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Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa , ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Tick-borne encephalitis is a vaccine-preventable disease of concern for public health in large parts of Europe, with EU notification rates increasing since 2018. It is caused by the orthoflavivirus tick-borne encephalitis virus (TBEV) and a diagnosis of infection is mainly based on serology due to its short viremic phase, often before symptom onset. The interpretation of TBEV serology is hampered by a history of orthoflavivirus vaccination and by previous infections with related orthoflaviviruses. Here, we sought to improve TBEV sero-diagnostics using an antigen combination of in-house expressed NS1 and EDIII in a multiplex, low-specimen-volume set-up for the detection of immune responses to TBEV and other clinically important orthoflaviviruses (i.e., West Nile virus, dengue virus, Japanese encephalitis virus, Usutu virus and Zika virus). We show that the combined use of NS1 and EDIII results in both a specific and sensitive test for the detection of TBEV IgG for patient diagnostics, vaccination responses and in seroprevalence studies. This novel approach potentially allows for a low volume-based, simultaneous analysis of IgG responses to a range of orthoflaviviruses with overlapping geographic circulations and clinical manifestations.

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland., (© 2023. The Author(s).)

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in 242 household members of different ages and with different symptom severity after SARS-CoV-2 exposure early in the pandemic (March-April 2020). Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable., (© 2022. The Author(s).)

Defining immune correlates of disease severity is important to better understand the immunopathogenesis in COVID-19. Here we made use of a protein microarray platform to detect IgG- and IgA-reactive antibodies in sera and saliva respectively, and assess cross-reactivity between SARS-CoV-2 and endemic coronaviruses (eCoVs). IgG responses against the full protein of spike, but not the S1 subunit, were significantly higher in convalescent sera of patients with severe disease compared to mild disease and healthy controls. In addition, we detected reactivity of secretory IgA to eCoVs in saliva of patients with severe disease, not present in patients with moderate disease or seropositive healthy controls. These heterologous immune responses are in line with non-protective cross-reactivity, and support a potential role for immune imprinting in the pathogenesis of severe COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Smit, van Tol, Wal, van Vulpen, la Grouw, van Lelyveld, Limonard, Bossink, Godeke, Shrestha, Reimerink, Eggink, Reusken, Heron and Thijsen.)

SARS-CoV-2 infection in children is less severe than it is in adults. We perform a longitudinal analysis of the early innate responses in children and adults with mild infection within household clusters. Children display fewer symptoms than adults do, despite similar initial viral load, and mount a robust anti-viral immune signature typical of the SARS-CoV-2 infection and characterized by early interferon gene responses; increases in cytokines, such as CXCL10 and GM-CSF; and changes in blood cell numbers. When compared with adults, the antiviral response resolves faster (within a week of symptoms), monocytes and dendritic cells are more transiently activated, and genes associated with B cell activation appear earlier in children. Nonetheless, these differences do not have major effects on the quality of SARS-CoV-2-specific antibody responses. Our findings reveal that better early control of inflammation as observed in children may be key for rapidly controlling infection and limiting the disease course., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Real-time reverse transcription-polymerase chain reaction (RT-PCR) on upper respiratory tract (URT) samples is the primary method to diagnose SARS-CoV-2 infections and guide public health measures, with a supportive role for serology. We reinforce previous findings on limited sensitivity of PCR testing, and solidify this fact by statistically utilizing a firm basis of multiple tests per individual. We integrate stratifications with respect to several patient characteristics such as severity of disease and time since onset of symptoms. Bayesian statistical modelling was used to retrospectively determine the sensitivity of RT-PCR using SARS-CoV-2 serology in 644 COVID-19-suspected patients with varying degrees of disease severity and duration. The sensitivity of RT-PCR ranged between 80% - 95%; increasing with disease severity, it decreased rapidly over time in mild COVID-19 cases. Negative URT RT-PCR results should be interpreted in the context of clinical characteristics, especially with regard to containment of viral transmission based on 'test, trace and isolate'. Keywords: SARS-CoV-2, RT-PCR, serology, sensitivity, public health., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging SARS-CoV-2 variants of concern (VOCs) pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three VOCs (B.1.1.7, B.1.351, and P.1) in cohorts of COVID-19 convalescent patients ( n = 69) and Pfizer-BioNTech vaccine recipients ( n = 50). Spike binding and neutralization against all three VOCs were substantially reduced in most individuals, with the largest four- to sevenfold reduction in neutralization being observed against B.1.351. While hospitalized patients with COVID-19 and vaccinees maintained sufficient neutralizing titers against all three VOCs, 39% of nonhospitalized patients exhibited no detectable neutralization against B.1.351. Moreover, monoclonal neutralizing antibodies show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1 but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOCs and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOCs.

Serology is a crucial part of the public health response to the ongoing SARS-CoV-2 pandemic. Here, we describe the development, validation and clinical evaluation of a protein micro-array as a quantitative multiplex immunoassay that can identify S and N-directed SARS-CoV-2 IgG antibodies with high specificity and sensitivity and distinguish them from all currently circulating human coronaviruses. The method specificity was 100% for SARS-CoV-2 S1 and 96% for N antigen based on extensive syndromic (n=230 cases) and population panel (n=94) testing that also confirmed the high prevalence of seasonal human coronaviruses. To assess its potential role for both SARS-CoV-2 patient diagnostics and population studies, we evaluated a large heterogeneous COVID-19 cohort (n=330) and found an overall sensitivity of 89% (≥ 21 days post onset symptoms (dps)), ranging from 86% to 96% depending on severity of disease. For a subset of these patients longitudinal samples were provided up to 56 dps. Mild cases showed absent or delayed, and lower SARS-CoV-2 antibody responses. Overall, we present the development and extensive clinical validation of a multiplex coronavirus serological assay for syndromic testing, to answer research questions regarding to antibody responses, to support SARS-CoV-2 diagnostics and to evaluate epidemiological developments efficiently and with high-throughput.

To understand SARS-CoV-2 immunity after natural infection or vaccination, functional assays such as virus neutralising assays are needed. So far, assays to detect SARS-CoV-2 neutralising antibodies rely on cell-culture based infection assays either using wild type SARS-CoV-2 or pseudotyped viruses. Such assays are labour-intensive, require appropriate biosafety facilities and are difficult to standardize. Recently, a new surrogate virus neutralisation test (sVNT) was described that uses the principle of an ELISA to measure the neutralisation capacity of anti-SARS-CoV-2 antibodies directed against the receptor binding domain. Here, we performed an independent evaluation of the robustness, specificity and sensitivity on an extensive panel of sera from 269 PCR-confirmed COVID-19 cases and 259 unmatched samples collected before 2020 and compared it to cell-based neutralisation assays. We found a high specificity of 99.2 (95%CI: 96.9-99.9) and overall sensitivity of 80.3 (95%CI: 74.9-84.8) for the sVNT. Clinical sensitivity increased between early (<14 days post symptom onset or post diagnosis, dpos/dpd) and late sera (>14 dpos/dpd) from 75.0 (64.7-83.2) to 83.1 (76.5-88.1). Also, higher severity was associated with an increase in clinical sensitivity. Upon comparison with cell-based neutralisation assays we determined an analytical sensitivity of 74.3 (56.4-86.9) and 98.2 (89.4-99.9) for titres ≥10 to <40 and ≥40 to <160, respectively. Only samples with a titre ≥160 were always positive in the sVNT. In conclusion, the sVNT can be used as an additional assay to determine the immune status of COVID-19 infected of vaccinated individuals but its value needs to be assessed for each specific context.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes., (© 2016 Blackwell Verlag GmbH.)

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified virus causing severe viral respiratory illness in people. Little is known about the reservoir in the Horn of Africa. In Kenya, where no human MERS cases have been reported, our survey of 335 dromedary camels, representing nine herds in Laikipia County, showed a high seroprevalence (46.9%) to MERS-CoV antibodies. Between herd differences were present (14.3%- 82.9%), but was not related to management type or herd isolation. Further research should focus on identifying similarity between MERS-CoV viral isolates in Kenya and clinical isolates from the Middle East and elsewhere.

We determined the presence of neutralizing antibodies to Middle East respiratory syndrome coronavirus in persons in Qatar with and without dromedary contact. Antibodies were only detected in those with contact, suggesting dromedary exposure as a risk factor for infection. Findings also showed evidence for substantial underestimation of the infection in populations at risk in Qatar.

Two of the earliest Middle East respiratory syndrome (MERS) cases were men who had visited the Doha central animal market and adjoining slaughterhouse in Qatar. We show that a high proportion of camels presenting for slaughter in Qatar show evidence for nasal MERS-CoV shedding (62/105). Sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of MERS-CoV circulation and a high-risk area for human exposure. No correlation between RNA loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure.

Background: The family Flaviviridae, genus Flavivirus, holds many of the world's most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods., Goal: We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray., Results: Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses., Conclusion: Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses.

We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula.

Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral RNA was detected in milk of five of these seven camels. The presence of MERS-CoV RNA in milk of camels actively shedding the virus warrants measures to prevent putative food-borne transmission of MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe lower respiratory tract infection in people. Previous studies suggested dromedary camels were a reservoir for this virus. We tested for the presence of MERS-CoV in dromedary camels from a farm in Qatar linked to two human cases of the infection in October, 2013., Methods: We took nose swabs, rectal swabs, and blood samples from all camels on the Qatari farm. We tested swabs with RT-PCR, with amplification targeting the E gene (upE), nucleocapsid (N) gene, and open reading frame (ORF) 1a. PCR positive samples were tested by different MERS-CoV specific PCRs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. We tested serum samples from the camels for IgG immunofluorescence assay, protein microarray, and virus neutralisation assay., Findings: We obtained samples from 14 camels on Oct 17, 2013. We detected MERS-CoV in nose swabs from three camels by three independent RT-PCRs and sequencing. The nucleotide sequence of an ORF1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the MERS-CoV from two human cases on the same farm and a MERS-CoV isolate from Hafr-Al-Batin. Eight additional camel nose swabs were positive on one or more RT-PCRs, but could not be confirmed by sequencing. All camels had MERS-CoV spike-binding antibodies that correlated well with the presence of neutralising antibodies to MERS-CoV., Interpretation: Our study provides virological confirmation of MERS-CoV in camels and suggests a recent outbreak affecting both human beings and camels. We cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible., Funding: European Union projects EMPERIE (contract number 223498), ANTIGONE (contract number 278976), and the VIRGO consortium., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Between June and September 2013, sera from 11 dromedary camels, 150 goats, 126 sheep and 91 cows were collected in Jordan, where the first human Middle-East respiratory syndrome (MERS) cluster appeared in 2012. All sera were tested for MERS-coronavirus (MERS-CoV) specific antibodies by protein microarray with confirmation by virus neutralisation. Neutralising antibodies were found in all camel sera while sera from goats and cattle tested negative. Although six sheep sera reacted with MERS-CoV antigen, neutralising antibodies were not detected.

Background: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock., Methods: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus., Findings: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies., Interpretation: MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection., Funding: European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The emergence of the A(H1N1) 2009 pandemic influenza virus was initially seen as a major world-wide health concern since a low degree of immunity to this virus strain was anticipated. However, age-specific infection attack rates and age-specific differences in seroresponse indicate that pre-existing immunity may have played a significant role in protection especially in older age groups. This study describes the use of a protein microarray as a multiplex analysis tool for detection of influenza virus H1 strain-specific memory B-cells before and after infection with A(H1N1)pdm09. The discrimination was based on detection of specific antibodies in culture supernatants from polyclonally stimulated B-cells against recombinant influenza virus HA1 proteins representing influenza virus subtypes H1 through H9. The protein microarray proved sensitive and specific for antibody detection in culture supernatants of B-cells, and with the potential to deduce a person's history of infection with particular influenza virus variants, including A(H1N1)pdm09. Blood samples obtained from different age groups prior to the pandemic in 2009 partly showed the presence of B-cells producing antibodies binding to the closely related A(H1N1) 1918 pandemic influenza virus, and of which the magnitude increased with age. These cross-reactive antibodies were produced by single memory B-cells present in these donors, and either bind to epitopes on HA1 which are shared within different H1 strains (homosubtypic response) or shared between different subtypes (heterosubtypic response)., (Copyright © 2013 Wiley Periodicals, Inc.)

We present a serological assay for the specific detection of IgM and IgG antibodies against the emerging human coronavirus hCoV-EMC and the SARS-CoV based on protein microarray technology. The assay uses the S1 receptor-binding subunit of the spike protein of hCoV-EMC and SARS-CoV as antigens. The assay has been validated extensively using putative cross-reacting sera of patient cohorts exposed to the four common hCoVs and sera from convalescent patients infected with hCoV-EMC or SARS-CoV.

The emergence of Schmallenberg virus (SBV), a novel orthobunyavirus, in ruminants in Europe triggered a joint veterinary and public health response to address the possible consequences to human health. Use of a risk profiling algorithm enabled the conclusion that the risk for zoonotic transmission of SBV could not be excluded completely. Self-reported health problems were monitored, and a serologic study was initiated among persons living and/or working on SBV-affected farms. In the study set-up, we addressed the vector and direct transmission routes for putative zoonotic transfer. In total, 69 sheep farms, 4 goat farms, and 50 cattle farms were included. No evidence for SBV-neutralizing antibodies was found in serum of 301 participants. The lack of evidence for zoonotic transmission from either syndromic illness monitoring or serologic testing of presumably highly exposed persons suggests that the public health risk for SBV, given the current situation, is absent or extremely low.

Western tourists are increasingly traveling to exotic locations often located in tropical or subtropical regions of the world. The magnitude of international travel and the constantly changing dynamics of arbovirus diseases across the globe demand up-to-date information about arbovirus threats to travelers and the countries they visit. In this review, the current knowledge on arbovirus threats to global travelers is summarized and prioritized per region. Based on most common clinical syndromes, currently known arboviruses can be grouped to develop diagnostic algorithms to support decision-making in diagnostics. This review systematically combines and structures the current knowledge on medically important travel-related arboviruses and illustrates the necessity of a detailed patient history (travel history, symptoms experienced, vaccination history, engaged activities, tick or mosquito bite and use of repellent and onset of symptoms), to guide the diagnosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays., (© 2011 European Society of Clinical Microbiology and Infectious Diseases. No claim to original US government works.)

Background: Antibody microarrays (Ab-array) represent a new, innovative proteomics platform for high-throughput protein expression profiling in body fluids. Because they allow for multiplexed measurements in small sample volumes, Ab-arrays are an interesting alternative to conventional indirect sandwich immunoassay (ELISA or DELFIA) tests in clinical or population screening if sets of markers are to be analyzed simultaneously. However, to allow implementation of Ab-arrays in clinical or population screening programs, it is of vital importance to establish that this method is both sensitive and quantitative., Methods: This study developed and optimized a duplex Ab-array for pregnancy-associated plasma protein A (PAPP-A) and human chorion gonadotropin (fβ-hCG), two serum biomarkers currently analyzed by conventional biochemical techniques in prenatal screening. Serum samples from pregnant women, representing the dynamic range of both markers, were analyzed on Ab-arrays, and validated to the, in prenatal screening routinely applied, AutoDelfia system., Results: Two different array hybridization conditions were tested, i.e., direct and indirect labeling, of which the indirect method displayed a sensitive and quantitative performance and a low intra- and inter-assay variation., Conclusions: Taken together, these findings indicate that Ab-array technology is a promising alternative for ELISA or DELFIA in population screening programs, allowing future quantitative analysis of multiple biomarkers simultaneously in small volumes of serum.

Hepatitis C virus (HCV) is believed to assemble by budding into membranes of the early secretory pathway, consistent with the membrane location where the viral envelope glycoproteins E1 and E2 accumulate when expressed. Coronavirus assembly also takes place at pre-Golgi membranes. Here, we generated coronavirus-like particles carrying in their envelope chimeric HCV glycoproteins composed of the ectodomains of E1 and E2, each fused to the transmembrane plus endodomain of the mouse hepatitis coronavirus spike glycoprotein. The chimeric particle system will enable structural and functional studies of the HCV glycoproteins.

West Nile virus (WNV) is an arthropod-borne flavivirus that is endemic in Africa, Europe, and Eastern Asia. The recent introduction and rapid dissemination of the virus in the United States as well as an increase in WNV outbreaks in Europe, has raised concerns for its spread in Europe. A surveillance system was developed to allow timely detection of an introduction of WNV infections in The Netherlands. This program focuses on cases presenting with neurological disease and includes the monitoring of hospital discharge diagnoses, trends in cerebrospinal fluid (CSF) diagnostic requests, laboratory testing of CSF, and monitoring of neurological disease in horses. Retrospective data from the hospital discharge records showed yearly peaks of unexplained meningitis and (meningo)encephalitis in the summer. A total of 781 CSF samples from humans and 71 serum and/or CSF samples from horses presenting with neurological disease of suspected viral etiology tested negative for the presence of specific antibodies to WNV. With a coverage rate of 59% in 2003, the probability that a cluster of five WNV cases presenting with neurological symptoms would have been detected was 99%. We conclude that, from 1999 to 2004, no evidence of WNV infection could be found in either humans or horses in The Netherlands.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP(2b) (previously named G(S)), GP(3), GP(4), and GP(5) (previously named G(L)). Proteins N, M, and GP(5) are major virion components, E occurs in virus particles in intermediate amounts, and GP(4), GP(3), and GP(2b) are minor structural proteins. The M and GP(5) proteins occur in virus particles as disulfide-linked heterodimers while the GP(4), GP(3), and GP(2b) proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP(2b), GP(3), or GP(4) proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP(2b), GP(3), and GP(4) proteins into viral particles. EAV particles lacking GP(2b), GP(3), GP(4), and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP(2b)/GP(3)/GP(4) heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.

Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.

The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material., (Copyright 2000 Academic Press.)

The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the virus-like particle (VLP) system that we developed earlier for the mouse hepatitis virus strain A59 (MHV-A59) and which we describe now also for the unrelated coronavirus feline infectious peritonitis virus (FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were constructed, consisting of the ectodomain of the one virus and the transmembrane and endodomain of the other. These proteins were tested for their incorporation into VLPs of either species. They were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated. Thus, the 64-terminal-residue sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active. This was indeed confirmed by incorporating one of the proteins into virions which thereby acquired a new host cell tropism, as will be reported elsewhere.

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly.

We have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric coronaviruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93.3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90.8% to 96.8% . The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M(r) (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells.

Infection with the mouse hepatitis coronavirus (MHV) provides an excellent model for the study of viral diseases of the central nervous system and the gastrointestinal tract. With the ultimate aim of studying mucosal immunity to MHV we have cloned the genes encoding the structural proteins of MHV strain A59 (MHV-A59) into the E3 region of a human adenovirus type 5 vector. Infection of HeLa cells with the resulting recombinant adenoviruses AdMHVS, AdMHVN and AdMHVM revealed the correct expression of the spike (S), nucleocapsid (N) and membrane (M) proteins, respectively. Intraperitoneal inoculation of BALB/c mice with the recombinant viruses elicited serum antibodies which specifically recognized the respective MHV proteins in an immunoprecipitation assay. Only antibodies to the S protein neutralized MHV-A59 in vitro but titres were low. When analysed by ELISA or by immunofluorescence only the antibody response to the N protein was significant; weak responses or no detectable response at all were found for S and M, respectively. Upon intracerebral challenge with a lethal dose of MHV-A59 we found that a significant fraction of animals vaccinated with adenovirus vectors expressing either the S protein or N protein were protected. This protective effect was significantly stronger when the animals were given a booster immunization with the same vector prior to challenge. No protection was induced by AdMHVM. Interestingly, enhanced protection resulted when AdMHVS and AdMHVN were applied in combination as compared to survival after single immunizations. The results indicate that both the N and S proteins generate a protective immune response and suggest that this response is enhanced by combined expression of the two proteins.