Hannah Romo

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

ABSTRACT Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

Experimental Evolution of an RNA Virus in Wild Birds: Evidence for Host-Dependent Impacts on Population Structure and Competitive Fitness

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Evidence for Co-evolution of West Nile Virus and House Sparrows in North America

West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains.

Transmission Incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika Virus

In late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed.

Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses

These birds are highly susceptible to strains circulating in Europe and, thus, may serve as surveillance sentinels.

Host Competence and Helicase Activity Differences Exhibited by West Nile Viral Variants Expressing NS3-249 Amino Acid Polymorphisms

A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.

Differential Neurovirulence of African and Asian Genotype Zika Virus Isolates in Outbred Immunocompetent Mice

Abstract. Although first isolated almost 70 years ago, Zika virus (ZIKV; Flavivirus, Flaviviridae) has only recently been associated with significant outbreaks of disease in humans. Several severe ZIKV disease manifestations have also been recently documented, including fetal malformations, such as microcephaly, and Guillain–Barré syndrome in adults. Although principally transmitted by mosquitoes, sexual transmission of ZIKV has been documented. Recent publications of several interferon receptor knockout mouse models have demonstrated ZIKV-induced disease. Herein, outbred immunocompetent CD-1/ICR adult mice were assessed for susceptibility to disease by intracranial (i.c.) and intraperitoneal (i.p.) inoculation with the Ugandan prototype strain (MR766; African genotype), a low-passage Senegalese strain (DakAr41524; African genotype) and a recent ZIKV strain isolated from a traveler infected in Puerto Rico (PRVABC59; Asian genotype). Morbidity was not observed in mice inoculated by the i.p. route with either MR766 or PRVABC59 for doses up to 6 log10 PFU. In contrast, CD-1/ICR mice inoculated i.c. with the MR766 ZIKV strain exhibited an 80–100% mortality rate that was age independent. The DakAr41524 strain delivered by the i.c route caused 30% mortality, and the Puerto Rican ZIKV strain failed to elicit mortality but did induce a serum neutralizing immune response in 60% of mice. These data provide a potential animal model for assessing neurovirulence determinants of different ZIKV strains as well as a potential immunocompetent challenge model for assessing protective efficacy of vaccine candidates.

Comparative vector competence of North American culex pipiens and culex quinquefasciatus for African and European lineage 2 west nile viruses

West Nile virus (WNV) is a mosquito-borne flavivirus that is phylogenetically separated into distinct lineages. Lineage 1 (L1) and lineage 2 (L2) encompass all WNV isolates associated with human and veterinary disease cases. Although L1 WNV is globally distributed, including North America, L2 WNV only recently emerged out of sub-Saharan Africa into Europe and Russia. The spread of L2 WNV throughout and beyond Europe depends, in part, on availability of competent vectors. The vector competence of mosquitoes within the Culex genus for WNV is well established for L1 WNV but less extensively studied for L2 WNV. Assessing the vector competence of North American Culex mosquitoes for L2 WNV will be critical for predicting the potential for L2 WNV emergence in North America. We address the vector competence of North American Culex pipiens and Culex quinquefasciatus for L2 WNV. Both mosquito species were highly competent for each of the L2 WNV strains assessed, but variation in infection, dissemination, and transmission was observed. An L2 WNV strain (NS10) isolated during the Greek outbreak in 2010 exhibited a reduced capacity to infect Cx. pipiens compared with other L2 WNV strains. In addition, a South African L2 WNV strain (SA89) displayed a significantly shorter extrinsic incubation period in Cx. quinquefasciatus compared with other L2 WNV strains. These results demonstrate that North American Culex mosquito species are competent vectors of African and European L2 WNV and that emergence of L2 WNV is unlikely to be hindered by poor competence of North American vectors.

Erratum for Weger-Lucarelli et al., “Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone”

Volume 91, no. 1, 2017, https://doi.org/10.1128/JVI.01765-16. Page 1, shaded box: The following footnote should be added: “J.W.-L. and N.K.D. contributed equally to this work.” Citation Weger-Lucarelli J, Duggal NK, BullardFeibelman K, Veselinovic M, Romo H, Nguyen C, Rückert C, Brault AC, Bowen RA, Stenglein M, Geiss BJ, Ebel GD. 2017. Erratum for WegerLucarelli et al., “Development and characterization of recombinant virus generated from a New World Zika virus infectious clone.” J Virol 91:e00172-17. https:// doi.org/10.1128/JVI.00172-17. Copyright