Zika Virus

The streptavidin alkaline phosphatase method was adapted to detect the viral antigen using a polyclonal anti-ZIKV antibody produced at the Evandro Chagas Institute^{2 (link)}. The biotin-streptavidin peroxidase method was used for immunostaining of tissues with antibodies specific for each marker studied. First, the tissue samples were deparaffinized in xylene and hydrated in a decreasing ethanol series (90%, 80%, and 70%). Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide for 45 min. Antigen retrieval was performed by incubation in citrate buffer, pH 6.0, or EDTA, pH 9.0, for 20 min at 90 °C. Nonspecific proteins were blocked by incubating the sections in 10% skim milk for 30 min. The histological sections were then incubated overnight with the primary antibodies diluted in 1% bovine serum albumin (Supplementary Table S1). After this period, the slides were immersed in 1 × PBS and incubated with the secondary biotinylated antibody (LSAB, DakoCytomation) in an oven for 30 min at 37 °C. The slides were again immersed in 1X PBS and incubated with streptavidin peroxidase (LSAB, DakoCytomation) for 30 min at 37 °C. The reactions were developed with 0.03% diaminobenzidine and 3% hydrogen peroxide as the chromogen solution. After this step, the slides were washed in distilled water and counterstained with Harris hematoxylin for 1 min. Finally, the sections were dehydrated in an increasing ethanol series and cleared in xylene.

The results were stored in electronic spreadsheets. Statistical analysis was performed with the GraphPad Prism 5.0 program. For univariate analysis, frequencies and measures of central tendency and dispersion were obtained. We highlight that for meninges only seven cases ZIKV-positive were included in the statistical analysis because presented structure and were compared to five controls. For space perivascular and parenchyma 10 cases ZIKV-positive were analized and compared to five controls. The hypotheses were tested using Student’s t (Supplementary Table S3) and Pearson’s correlation tests (Supplementary Tables S4–S6). A level of significance of 5% (p ≤ 0.05) was adopted for all tests.

Biological samples of patients were obtained and processed in the context of the emergency definition of the Ministry of Health during surveillance activities of the ZIKV epidemic in Brazil. This study was approved (opinion number 1.888.946) by the Research Ethics Committee (CEP) of the Evandro Chagas Institute (IEC). All steps and methods of the study followed the recommendations established by legislation in force in Brazil.

To determine whether classification differences were consistent across growth curves, we classified HC using three different growth curves. We used growth curves that are either commonly used (Hadlock and Intergrowth-21st) and/or were designed to be representative of the U.S. population (Intergrowth-21st and National Institute of Child Health and Human Development [NICHD]) to determine the proportion of female and male fetuses classified as having microcephaly (<3rd percentile, z-score < −1.88) or macrocephaly (>97th percentile, z-score >1.88) in the data subset of our sample described above. While the Society for Maternal-Fetal Medicine (SMFM) provides recommendations for standardizing the evaluation of fetal HC in the context of Zika virus exposure (5 ), there are no universal definitions for microcephaly and macrocephaly; the 3rd and 97th percentiles were chosen because they are commonly used and because the information provided in the NICHD curves does not allow direct calculation of other potential cutpoints for microcephaly and macrocephaly. All three evaluated growth curves are sex-neutral, using a single set of curves for both sexes.
We used cubic interpolation to calculate values of the 3rd and 97th percentiles for integer values of GA in days (17 (link)). NICHD percentiles were published separately for four specific race/ethnicity groups: Asian/Pacific Islander, Hispanic, Black non-Hispanic, and White non-Hispanic. There were no published NICHD percentiles that were nonspecific for race/ethnicity. We used the mean of the four race/ethnicity-specific values at each GA to create percentiles for a fifth group, deemed “Uncategorized.” For the NICHD analyses only, we excluded the small number of ultrasounds that could not be linked with maternal data. Women with a recorded race/ethnicity that did not fit in these categories, or whose maternal data were available but missing race/ethnicity data, were evaluated using the “Uncategorized” percentiles. Although there are limits of the reliability and precision of race/ethnicity data, the EHR was the only potential source of race/ethnicity data for this population and therefore the only way to evaluate our data compared to the US-based NICHD percentiles.

Acute-phase serum or plasma samples were collected during the initial visit for study enrollment and transported to the IICS-UNA laboratory. Samples were tested for DENV NS1 antigen using the Standard Q Dengue Duo rapid immunochromatographic test (SD Biosensor, Suwon, South Korea) according to manufacturer recommendations. Qualitative antibody data acquired using this method was not evaluated in this study, see antibody section below. Primary samples were then aliquoted and stored at −80°C until later use or shipment on dry ice to Emory University for additional testing. For molecular testing, total nucleic acids were extracted from 200μL of sample on an EMAG instrument and eluted into 50μL of buffer. Samples were tested for Zika virus, chikungunya virus and DENV by real-time RT-PCR (rRT-PCR) using a validated and published multiplex assay (the ZCD assay) [60 (link)], and DENV serotype and viral load were determined with a published DENV multiplex assay [61 (link), 62 (link)]. Both rRT-PCRs were performed as previously described [60 (link)–62 (link)].
Serologic testing was performed on acute-phase samples using two different methods. First, anti-DENV IgG and IgM were analyzed using commercial ELISA kits [Dengue ELISA IgG (G1018) and Dengue ELISA IgM Capture (M1018), Vircell Microbiologists, Granada, Spain] according to manufacturer recommendations (interpretation: IgM or IgG index >11 positive, 9–11 indeterminate, <9 negative). Second, a 5μL aliquot of serum from 139 participants with sufficient sample was tested in the pGOLD assay (Nirmidas Biotech, Inc, Palo Alto, CA), which is a multiplex serological assay for IgM and IgG against DENV (DENV-2 whole virus antigen) and ZIKV (NS1 antigen). The pGOLD assay was performed as previously described [59 (link), 63 (link)]. In each well of the pGOLD slide, antigens are spotted in triplicate, and average signals are used during analysis. For IgG, the negative control signal was subtracted from the sample signal, and the difference was divided by the average signal of four IgG control spots included in each well. For IgM, a similar calculation was performed using the signal from a known anti-DENV IgM positive control sample included on each run. A positive threshold ratio of 0.1 was established for each isotype, which was ≥ 3 standard deviations above the mean of the negative control.
Chymase and LBP levels were determined using commercial ELISA kits (G-Biosciences, St. Louis, MO, USA), following the manufacturer’s instructions. Complete blood counts and chemistries were performed at the clinical site at the discretion of the care team, and results were included if the sample was obtained within ±1 day of enrollment.

With data gleaned from the Oxford COVID-19 Government Response Tracker (OxCGRT), the World Health Organization, the World Bank, and the study of Pifarré i Arolas et al., we constructed a dataset of 80 countries on the basis of availability of data for a cross-country comparative configurational analysis. Our study comprises YLL rate as the outcome of interest and five explanatory conditions: a delayed public-health response, past epidemic experience, proportion of elderly in population, population density, and national income per capita. Table 1 presents the descriptive statistics of the five conditions and the outcome, and the dataset of 80 countries is provided in S1 Table.
YLL rate was computed as the number of years of life lost due to COVID-19 per 100,000 population as of January 6, 2021, obtained directly from the study of Pifarré i Arolas et al. [5 (link)]. With a focus on early COVID-19 mortality impact, this study used January 6, 2021 as the cutoff point to isolate the effects of vaccination rollout [44 (link)], the availability of more effective treatments, and the emergence of new virus variants on overall COVID-19 mortality impact.
A delayed public-health response concerned the number of days from January 1, 2020 until a country first implemented any of nine containment and closure policies [45 (link)]. Specifically, OxCGRT provides researchers with nine indicators recording information on the enactment of containment and closure policies: school closing, workplace closing, cancel public events, restrictions on gatherings, close public transport, stay at home requirements, restrictions on internal movement, international travel controls, and facial coverings [46 (link)].
Past epidemic experience was a binary condition (0 or 1) showing whether a country had experienced one of four recent epidemics: SARS, MERS, Zika, and Ebola [47 (link)]. The epidemiological records on the above four pandemics were all collected from the World Health Organization.
Proportion of elderly in population was computed as the population aged 65 years and above as a percentage of the total population in a country [48 ]. Population density was midyear population divided by land area in square kilometers [49 ]. National income per capita referred to the gross national income, converted to U.S. dollars using the World Bank Atlas method, divided by the midyear population [50 ]. All of these three conditions were obtained directly from the World Bank.