Comparing the Performance of Three Models Incorporating Weather Data to Forecast Dengue Epidemics in Reunion Island, 2018-2019.

We developed mathematical models to analyze a large dengue virus (DENV) epidemic in Reunion Island in 2018-2019. Our models captured major drivers of uncertainty including the complex relationship between climate and DENV transmission, temperature trends, and underreporting. Early assessment correctly concluded that persistence of DENV transmission during the austral winter 2018 was likely and that the second epidemic wave would be larger than the first one. From November 2018, the detection probability was estimated at 10%-20% and, for this range of values, our projections were found to be remarkably accurate. Overall, we estimated that 8% and 18% of the population were infected during the first and second wave, respectively. Out of the 3 models considered, the best-fitting one was calibrated to laboratory entomological data, and accounted for temperature but not precipitation. This study showcases the contribution of modeling to strengthen risk assessments and planning of national and local authorities.

Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

COVID-19 convalescent plasma: Evolving strategies for serological screening in France.

Quantitation of anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) neutralizing antibodies (Nabs) is a key parameter in determining the effective dose for treatment with COVID-19 convalescent plasma (CCP). Interpretation of results from clinical trials conducted worldwide requires comparison of Nabs titres obtained from different methods. As virus neutralization tests (VNTs) are not standardized scalable or commercially available, strategies based on intensity of ELISA (Enzyme Linked Immunosorbent Assay) or chemiluminescent binding serological tests were implemented to allow comparisons and establish criteria for determining 'high-titres' of anti-SARS-CoV-2 antibodies (Abs). To this end, the FDA (Food and Drug Administration) has proposed criteria to define high-titre plasmas using different serological assays, including the one used in France for the CCP SARS-CoV-2 Abs screening (Euroimmun anti-S1 IgG). A retrospective study revealed that when using the FDA criteria (ELISA signal-to-cut-off [S/C ratio] ≥3.5), 91% of CCP had Nabs titres ≥40 as assessed with an in-house VNT. French strategy to ensure sufficient stocks of CCP of increasing titre has evolved over time. Recently, we improved our strategy by collecting only plasma from vaccinated convalescent donors as we confirmed that the mean IgG antibody level (ELISA S/C ratio) was significantly higher in plasma from vaccinated convalescent donors compared to donations from unvaccinated convalescent donors: 9.31 (CI 95%: 8.46-10.16) versus 3.22 (CI 95%: 3.05-3.39) (p < 0.001).

ZIKA Virus Neutralizing Antibody Kinetics in Antenatally Exposed Infants.

BACKGROUND: Zika virus (ZIKV) is associated with severe congenital abnormalities and laboratory diagnosis of antenatal infection is difficult. Here we evaluated ZIKV neutralizing antibody (nAb) kinetics in infants born to mothers with PCR-confirmed ZIKV infection during pregnancy. METHODS: Neonates (n = 98) had serum specimens tested repeatedly for ZIKV nAb over the first 2 years of life using virus neutralization test (VNT). ZIKV neonatal infection was confirmed by RT-PCR in blood or urine and/or presence of ZIKV IgM antibodies, and results were correlated with infant clinical features. RESULTS: Postnatal laboratory evidence of ZIKV vertical transmission was obtained for 60.2% of children, while 32.7% exhibited clinical abnormalities. Congenital abnormalities were found in 37.3% of children with confirmed ZIKV infection and 31.0% of children without confirmed infection (P = .734). All but 1 child displayed a physiologic decline in ZIKV nAb, reflecting maternal antibody decay, despite an early ZIKV-IgM response in one-third of infants. CONCLUSIONS: Infants with antenatal ZIKV exposure do not develop ZIKV nAb despite an early IgM response. Therefore, ZIKV VNT in children is not useful for diagnosis of congenital infection. In light of these findings, it remains to be determined if children infected in utero are potentially susceptible to reinfection.

Prolonged Infectivity of SARS-CoV-2 in Fomites.

We spotted severe acute respiratory syndrome coronavirus 2 on polystyrene plastic, aluminum, and glass for 96 hours with and without bovine serum albumin (3 g/L). We observed a steady infectivity (<1 log10 drop) on plastic, a 3.5 log10 decrease on glass, and a 6 log10 drop on aluminum. The presence of proteins noticeably prolonged infectivity.

Zika Virus Circulation in Mali.

The circulation of Zika virus (ZIKV) in Mali has not been clearly characterized. Therefore, we conducted a serologic survey of 793 asymptomatic volunteers >15 years of age (2016), and 637 blood donors (2013) to assess the seroprevalence of ZIKV infection in 2 ecoclimatic regions of Mali, tropical savannah and warm semiarid region, using ELISA and seroneutralization assays. The overall seroprevalence was ≈12% and increased with age, with no statistical difference between male and female participants. In the warm semiarid study sites we detected immunological markers of an outbreak that occurred in the late 1990s in 18% (95% CI 13%-23%) of participants. In tropical savannah sites, we estimated a low rate of endemic transmission, with 2.5% (95% CI 2.0%-3.1%) of population infected by ZIKV annually. These data demonstrate the circulation of ZIKV in Mali and provide evidence of a previously unidentified outbreak that occurred in the late 1990s.

Correction to: Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies.

In the original publication of this article [1], Table 1 has some errors.

Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies.

Here we propose a strategy allowing implementing efficient and practicable large-scale seroepidemiological studies for Zika Virus (ZIKV). It combines screening by a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (CPE-based VNT). In post-epidemic samples from Martinique Island blood donors (a population with a dengue seroprevalence above 90%), this strategy allowed reaching specificity and sensitivity values over 98%. The CPE-based VNT consists of recording CPE directly under the optical microscope, which is easy to identify with ZIKV strain H/PF/2013 at day 5 pi. Overall, considered that CPE-based VNT is cost effective and widely automatable, the NS1 protein-based Zika IgG ELISA+CPE-based VNT combination strategy represents a convenient tool to expedite ZIKV seroprevalence studies.

Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral.

OBJECTIVES: T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus. METHODS: The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir. RESULTS: Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general. CONCLUSIONS: This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals.

Structure-activity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication.

Chikungunya virus (CHIKV), a mosquito-borne arthrogenic Alphavirus, causes an acute febrile illness in humans, that is, accompanied by severe joint pains. In many cases, the infection leads to persistent arthralgia, which may last for weeks to several years. The re-emergence of this infection in the early 2000s was exemplified by numerous outbreaks in the eastern hemisphere. Since then, the virus is rapidly spreading. Currently, no drugs have been approved or are in development for the treatment of CHIKV, which makes this viral infection particularly interesting for academic medicinal chemistry efforts. Several molecules have already been identified that inhibit CHIKV replication in phenotypic virus-cell-based assays. One of these is arbidol, a molecule that already has been licensed for the treatment of influenza A and B virus infections. For structural optimization, a dedicated libraries of 43 indole-based derivatives were evaluated leading to more potent analogues (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities higher than those of the other derivatives, including the lead compound, and with a selective index of inhibition 13.2 and 14.6, respectively, higher than that of ARB (4.6).

Current status of pathogen handling in European laboratories: focus on viral inactivation process.

For handling safely infectious agents, European laboratories must comply with specific EC Directives, national regulations and recommendations from the World Health Organization (WHO). To prevent laboratory acquired infections (LAIs) and pathogens dissemination, a key biosafety rule requires that any infectious material (clinical specimens or research samples) manipulated outside a biosafety cabinet (BSC) must be inactivated unless the lack of infectivity is proven. This inactivation process is a crucial step for biosafety and must be guided by a rigorous experimental qualification and validation procedure. However, for diagnostic or research laboratories, this process is not harmonized with common standard operation procedures (SOPs) but based on individual risk assessment and general international guidelines which can pose problems in emergency situations such as major outbreaks or pandemics. This review focuses on viral inactivation method, outlining the current regulatory framework, its limitations and a number of ways in which biosafety can be improved.

SARS-CoV-2 IgG seroprevalence surveys in blood donors before the vaccination campaign, France 2020-2021.

We conducted a cross-sectional study for SARS-CoV-2 anti-S1 IgG prevalence in French blood donors (n = 32605), from March-2020 to January-2021. A mathematical model combined seroprevalence with a daily number of hospital admissions to estimate the probability of hospitalization upon infection and determine the number of infections while correcting for antibody decay. There was an overall seroprevalence increase over the study period and we estimate that ∼15% of the French population had been infected by SARS-CoV-2 by January-2021. The infection/hospitalization ratio increased with age, from 0.31% (18-30yo) to 4.5% (61-70yo). Half of the IgG-S1 positive individuals had no detectable antibodies 4 to 5 months after infection. The seroprevalence in group O donors (7.43%) was lower (p = 0.003) than in A, B, and AB donors (8.90%). We conclude, based on seroprevalence data and mathematical modeling, that a large proportion of the French population was unprotected against severe disease prior to the vaccination campaign.

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug.

OBJECTIVES: Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. METHODS: Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. RESULTS: Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. CONCLUSIONS: The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5.

The replacement of the Omicron BA.1 variant of SARS-CoV-2 by the BA.2 and the rapid growth of the BA.5 sub lineage, which have both different sets of mutations in the spike glycoprotein, alters the spectrum of activity of therapeutic antibodies currently licensed in the European Union. Using clinical strains of the Omicron BA.2 and BA.5 variants, we compared the neutralising power of monoclonal antibodies against the Omicron BA.1, BA.2 and BA.5 variants, using an ancestral strain (lineage B.1, D614G) and a Delta variant strain as reference. Sotrovimab/Vir-7831 is less active against BA.2 than against BA.1 (fold change reduction ~ 1,4) and even less active against BA.5 (fold change reduction ~ 2.7). Within the Evusheld /AZD7442 cocktail, Cilgavimab/AZD1061 is more active against BA.2 and BA.5 than against BA.1 (fold change increase ~ 32), whilst the very low activity of Tixagevimab/AZD8895 against BA.1 is not enhanced against BA.2 nor BA.5. In total, compared to BA.1, the activity of the Evusheld/AZD7442 is significantly improved against BA.2 while BA.5 is intermediate but closer to BA.2.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

Identification of cellular proteome modifications in response to West Nile virus infection.

Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.

Model-based assessment of Chikungunya and O'nyong-nyong virus circulation in Mali in a serological cross-reactivity context.

Serological surveys are essential to quantify immunity in a population but serological cross-reactivity often impairs estimates of the seroprevalence. Here, we show that modeling helps addressing this key challenge by considering the important cross-reactivity between Chikungunya (CHIKV) and O'nyong-nyong virus (ONNV) as a case study. We develop a statistical model to assess the epidemiology of these viruses in Mali. We additionally calibrate the model with paired virus neutralization titers in the French West Indies, a region with known CHIKV circulation but no ONNV. In Mali, the model estimate of ONNV and CHIKV prevalence is 30% and 13%, respectively, versus 27% and 2% in non-adjusted estimates. While a CHIKV infection induces an ONNV response in 80% of cases, an ONNV infection leads to a cross-reactive CHIKV response in only 22% of cases. Our study shows the importance of conducting serological assays on multiple cross-reactive pathogens to estimate levels of virus circulation.

A derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro.

A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (α-l-daunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC(50)] = 0.34 ± 0.20 µg/ml [0.52 ± 0.31 µM]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of ∼100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC(50) = 3.1 ± 1.0 µg/ml [4.8 ± 1.5 µM]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) (∼2 to 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with ≥5 µg/ml SA-17 resulted in 100% inhibition of viral infectivity (≥3 log reduction). SA-17, however, did not prove virucidal.

Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples.

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.