Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020.

BackgroundThe ongoing coronavirus disease (COVID-19) pandemic has major impacts on health systems, the economy and society. Assessing infection attack rates in the population is critical for estimating disease severity and herd immunity which is needed to calibrate public health interventions. We have previously shown that it is possible to achieve this in real time to impact public health decision making.AimOur objective was to develop and evaluate serological assays applicable in large-scale sero-epidemiological studies.MethodsWe developed an ELISA to detect IgG and IgM antibodies to the receptor-binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated its sensitivity and specificity in combination with confirmatory microneutralisation (MN) and 90% plaque reduction neutralisation tests (PRNT90) in 51 sera from 24 patients with virologically confirmed COVID-19 and in age-stratified sera from 200 healthy controls.ResultsIgG and IgM RBD ELISA, MN and PRNT90 were reliably positive after 29 days from illness onset with no detectable cross-reactivity in age-stratified controls. We found that PRNT90 tests were more sensitive in detecting antibody than MN tests carried out with the conventional 100 tissue culture infectious dose challenge. Heparinised plasma appeared to reduce the infectivity of the virus challenge dose and may confound interpretation of neutralisation test.ConclusionUsing IgG ELISA based on the RBD of the spike protein to screen sera for SARS-CoV-2 antibody, followed by confirmation using PRNT90, is a valid approach for large-scale sero-epidemiology studies.

Middle East respiratory syndrome coronavirus (MERS-CoV) neutralising antibodies in a high-risk human population, Morocco, November 2017 to January 2018.

BackgroundMiddle East respiratory syndrome coronavirus (MERS-CoV) remains a major concern for global public health. Dromedaries are the source of human zoonotic infection. MERS-CoV is enzootic among dromedaries on the Arabian Peninsula, the Middle East and in Africa. Over 70% of infected dromedaries are found in Africa. However, all known zoonotic cases of MERS have occurred in the Arabian Peninsula with none being reported in Africa.AimWe aimed to investigate serological evidence of MERS-CoV infection in humans living in camel-herding areas in Morocco to provide insights on whether zoonotic transmission is taking place.MethodsWe carried out a cross sectional seroprevalence study from November 2017 through January 2018. We adapted a generic World Health Organization MERS-CoV questionnaire and protocol to assess demographic and risk factors of infection among a presumed high-risk population. ELISA, MERS-CoV spike pseudoparticle neutralisation tests (ppNT) and plaque neutralisation tests (PRNT) were used to assess MERS-CoV seropositivity.ResultsSerum samples were collected from camel slaughterhouse workers (n = 137), camel herders (n = 156) and individuals of the general population without occupational contact with camels but living in camel herding areas (n = 186). MERS-CoV neutralising antibodies with ≥ 90% reduction of plaque numbers were detected in two (1.5%) slaughterhouse workers, none of the camel herders and one individual from the general population (0.5%).ConclusionsThis study provides evidence of zoonotic transmission of MERS-CoV in Morocco in people who have direct or indirect exposure to dromedary camels.

Lack of serological evidence of Middle East respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in Nigeria, 2016.

BackgroundMiddle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic threat of global public health concern and dromedary camels are the source of zoonotic infection. Although MERS-CoV is enzootic in dromedaries in Africa as well as the Middle East, zoonotic disease has not been reported in Africa. Methods: In an abattoir in Kano, Nigeria, we tested nasal swabs from camels and investigated 261 humans with repeated occupational exposure to camels, many of whom also reported drinking fresh camel milk (n = 138) or urine (n = 94) or using camel urine for medicinal purposes (n = 96). Results: Weekly MERS-CoV RNA detection in January-February 2016 ranged from 0-8.4% of camels sampled. None of the abattoir workers with exposure to camels had evidence of neutralising antibody to MERS-CoV. Conclusion: There is a need for more studies to investigate whether or not zoonotic transmission of MERS-CoV does take place in Africa.

Risk factors for MERS coronavirus infection in dromedary camels in Burkina Faso, Ethiopia, and Morocco, 2015.

Understanding Middle East respiratory syndrome coronavirus (MERS-CoV) transmission in dromedary camels is important, as they consitute a source of zoonotic infection to humans. To identify risk factors for MERS-CoV infection in camels bred in diverse conditions in Burkina Faso, Ethiopia and Morocco, blood samples and nasal swabs were sampled in February-March 2015. A relatively high MERS-CoV RNA rate was detected in Ethiopia (up to 15.7%; 95% confidence interval (CI): 8.2-28.0), followed by Burkina Faso (up to 12.2%; 95% CI: 7-20.4) and Morocco (up to 7.6%; 95% CI: 1.9-26.1). The RNA detection rate was higher in camels bred for milk or meat than in camels for transport (p = 0.01) as well as in younger camels (p = 0.06). High seropositivity rates (up to 100%; 95% CI: 100-100 and 99.4%; 95% CI: 95.4-99.9) were found in Morocco and Ethiopia, followed by Burkina Faso (up to 84.6%; 95% CI: 77.2-89.9). Seropositivity rates were higher in large/medium herds (≥51 camels) than small herds (p = 0.061), in camels raised for meat or milk than for transport (p = 0.01), and in nomadic or sedentary herds than in herds with a mix of these lifestyles (p < 0.005).

Serum 25-Hydroxyvitamin D Was Not Associated with Influenza Virus Infection in Children and Adults in Hong Kong, 2009-2010.

BACKGROUND: Some studies have hypothesized that vitamin D may have a role to play in protection against influenza virus infections and illnesses, and that seasonal fluctuation in serum 25-hydroxyvitamin D [25(OH)D] may affect seasonal patterns of influenza virus infections. OBJECTIVE: We aimed to investigate whether serum 25(OH)D concentrations were associated with the incidence of influenza virus infections and illnesses in children and adults in Hong Kong. METHODS: In 2009-2010, 3030 children and adults of all ages from 796 households in Hong Kong were followed up to identify acute respiratory illnesses. Sera from 2694 participants were collected at baseline and after ∼1 mo, 6 mo, and 12 mo. Influenza virus infections were confirmed by reverse transcriptase-polymerase chain reaction performed on nasal and throat swab samples collected during illness episodes. Serologic evidence of influenza virus infection was measured by hemagglutination inhibition assays in unvaccinated participants. The serum 25(OH)D concentrations were measured after collection of all specimens. Each individual's baseline serum 25(OH)D concentration on 1 January 2010 was predicted by a random-effects linear regression model. RESULTS: We found that, in children and adults who had not received a seasonal influenza vaccine, baseline serum 25(OH)D concentrations (<50 nmol/L compared with ≥50 nmol/L) were not statistically significantly associated with serologic evidence of influenza A(H1N1)pdm09 (RR, 1.18; 95% CI: 0.85, 1.65) or seasonal influenza virus infections [including A(H3N2) and B virus] (RR, 1.13; 95% CI: 0.86, 1.49). In all participants, baseline serum 25(OH)D concentrations were not statistically significantly associated with polymerase chain reaction-confirmed influenza virus infection (RR, 1.15; 95% CI: 0.73, 1.83) and influenza-like illness (RR, 1.18; 95% CI: 0.98, 1.43). CONCLUSIONS: These findings indicate that lower serum vitamin D concentrations may not contribute to the seasonality of influenza and are not associated with an increased risk of influenza virus infections in persons of all ages in Hong Kong.

Surface-aerosol stability and pathogenicity of diverse MERS-CoV strains from 2012 - 2018.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a coronavirus that infects both humans and dromedary camels and is responsible for an ongoing outbreak of severe respiratory illness in humans in the Middle East. While some mutations found in camel-derived MERS-CoV strains have been characterized, the majority of natural variation found across MERS-CoV isolates remains unstudied. Here we report on the environmental stability, replication kinetics and pathogenicity of several diverse isolates of MERS-CoV as well as SARS-CoV-2 to serve as a basis of comparison with other stability studies. While most of the MERS-CoV isolates exhibited similar stability and pathogenicity in our experiments, the camel derived isolate, C/KSA/13, exhibited reduced surface stability while another camel isolate, C/BF/15, had reduced pathogenicity in a small animal model. These results suggest that while betacoronaviruses may have similar environmental stability profiles, individual variation can influence this phenotype, underscoring the importance of continual, global viral surveillance.

Cross-reactive antibody-dependent cellular cytotoxicity antibodies are increased by recent infection in a household study of influenza transmission.

OBJECTIVES: Influenza causes a spectrum of disease from asymptomatic infection to fatal outcome, and pre-existing immunity can alter susceptibility and disease severity. In a household transmission study, we recruited outpatients with confirmed influenza virus infection and prospectively identified secondary infections in their household contacts, therefore identifying infection cases with baseline samples for determining immune-mediated protection from influenza infection. METHODS: We examined baseline broadly reactive immune correlates of relevance to universal vaccine development, specifically antibody-dependent cytotoxic (ADCC) antibodies and T-cell responses in functional assays. Antibodies were assessed in a cell-based NK cell degranulation assay by flow cytometry, and T-cell responses were assessed by IFN-γ intracellular cytokine staining flow cytometry assay. RESULTS: The magnitude of antibody responses and ADCC function for multiple influenza-specific proteins was lower in participants who became infected, consolidating the role of pre-existing antibodies in protection from seasonal influenza virus infection. Among H1N1-infected contacts, we found that higher levels of pre-existing H1-haemagglutinin ADCC responses correlated with reduced symptom severity. Recent infection boosted the titre and magnitude of haemagglutinin-, neuraminidase- and nucleoprotein-specific ADCC antibodies. Limited T-cell samples precluded conclusions on the role of pre-existing T-cell responses. CONCLUSIONS: Overall, ADCC responses are a protective correlate against influenza virus infection that should be considered in future vaccine development and evaluation.Influenza-specific ADCC responses are elevated in uninfected subjects, associated with reduced symptoms and boosted by recent infection, whilst HA stem and NA IgG are also elevated in uninfected participants irrespective of ADCC function.

Middle East respiratory syndrome coronavirus infection in non-camelid domestic mammals.

Dromedary camels are natural host of the Middle East respiratory syndrome coronavirus (MERS-CoV). However, there are limited studies of MERS-CoV infection of other domestic mammals exposed to infected dromedaries. We expanded our surveillance among camels in Egypt, Tunisia, and Senegal to include other domestic mammalian species in contact with infected camels. A total of 820 sera and 823 nasal swabs from cattle, sheep, goats, donkeys, buffaloes, mules, and horses were collected. Swabs were tested using RT-PCR and virus RNA-positive samples were genetically sequenced and phylogenetically analysed. Sera were screened using virus microneutralization tests and positive sera (where available) were confirmed using plaque reduction neutralization tests (PRNT). We detected 90% PRNT confirmed MERS-CoV antibody in 35 (55.6%) of 63 sera from sheep collected from Senegal, two sheep (1.8%) of 114 in Tunisia and a goat (0.9%) of 107 in Egypt, with titres ranging from 1:80 to ≥1:320. We detected MERS-CoV RNA in swabs from three sheep (1.2%) of 254 and five goats (4.1%) of 121 from Egypt and Senegal, as well as one cow (1.9%) of 53 and three donkeys (7.1%) of 42 from Egypt. Partial sequences of the RT-PCR amplicons confirmed specificity of the results. This study showed that domestic livestock in contact with MERS-CoV infected camels may be at risk of infection. We recommend expanding current MERS-CoV surveillance in animals to include other livestock in close contact with dromedary camels. The segregation of camels from other livestock in farms and live animal markets may need to be considered.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Dromedary Camels in Africa and Middle East.

: Dromedary camels are the natural reservoirs of the Middle East respiratory syndrome coronavirus (MERS-CoV). Camels are mostly bred in East African countries then exported into Africa and Middle East for consumption. To understand the distribution of MERS-CoV among camels in North Africa and the Middle East, we conducted surveillance in Egypt, Senegal, Tunisia, Uganda, Jordan, Saudi Arabia, and Iraq. We also performed longitudinal studies of three camel herds in Egypt and Jordan to elucidate MERS-CoV infection and transmission. Between 2016 and 2018, a total of 4027 nasal swabs and 3267 serum samples were collected from all countries. Real- time PCR revealed that MERS-CoV RNA was detected in nasal swab samples from Egypt, Senegal, Tunisia, and Saudi Arabia. Microneutralization assay showed that antibodies were detected in all countries. Positive PCR samples were partially sequenced, and a phylogenetic tree was built. The tree suggested that all sequences are of clade C and sequences from camels in Egypt formed a separate group from previously published sequences. Longitudinal studies showed high seroprevalence in adult camels. These results indicate the widespread distribution of the virus in camels. A systematic active surveillance and longitudinal studies for MERS-CoV are needed to understand the epidemiology of the disease and dynamics of viral infection.

Longitudinal study of Middle East Respiratory Syndrome coronavirus infection in dromedary camel herds in Saudi Arabia, 2014-2015.

Two herds of dromedary camels were longitudinally sampled with nasal and rectal swabs and serum, between September 2014 and May 2015, and the samples were tested for Middle East Respiratory Syndrome (MERS) coronavirus RNA and antibodies. Evidence of MERS-CoV infection was confirmed in one herd on the basis of detection of virus RNA in nasal swabs from three camels and significant increases in the antibody titers from three others. The three viruses were genetically identical, thus indicating introduction of a single virus into this herd. There was evidence of reinfection of camels that were previously seropositive, thus suggesting that prior infection does not provide complete immunity from reinfection, a finding that is relevant to camel vaccination strategies as a means to prevent zoonotic transmission.