Flavivirus

RNA was extracted from patient samples that demonstrated the highest concentration of ZIKV RNA determined by the real-time assay, and for which sufficient sample volume was available (patients 824, 037, 830a, and 958). Briefly, RNA was extracted from 150 μL of serum by using the QIAamp Viral RNA Mini Kit (QIAGEN), and RNA was eluted with 75 μL of RNase-free water. A series of RT-PCRs was performed with each RNA preparation by using primer pairs designed to generate overlapping DNA fragments that spanned the entire polyprotein coding region of the virus. Primers were designed by using the ZIKV MR 766 prototype virus coding region sequence (GenBank accession no. AY632535) and the PrimerSelect software module of the LaserGene package (DNASTAR Inc., Madison, WI, USA). Several primers initially failed to amplify because of sequence mismatches between ZIKV MR 766 and ZIKV Yap 2007. Therefore, primers were redesigned by using newly generated DNA sequence data, and a “genome walking” approach was used to derive complete coding region sequence data. The complete list of amplification and sequencing primers is available upon request.
All RT-PCRs were performed with 10 μL of RNA by using the OneStep RT-PCR Kit (QIAGEN) following the manufacturer’s protocol. DNAs were analyzed by 2% agarose gel electrophoresis, and bands of the predicted size were excised from the gel and purified by using the QIAquick Gel Extraction Kit (QIAGEN). Purified DNAs were subjected to nucleic acid sequence analysis with sequencing primers spaced ≈500 bases apart on both strands of the DNA fragments by using the ABI BigDye Terminator V3.1 Ready Reaction Cycle Sequencing Mixture (Applied Biosystems). Nucleotide sequence was determined by capillary electrophoresis by using the ABI 3130 genetic analyzer (Applied Biosystems) following the manufacturer’s protcol. Raw sequence data were aligned and edited by using the SeqMan module of LaserGene (DNASTAR Inc.). Because of insufficient sample volume, no patient RNA was sufficient to generate DNA that included the entire coding region. Therefore, DNA data obtained from 4 patients was combined to generate a consensus sequence heretofore designated the ZIKV 2007 epidemic consensus (EC) sequence (GenBank accession no. EU545988).
The complete coding region of ZIKV 2007 EC or the nonstructural protein 5 (NS5) gene subregion was aligned with all available flavivirus sequences in GenBank by using the Clustal W algorithm within the MEGA version 4 software package (www.megasoftware.net). Phylogenetic trees were constructed by using either the complete coding region or the NS5 region because a large number of NS5 sequences were available in GenBank and trees for the NS5 region have been constructed (16 (link)). Additional ZIKV strains from the CDC/World Health Organization reference collection (strains 41662, 41524, and 41525) isolated from Aedes spp. mosquitoes collected in Senegal in 1984 were also amplified by RT-PCR in the NS5 region and subjected to nucleic acid sequencing as described above and included in the NS5 region analysis. Trees were constructed from coding region data or from NS5 data by MEGA 4 from aligned nucleotide sequences. We used maximum parsimony, neighbor-joining, or minimum evolution algorithms with 2,000 replicates for bootstrap support of tree groupings. All trees generated nearly identical topology; only the neighbor-joining NS5 tree is shown (Figure 1).

Details of the epidemic, including clinical and laboratory findings for all patients, will be reported elsewhere (M.R. Duffy et al., unpub. data). A subset of ZIKV-infected patients for whom acute- and convalescent-phase paired serum specimens had been collected was analyzed by using several serologic assays to evaluate the extent of cross-reactivity to several related flaviviruses. Patients were classified as primary flavivirus/ZIKV infected or secondary flavivirus/ZIKV probable infected. Primary flavivirus/ZIKV–infected patients were those in whom acute-phase serum specimens (<10 days) had no detectible antibodies (by IgG ELISA and plaque-reduction neutralization test [PRNT]) to any of the heterologous flaviviruses tested (Tables 1, 2) and were either IgM-positive in their acute-phase specimen or IgM and IgG positive for ZIKV in a convalescent-phase specimen (seroconversion). Secondary flavivirus/ZIKV probable–infected patients were those who had detectable antibodies to >1 heterologous flaviviruses in their acute-phase specimen and were also IgM positive for ZIKV in their acute-phase specimen, or IgM and IgG positive for ZIKV in their convalescent-phase specimen. The designation “ZIKV probable” was used because secondary flavivirus infections demonstrate extensive cross-reactivity with other flaviviruses, and in some cases, higher serologic reactivity to the original infecting flavivirus (“original antigenic sin” phenomenon). Thus, in secondary flavivirus infections shown in Tables 1 and 2, serologic data alone is insufficient to confirm ZIKV as the recently infecting flavivirus. However, these secondary flavivirus/ZIKV probable infections were likely recent ZIKV infections because ZIKV was the only virus detected during the epidemic in Yap, a relatively small and isolated island (11 ).

Viral strains used were provided by WHO Collaborating Center for arboviruses and viral hemorrhagic fever (CRORA) at the Institut Pasteur de Dakar. ZIKV and other flaviviruses strains isolated from mosquitoes and non-human vertebrates used in this study are described in Tables 1 and 2. Viral stocks were prepared by inoculating viral strains into AP 61 monolayer continuous cell lines in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% foetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% tryptose phosphate, penicillin-streptomycin and fungizone (Sigma, Gmbh, Germany). After 7 days of propagation, viral infection was tested by an indirect immunofluorescence assay (IFA) using specific hyperimmune mouse ascitic fluids as previously described [23 (link)] and supernatants from infected cells were collected as stocks for virus RNA isolation. ZIKV stocks were used for sequencing and evaluation of the sensitivity of the rRT-PCR assay. Other flaviviruses were used to evaluate the specificity of the assay.

Virus isolation was performed using C6/36 Aedes albopictus cells. Mosquito homogenates were centrifuged at 4°C for 20 min at 12,000 rpm in a high-speed refrigerated centrifuge, and 40 μL of supernatant was aspirated and inoculated into a single layer of C6/36 cells in a 24-well plate and grown for 1 day (2 wells/sample). Two wells per plate of cell controls were set up and incubated at 32°C. Cytopathic effects (CPE) were observed and recorded continuously for 4–7 days. Isolates with obvious or suspected CPE were freeze-thawed once and transferred to 25-cm² cell bottles for blind transmission to 3–6 generations. Viral isolates with regular CPE were freeze-thawed once and then aspirated into a freezing tube at −40°C for identification. During virus isolation, the group of C6/36 Aedes albopictus cell without mosquito supernatant was cultured as negative control.
Virus-specific RT-PCR was performed to identify the viral isolates. The viral nucleic acid was extracted using the ZYBIO Magnetic Beads Virus DNA/RNA Extraction Kit (ZYBIO, Chongqing, China) and the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using M-MLV Reverse Transcriptase (Promega, Madison, WI, United States). Based on the literature, we synthesized PCR, detection, and Sanger sequencing primers (Supplementary Table S2) for a variety of arboviruses, such as flaviviruses, alphaviruses, and viruses in the family Reoviridae, and then designed primers from newly discovered viral sequences from Yunnan and Guangxi provinces published in recent years (GoTaq® Green Master Mix, Promega). Next, next-generation sequencing (NGS) was performed to detect viruses in the isolates with CEP, but negative for RT-PCR using the designed primers. A sequencing library was constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina, San Diego, CA, United States). The library was subsequently sequenced on an Illumina NovaSeq 6000 (PE150) sequencing platform. Trimmomatic v0.36 (Bolger et al., 2014 (link)) was used to remove low-quality and short reads, and SPAdes v3.13.0 (Nurk et al., 2013 (link)) was used for metagenomic assembly. Blastn and Blastx were used to search for viral contigs. PCR detection and Sanger sequencing were performed using redesigned primers based on the viruses detected in samples with unknown viruses.

Healthy adults aged 18–60 years were eligible for enrollment in the study. Exclusion criteria included fever (≥38°C) within three days of vaccination; known hypersensitivity or allergy to the vaccine or components; contraindications to YF-17D vaccine receipt; any serious chronic or progressive disease; altered immune function; body mass index ≥ 35kg/m²; pregnancy or breastfeeding; participation in another clinical trial within 30 days; receipt of vaccines prior to enrollment (14 days for inactivated, 28 days for live vaccines); previous vaccination against any flavivirus or tick-borne encephalitis; current or previous flavivirus infection and ≥1 year living in a dengue-endemic area.

Vero cells were seeded 1 day prior to infection at a minimum concentration of 1 × 10⁵ cells/ml in cell culture flasks. Cells were infected with ZIKV (MR766 strain) at a multiplicity of infection (MOI) of 1. Adsorption was carried out for 1 h at 4 °C to prevent virus entry and synchronize infection of cells. Cells were washed twice with a cold medium to remove unbound virus and replenished with a prewarmed medium. The coverslips were taken out of the culture at 12, 48 and 72 h post-infection (h.p.i.) under sterile conditions, washed with 0.01 M phosphate buffer saline pH 7.2 (PBS) and fixed in ice-cold acetone for 10 min at − 20 °C for experiments. The fixed cells were washed with PBS and blocked with 1 % bovine serum albumin (BSA) in PBS for 30 min. For dual staining, NTDyn was labelled with our labmade polyclonal antibody (rabbit) and the E protein using the pan-flavivirus monoclonal antibody 4G2. The primary antibodies were added simultaneously and incubated for 1 h followed by washing with PBS. This was followed by incubation with appropriate secondary antibodies conjugated to fluorescent dyes, Alexa 488 or Alexa 594 added simultaneously. DAPI (4, 6 diamino-2-phenylindole) was used to stain the nucleus in all experiments. At the end of the staining process, coverslips were washed and mounted onto slides using a mounting medium. Control samples were non-infected cells processed with the same procedures described previously and were included in all experiments as mock-infected cultures. Cells were observed and images were digitalized in a Zeiss 700 Confocal microscope.