Immunogenicity and Protection After Vaccination With a Modified Vaccinia Virus Ankara-Vectored Yellow Fever Vaccine in the Hamster Model

The highly efficacious live-attenuated 17D yellow fever (YF) vaccine is occasionally associated with rare life-threatening adverse events. Modified vaccinia virus Ankara (MVA), a non-replicating poxvirus, has been used as a vaccine platform to safely deliver various antigens. A MVA-based YF vaccine (MVA-BN-YF) was tested with and without a non-mineral oil adjuvant in a hamster model of lethal YF disease and protective efficacy of this vaccine was compared with the 17D vaccine. The vaccine candidate MVA-BN-YF generated a protective response in hamsters infected with YFV that was comparable to protection by the live 17D vaccine. Similar levels of neutralizing antibody were observed in animals vaccinated with either vaccine alone or vaccine with adjuvant. Significant improvement in survival, weight change, and serum alanine aminotransferase levels were observed in vaccinated hamsters when administered 42 and 14 days prior to challenge with Jimenez YF virus (YFV). Neutralizing antibodies induced by MVA-BN-YF were transferred to naïve hamsters prior to virus challenge. Passive administration of neutralizing antibody 24 h prior to virus infection resulted in significantly improved survival and weight change. A trend toward reduced liver enzyme levels was also observed. MVA-BN-YF, therefore, represents a safe alternative to vaccination with live-attenuated YFV

Small-Animal Models of Zika Virus

Zika virus (ZIKV) infection can result in serious consequences, including severe congenital manifestations, persistent infection in the testes and neurologic sequelae. After a pandemic emergence, the virus has spread to much of the new world and has been introduced to many countries outside of endemic areas as infected travelers return to their home countries. Rodent models have been important in gaining a better understanding of the wide range of disease etiologies associated with ZIKV infection and for the initial phase of developing countermeasures to prevent or treat viral infections. We discuss herein the advantages and disadvantages of small animal models that have been developed to replicate various aspects of disease associated with ZIKV infection

Zika Virus-induced Acute Myelitis and Motor Deficits in Adult Interferon αβ/γ Receptor Knockout Mice

Zika virus (ZIKV) has received widespread attention because of its effect on the developing fetus. It is becoming apparent, however, that severe neurological sequelae, such as Guillian-Barrë syndrome (GBS), myelitis, encephalitis, and seizures can occur after infection of adults. This study demonstrates that a contemporary strain of ZIKV can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (AG129 strain) and cause progressive hindlimb paralysis, as well as severe seizure-like activity during the acute phase of disease. The severity of hindlimb motor deficits correlated with increased numbers of ZIKV-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. Electrophysiological compound muscle action potential (CMAP) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. ZIKV immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. Infection in the brain and spinal cord was also associated with astrogliosis as well as T cell and neutrophil infiltration. CMAP and histological analysis indicated that peripheral nerve and muscle functions were intact. Consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a GBS-like syndrome. Thus, acute ZIKV infection of adult AG129 mice may be a useful model for ZIKV-induced myelitis, encephalitis, and seizure activity

Evolutionary Selection Against Short Nucleotide Sequences in Viruses and Their Related Hosts

Viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. Understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. These sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. We specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. In addition, we show that more sequences tend to be under-represented in dsDNA viruses than in other viral groups. Finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated Zika virus strains

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

Zika Virus Associated Pathology and Antigen Presence in the Testicle in the Absence of Sexual Transmission During Subacute to Chronic Infection in a Mouse Model

Zika virus (ZIKV) is an arboviral infection that has been shown to be sexually transmitted. The study outlined herein aims to determine if accessory sex glands and epididymal epithelial cells are sources of viral persistence in subacute and chronic ZIKV infection, and if infection of these organs is important in sexual transmission during long-term (chronic) infection. Male interferon type I receptor knockout (Ifnar−/−) mice were challenged with ZIKV and reproductive tissues were harvested 14 and 35 days post infection (DPI) for inoculation studies and 14, 35 and 70 DPI for histopathology. Artificial insemination fluid derived from epididymal flush and seminal plasma from the prostate and seminal vesicle was obtained from ZIKV inoculated and sham-infected males. Naïve interferon type I and II receptor knockout (AG129) female mice were pre-treated with progesterone and inoculated intravaginally with artificial insemination fluid from ZIKV-infected males. ZIKV RNA was detected in the artificial insemination fluid generated from epididymal flush or seminal plasma from ZIKV infected males at 14 and 35 DPI. ZIKV antigens were only detected in seminiferous tubules at 14 DPI. Epididymal epithelial cells did not show ZIKV antigen immunoreactivity at 14, 35 or 70 DPI. Severe fibrosing orchitis (end stage orchitis) was observed at 35 and 70 DPI. Mild inflammation and peri-tubular fibrosis were observed in the epididymis following clearance of virus. Viral RNA was not detected by PCR in whole blood samples of females from any intravaginal experimental group and only detected in 20% of subcutaneously inoculated animals (derived from 1 experimentally infected male) at 35 DPI. While ZIKV RNA and antigens can be detected in the male reproductive tract at 14 DPI and RNA can also be detected at 35 DPI, intravaginal inoculation of artificial insemination fluid from these time-points failed to result in viremia in naïve females inoculated intravaginally. These studies support the hypothesis that epididymal epithelial cells are critical to sexual transmission in immunocompromised mice. Additionally, acute but not chronic male reproductive tract infection with ZIKV results in infectious virus capable of being sexually transmitted in mice

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)

Efficacy of a ML336 Derivative Against Venezuelan and Eastern Equine Encephalitis Viruses

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase

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

Human Polyclonal Antibodies Produced from Transchromosomal Bovine Provides Prophylactic and Therapeutic Protections Against Zika Virus Infection in STAT2 KO Syrian Hamsters

Zika virus (ZIKV) infection can cause severe congenital diseases, such as microcephaly, ocular defects and arthrogryposis in fetuses, and Guillain–Barré syndrome in adults. Efficacious therapeutic treatments for infected patients, as well as prophylactic treatments to prevent new infections are needed for combating ZIKV infection. Here, we report that ZIKV-specific human polyclonal antibodies (SAB-155), elicited in transchromosomal bovine (TcB), provide significant protection from infection by ZIKV in STAT2 knockout (KO) golden Syrian hamsters both prophylactically and therapeutically. These antibodies also prevent testicular lesions in this hamster model. Our data indicate that antibody-mediated immunotherapy is effective in treating ZIKV infection. Because suitable quantities of highly potent human polyclonal antibodies can be quickly produced from the TcB system against ZIKV and have demonstrated therapeutic efficacy in a small animal model, they have the potential as an effective countermeasure against ZIKV infection