.

Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands.

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.

Age-seroprevalence curves for the multi-strain structure of influenza A virus.

The relationship between age and seroprevalence can be used to estimate the annual attack rate of an infectious disease. For pathogens with multiple serologically distinct strains, there is a need to describe composite exposure to an antigenically variable group of pathogens. In this study, we assay 24,402 general-population serum samples, collected in Vietnam between 2009 to 2015, for antibodies to eleven human influenza A strains. We report that a principal components decomposition of antibody titer data gives the first principal component as an appropriate surrogate for seroprevalence; this results in annual attack rate estimates of 25.6% (95% CI: 24.1% - 27.1%) for subtype H3 and 16.0% (95% CI: 14.7% - 17.3%) for subtype H1. The remaining principal components separate the strains by serological similarity and associate birth cohorts with their particular influenza histories. Our work shows that dimensionality reduction can be used on human antibody profiles to construct an age-seroprevalence relationship for antigenically variable pathogens.

Seasonal coronavirus-specific B cells with limited SARS-CoV-2 cross-reactivity dominate the IgG response in severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing "original antigenic sin."

Using NS1 Flavivirus Protein Microarray to Infer Past Infecting Dengue Virus Serotype and Number of Past Dengue Virus Infections in Vietnamese Individuals.

BACKGROUND: In recent years, researchers have had an increased focus on multiplex microarray assays, in which antibodies are measured against multiple related antigens, for use in seroepidemiological studies to infer past transmission. METHODS: We assess the performance of a flavivirus microarray assay for determining past dengue virus (DENV) infection history in a dengue-endemic setting, Vietnam. We tested the microarray on samples from 1 and 6 months postinfection from DENV-infected patients (infecting serotype was determined using reverse-transcription polymerase chain reaction during acute, past primary, and secondary infection assessed using plaque reduction neutralization tests 6 months postinfection). RESULTS: Binomial models developed to discriminate past primary from secondary infection using the protein microarray (PMA) titers had high area under the curve (0.90-0.97) and accuracy (0.84-0.86). Multinomial models developed to identify most recent past infecting serotype using PMA titers performed well in those with past primary infection (average test set: κ = 0.85, accuracy of 0.92) but not those with past secondary infection (κ = 0.24, accuracy of 0.45). CONCLUSIONS: Our results suggest that the microarray will be useful in seroepidemiological studies aimed at classifying the past infection history of individuals (past primary vs secondary and serotype of past primary infections) and thus inferring past transmission intensity of DENV in dengue-endemic settings. Future work to validate these models should be undertaken in different transmission settings and with samples later after infection.

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients.

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Bearing the brunt: Mongolian khulan (Equus hemionus hemionus) are exposed to multiple influenza A strains.

The majority of influenza A virus strains are hosted in nature by avian species in the orders of Anseriformes and Charadriformes. A minority of strains have been able to cross species boundaries and establish themselves in novel non-avian hosts. Influenza viruses of horses, donkeys, and mules represent such successful events of avian to mammal influenza virus adaptation. Mongolia has over 3 million domestic horses and is home to two wild equids, the Asiatic wild ass or khulan (Equus hemionus hemionus), and Przewalski's horse (Equus ferus przewalskii). Domestic and wild equids are sympatric across most of their range in Mongolia. Epizootic influenza A virus outbreaks among Mongolian domestic horses have been frequently recorded. However, the exposure, circulation and relation to domestic horse influenza A virus outbreaks among wild equids is unknown. We evaluated serum samples of Asiatic wild asses in Mongolia for antibodies against influenza A viruses, using modified protein microarray technique. We detected antibodies against hemagglutinin (H) H1, H3, H5, H7, H8 and H10 influenza A viruses. Asiatic wild asses may represent a previously unidentified influenza A virus reservoir in an ecosystem shared with populations of domestic horses in which influenza strains circulate.

Diet May Drive Influenza A Virus Exposure in African Mammals.

BACKGROUND: Influenza A viruses (IAVs) represent repeatedly emerging pathogens with near worldwide distribution and an unclear nonavian-host spectrum. While the natural hosts for IAV are among waterfowl species, certain mammals can be productively infected. Southern Africa is home to diverse avian and mammalian fauna for which almost no information exists on IAV dynamics. METHODS: We evaluated 111 serum samples from 14 mammalian species from Namibia for the presence of IAV-specific antibodies and tested whether host phylogeny, sociality, or diet influence viral prevalence and diversity. RESULTS: Free-ranging African mammals are exposed to diverse IAV subtypes. Herbivores developed antibodies against 3 different hemagglutinin (HA) subtypes, at low prevalence, while carnivores showed a higher prevalence and diversity of HA-specific antibody responses against 11 different subtypes. Host phylogeny and sociality were not significantly associated with HA antibody prevalence or subtype diversity. Both seroprevalence and HA diversity were significantly increased in carnivores regularly feeding on birds. CONCLUSIONS: The risk of infection and transmission may be driven by diet and ecological factors that increase contact with migratory and resident waterfowl. Consequently, wild mammals, particularly those that specialize on hunting and scavenging birds, could play an important but overlooked role in influenza epizootics.

MERS-CoV in Camels but Not Camel Handlers, Sudan, 2015 and 2017.

We tested samples collected from camels, camel workers, and other animals in Sudan and Qatar in 2015 and 2017 for evidence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV antibodies were abundant in Sudan camels, but we found no evidence of MERS-CoV infection in camel workers, other livestock, or bats.

Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.