A Reverse Genetics Platform That Spans the Zika Virus Family Tree

Abstract

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics.IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design.

FIG 1

(A) Schematic diagram of ZIKV MR766 infectious clone. The genome of the virus is divided into 4 fragments using the diagrammed restriction endonucleases and cloned into high-copy-number vectors. A T7 promoter is placed before the first nucleotide of the ZIKV genome, and a hepatitis delta virus ribozyme is placed after the final genomic nucleotide for RNA stability. Four restriction endonuclease sites shown beneath the genome (two PflMI, one SmaI, and one BstAPI) were removed using synonymous changes. Sizes of each fragment are shown, and the larger size is listed along with the location of the N154 allelic mutations (green star). (B) Amino acid sequences of the 3 allelic MR766 variants in the region of the N154 glycosylation site on E. Numbers (440 and 460) correspond to amino acid positions in the complete ZIKV genome. (C) Virus focus images on Vero-81 cells. (D) Growth curves of MR766 isolate and 3 infectious clones done at 37°C. MR766 +gly IC had significantly higher early growth than the other 3 strains by 2-way analysis of variance with Tukey’s test. (E) After reverse transcriptase-PCR, viral amplicons were subjected to SmaI digestion; only the DNA from the natural isolate was cut. (F) Western blot of E protein from MR766 isolate and 3 infectious clones performed using the pan-flavivirus MAb 4G2. Size differences of the E protein are shown based on their sequence and glycosylation status.

FIG 2

(A) Schematic diagram of ZIKV H/PF/2013 infectious clone. The genome of the virus is divided into 4 fragments using diagrammed restriction endonucleases and cloned into high-copy-number vectors. A T7 promoter and a hepatitis delta virus ribozyme flank the genome. Sizes of each fragment are shown. (B) H/PF/2013 virus isolate was harvested 4 days after infection of C6/36 cells at an MOI of 0.1. Recombinant virus was harvested 4 to 7 days after electroporation of RNA into C6/36 cells. Titrations were performed in triplicate. (C) Virus focus images on Vero-81 cells. (D) Growth curves of H/PF/2013 natural isolate and infectious clone at 32°C and 37°C. At both temperatures, the isolate grew significantly better than the infectious clone by 2-way analysis of variance.

FIG 3

(A and B) Diagrams of ZIKV SPH2015 and BeH819015 infectious clones. (A) Diagram shows changes introduced into the first 20 nt to recover SPH2015 infectious clone virus. (B) Diagram of infectious clone BeH819015. Six nucleotide changes made to convert SPH2015 clone into BeH819015 are shown (red arrows). (C) Virus focus images on Vero-81 cells. (D) Growth curves of PRVABC59 natural isolate and infectious clones of SPH2015 and BeH819015 at 37°C. Although they differ by only 6 nucleotides, growth of SPH2015 was significantly different from that of BeH819015 by 2-way analysis of variance with Tukey’s test.

FIG 4

Phylogenetic tree of Brazilian ZIKV full-length sequences. ZIKV sequences were acquired from GenBank and this study (see Materials and Methods), and the 5′ UTR was removed from each lineage. Multiple sequence alignment was performed using MAFFT, and the phylogenetic tree was generated using maximum likelihood (RAxML software) with 100 bootstrap replicates. Only bootstrap support values of >50 are displayed at each node. GenBank accession number and strain name are indicated on each branch. Two distinct clades of Brazilian ZIKV sequences are highly supported (green box). SPH2015 and BeH819015 are boxed in blue and red, respectively.

FIG 5

Antibody binding to DENV and ZIKV clone viruses. (A) The highly DENV serotype-specific MAbs 1F4, 2D22, 5J7, and 5H2 clearly bind only the appropriate DENV serotype but not ZIKV. (B) The DENV serotype-cross-reactive MAbs 1C19 and 1M7 bound all DENV and ZIKV strains tested, while 1B22, a prM-specific MAb, bound only the 4 DENV serotypes. DT000 is serum isolated from a traveler with repeat flavivirus vaccinations and infections and is used to control protein loading.

FIG 6

In vivo pathogenesis studies. ZIKV H/PF/2013 isolate (n = 9), infectious clone virus (n = 9), or PBS (n = 7) was inoculated into IFNGR-knockout mice by footpad inoculation with 10² FFU (A and B). In parallel, Ifnar1^−/− and Ifngr1^−/− mice were inoculated by footpad injection with 10³ FFU of BeH819015 (n = 6) or SPH2015 (n = 6) infectious clone or PBS (n = 3) (C and D) or 10⁵ FFU of recombinant SPH2015 (n = 5), BeH819015 (n = 5), or H/PF/2013 (n = 5) infectious clone (E), respectively. Weight loss and mortality were recorded through 25 days postinfection or until all animals had succumbed to infection.