Mice as an Animal Model for Japanese Encephalitis Virus Research: Mouse Susceptibility, Infection Route, and Viral Pathogenesis.

Japanese encephalitis virus (JEV), a zoonotic flavivirus, is principally transmitted by hematophagous mosquitoes, continually between susceptible animals and incidentally from those animals to humans. For almost a century since its discovery, JEV was geographically confined to the Asia-Pacific region with recurrent sizable outbreaks involving wildlife, livestock, and people. However, over the past decade, it has been detected for the first time in Europe (Italy) and Africa (Angola) but has yet to cause any recognizable outbreaks in humans. JEV infection leads to a broad spectrum of clinical outcomes, ranging from asymptomatic conditions to self-limiting febrile illnesses to life-threatening neurological complications, particularly Japanese encephalitis (JE). No clinically proven antiviral drugs are available to treat the development and progression of JE. There are, however, several live and killed vaccines that have been commercialized to prevent the infection and transmission of JEV, yet this virus remains the main cause of acute encephalitis syndrome with high morbidity and mortality among children in the endemic regions. Therefore, significant research efforts have been directed toward understanding the neuropathogenesis of JE to facilitate the development of effective treatments for the disease. Thus far, multiple laboratory animal models have been established for the study of JEV infection. In this review, we focus on mice, the most extensively used animal model for JEV research, and summarize the major findings on mouse susceptibility, infection route, and viral pathogenesis reported in the past and present, and discuss some unanswered key questions for future studies.

Development and characterization of type I interferon receptor knockout sheep: A model for viral immunology and reproductive signaling.

Type I interferons (IFNs) initiate immune responses to viral infections. Their effects are mediated by the type I IFN receptor, IFNAR, comprised of two subunits: IFNAR1 and IFNAR2. One or both chains of the sheep IFNAR were disrupted in fetal fibroblast lines using CRISPR/Cas9 and 12 lambs were produced by somatic cell nuclear transfer (SCNT). Quantitative reverse transcription-polymerase chain reaction for IFN-stimulated gene expression showed that IFNAR deficient sheep fail to respond to IFN-alpha. Furthermore, fibroblast cells from an IFNAR2 -/- fetus supported significantly higher levels of Zika virus (ZIKV) replication than wild-type fetal fibroblast cells. Although many lambs have died from SCNT related problems or infections, one fertile IFNAR2 -/- ram lived to over 4 years of age, remained healthy, and produced more than 80 offspring. Interestingly, ZIKV infection studies failed to demonstrate a high level of susceptibility. Presumably, these sheep compensated for a lack of type I IFN signaling using the type II, IFN-gamma and type III, IFN-lambda pathways. These sheep constitute a unique model for studying the pathogenesis of viral infection. Historical data supports the concept that ruminants utilize a novel type I IFN, IFN-tau, for pregnancy recognition. Consequently, IFNAR deficient ewes are likely to be infertile, making IFNAR knockout sheep a valuable model for studying pregnancy recognition. A breeding herd of 32 IFNAR2 +/- ewes, which are fertile, has been developed for production of IFNAR2 -/- sheep for both infection and reproduction studies.

Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses.

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals.

Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection.

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and genetically divergent ZIKVs: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015). Using the three molecularly cloned ZIKVs, together with 13 ZIKV region-specific polyclonal antibodies covering nearly the entire viral protein-coding region, we made three conceptual advances: (i) We created a comprehensive genome-wide portrait of ZIKV gene products and their related species, with several previously undescribed gene products identified in the case of all three molecularly cloned ZIKVs. (ii) We found that ZIKV has a broad cell tropism in vitro, being capable of establishing productive infection in 16 of 17 animal cell lines from 12 different species, although its growth kinetics varied depending on both the specific virus strain and host cell line. More importantly, we identified one ZIKV-non-susceptible bovine cell line that has a block in viral entry but fully supports the subsequent post-entry steps. (iii) We showed that in mice, the three molecularly cloned ZIKVs differ in their neuropathogenicity, depending on the particular combination of viral and host genetic backgrounds, as well as in the presence or absence of type I/II interferon signaling. Overall, our findings demonstrate the impact of viral and host genetic variations on the replication kinetics and neuropathogenicity of ZIKV and provide multiple avenues for developing and testing medical countermeasures against ZIKV.

Complete Genome Sequences of Three Historically Important, Spatiotemporally Distinct, and Genetically Divergent Strains of Zika Virus: MR-766, P6-740, and PRVABC-59.

Here, we report the 10,807-nucleotide-long consensus RNA genome sequences of three spatiotemporally distinct and genetically divergent Zika virus strains, with the functionality of their genomic sequences substantiated by reverse genetics: MR-766 (African lineage, Uganda, 1947), P6-740 (Asian lineage, Malaysia, 1966), and PRVABC-59 (Asian lineage-derived American strain, Puerto Rico, 2015).