Epididymal epithelium propels early sexual transmission of Zika virus in the absence of interferon signaling.

Recognition of Zika virus (ZIKV) sexual transmission (ST) among humans challenges our understanding of the maintenance of mosquito-borne viruses in nature. Here we dissected the relative contributions of the components of male reproductive system (MRS) during early male-to-female ZIKV transmission by utilizing mice with altered antiviral responses, in which ZIKV is provided an equal opportunity to be seeded in the MRS tissues. Using microRNA-targeted ZIKV clones engineered to abolish viral infectivity to different parts of the MRS or a library of ZIKV genomes with unique molecular identifiers, we pinpoint epithelial cells of the epididymis (rather than cells of the testis, vas deferens, prostate, or seminal vesicles) as a most likely source of the sexually transmitted ZIKV genomes during the early (most productive) phase of ZIKV shedding into the semen. Incorporation of this mechanistic knowledge into the development of a live-attenuated ZIKV vaccine restricts its ST potential.

Virus infection of the CNS disrupts the immune-neural-synaptic axis via induction of pleiotropic gene regulation of host responses.

Treatment for many viral infections of the central nervous system (CNS) remains only supportive. Here we address a remaining gap in our knowledge regarding how the CNS and immune systems interact during viral infection. By examining the regulation of the immune and nervous system processes in a nonhuman primate model of West Nile virus neurological disease, we show that virus infection disrupts the homeostasis of the immune-neural-synaptic axis via induction of pleiotropic genes with distinct functions in each component of the axis. This pleiotropic gene regulation suggests an unintended off-target negative impact of virus-induced host immune responses on the neurotransmission, which may be a common feature of various viral infections of the CNS.

Two Sides of a Coin: a Zika Virus Mutation Selected in Pregnant Rhesus Macaques Promotes Fetal Infection in Mice but at a Cost of Reduced Fitness in Nonpregnant Macaques and Diminished Transmissibility by Vectors.

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.

Zika virus tropism during early infection of the testicular interstitium and its role in viral pathogenesis in the testes.

Sexual transmission and persistence of Zika virus (ZIKV) in the testes pose new challenges for controlling virus outbreaks and developing live-attenuated vaccines. It has been shown that testicular infection of ZIKV is initiated in the testicular interstitium, followed by spread of the virus in the seminiferous tubules. This leads to testicular damage and/or viral dissemination into the epididymis and eventually into semen. However, it remains unknown which cell types are targeted by ZIKV in the testicular interstitium, and what is the specific order of infectious events leading to ZIKV invasion of the seminiferous tubules. Here, we demonstrate that interstitial leukocytes expressing mir-511-3p microRNA are the initial targets of ZIKV in the testes, and infection of mir-511-3p-expressing cells in the testicular interstitium is necessary for downstream infection of the seminiferous tubules. Mir-511-3p is expressed concurrently with CD206, a marker of lineage 2 (M2) macrophages and monocyte derived dendritic cells (moDCs). Selective restriction of ZIKV infection of CD206-expressing M2 macrophages/moDCs results in the attenuation of macrophage-associated inflammatory responses in vivo and prevents the disruption of the Sertoli cell barrier in vitro. Finally, we show that targeting of viral genome for mir-511-3p significantly attenuates early ZIKV replication not only in the testes, but also in many peripheral organs, including spleen, epididymis, and pancreas. This incriminates M2 macrophages/moDCs as important targets for visceral ZIKV replication following hematogenous dissemination of the virus from the site of infection.

Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability.

Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.

Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, attempts to develop of a live attenuated virus (LAV) vaccine against TBEV with acceptable safety and immunogenicity characteristics have not been successful. To overcome this impasse, we generated a chimeric TBEV that was highly immunogenic in nonhuman primates (NHPs). The chimeric virus contains the prM/E genes of TBEV, which are expressed in the genetic background of an antigenically closely related, but less pathogenic member of the TBEV complex-Langat virus (LGTV), strain T-1674. The neurovirulence of this chimeric virus was subsequently controlled by robust targeting of the viral genome with multiple copies of central nervous system-enriched microRNAs (miRNAs). This miRNA-targeted T/1674-mirV2 virus was highly stable in Vero cells and was not pathogenic in various mouse models of infection or in NHPs. Importantly, in NHPs, a single dose of the T/1674-mirV2 virus induced TBEV-specific neutralizing antibody (NA) levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent wild-type TBEV challenge in mice and against challenge with a parental (not miRNA-targeted) chimeric TBEV/LGTV in NHPs. Thus, this highly attenuated and immunogenic T/1674-mirV2 virus is a promising LAV vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in NHPs prior to entering clinical trials in humans.IMPORTANCE Tick-borne encephalitis virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV.

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice.

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.

Flaviviruses and the Central Nervous System: Revisiting Neuropathological Concepts.

Flaviviruses are major emerging human pathogens on a global scale. Some flaviviruses can infect the central nervous system of the host and therefore are regarded as neurotropic. The most clinically relevant classical neurotropic flaviviruses include Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. In this review, we focus on these flaviviruses and revisit the concepts of flaviviral neurotropism, neuropathogenicity, neuroinvasion, and resultant neuropathogenesis. We attempt to synthesize the current knowledge about interactions between the central nervous system and flaviviruses from the neuroanatomical and neuropathological perspectives and address some misconceptions and controversies. We hope that revisiting these neuropathological concepts will improve the understanding of flaviviral neuroinfections. This, in turn, may provide further guiding foundations for relevant studies of other emerging or geographically expanding flaviviruses with neuropathogenic potential, such as Zika virus and dengue virus, and pave the way for intelligent therapeutic strategies harnessing potentially beneficial, protective host responses to interfere with disease progression and outcome.

Decreased accumulation of subgenomic RNA in human cells infected with vaccine candidate DEN4Δ30 increases viral susceptibility to type I interferon.

The NIH has developed live attenuated dengue virus (DENV) vaccine candidates by deletion of 30 nucleotides (Δ30) from the untranslated region of the viral genome. Although this attenuation strategy has proven to be effective in generating safe and immunogenic vaccine strains, the molecular mechanism of attenuation is largely unknown. To examine the mediators of the observed attenuation phenotype, differences in translation efficiency, genome replication, cytotoxicity, and type I interferon susceptibility were compared between wild type parental DENV and DENVΔ30 attenuated vaccine candidates. We observed that decreased accumulation of subgenomic RNA (sfRNA) from the vaccine candidates in infected human cells causes increased type I IFN susceptibility and propose this as one of the of attenuation mechanisms produced by the 3' UTR Δ30 mutation.

Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

Synergistic Internal Ribosome Entry Site/MicroRNA-Based Approach for Flavivirus Attenuation and Live Vaccine Development.

The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Using another flavivirus (Langat virus [LGTV]) that belongs to the group of tick-borne flaviviruses as a model, we describe a dual strategy for virus attenuation which synergistically accesses the specificity of microRNA (miRNA) genome targeting and the effectiveness of internal ribosome entry site (IRES) insertion. To increase the stability and immunogenicity of bicistronic LGTVs, we developed a novel approach in which the capsid (C) protein gene was relocated into the 3' noncoding region (NCR) and expressed under translational control from an IRES. Engineered bicistronic LGTVs carrying multiple target sequences for brain-specific miRNAs were stable in Vero cells and induced adaptive immunity in mice. Importantly, miRNA-targeted bicistronic LGTVs were not pathogenic for either newborn mice after intracranial inoculation or adult immunocompromised mice (SCID or type I interferon receptor knockout) after intraperitoneal injection. Moreover, bicistronic LGTVs were restricted for replication in tick-derived cells, suggesting an interruption of viral transmission in nature by arthropod vectors. This approach is suitable for reliable attenuation of many flaviviruses and may enable development of live attenuated flavivirus vaccines.IMPORTANCE The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Allied separately attenuating approaches based on (i) microRNA genome targeting and (ii) internal ribosome entry site insertion are not sufficient for relievable attenuation of neurotropic flavivirus pathogenesis. Here, we describe a novel dual strategy that combines the specificity of miRNA-based and the effectiveness of IRES-based attenuating approaches, allowing us to overcome these critical limitations. This developed approach provides a robust platform for reliable attenuation of many flaviviruses and may enable development of live flavivirus vaccines.

Chemokine Receptor Ccr7 Restricts Fatal West Nile Virus Encephalitis.

West Nile virus (WNV) is a mosquito-transmitted flavivirus that can cause debilitating encephalitis. To delineate the mechanisms behind this pathology, we studied Ccr7-deficient mice, which afforded us the capacity to study infection in mice with disrupted peripheral cellular trafficking events. The loss of Ccr7 resulted in an immediate pan-leukocytosis that remained elevated throughout the infection. This leukocytosis resulted in a significant enhancement of leukocyte accumulation within the central nervous system (CNS). Despite an excess of virus-specific T cells in the CNS, Ccr7-deficient mice had significantly higher CNS viral loads and mortality rates than wild-type animals. Mechanistically, the elevated trafficking of infected myeloid cells into the brain in Ccr7-deficient mice resulted in increased levels of WNV in the CNS, thereby effectively contributing to neuroinflammation and lowering viral clearance. Combined, our experiments suggest that during WNV infection, Ccr7 is a gatekeeper for nonspecific viral transference to the brain.IMPORTANCE In this study, we show that Ccr7 is required for the sufficient migration of dendritic cells and T cells into the draining lymph node immediately following infection and for the restriction of leukocyte migration into the brain. Further, the severe loss of dendritic cells in the draining lymph node had no impact on viral replication in this organ, suggesting that WNV may migrate from the skin into the lymph node through another mechanism. Most importantly, we found that the loss of Ccr7 results in a significant leukocytosis, leading to hypercellularity within the CNS, where monocytes/macrophages contribute to CNS viremia, neuroinflammation, and increased mortality. Together, our data point to Ccr7 as a critical host defense restriction factor limiting neuroinflammation during acute viral infection.

A Live Attenuated Chimeric West Nile Virus Vaccine, rWN/DEN4Δ30, Is Well Tolerated and Immunogenic in Flavivirus-Naive Older Adult Volunteers.

West Nile virus (WNV) is a major cause of mosquito-borne illness in the United States. Human disease ranges from mild febrile illness to severe fatal neurologic infection. Adults aged >60 years are more susceptible to neuroinvasive disease accompanied by a high mortality rate or long-lasting neurologic sequelae. A chimeric live attenuated West Nile virus vaccine, rWN/DEN4Δ30, was shown to be safe and immunogenic in healthy adults aged 18-50 years. This study evaluated rWN/DEN4Δ30 in flavivirus-naive adults aged 50-65 years and found it to be safe and immunogenic. Outbreaks of WNV infection tend to be unpredictable, and a safe and effective vaccine will be an important public health tool.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System.

BACKGROUND: During recent West Nile virus (WNV) outbreaks in the US, half of the reported cases were classified as neuroinvasive disease. WNV neuroinvasion is proposed to follow two major routes: hematogenous and/or axonal transport along the peripheral nerves. How virus spreads once within the central nervous system (CNS) remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using immunohistochemistry, we examined the expression of viral antigens in the CNS of rhesus monkeys that were intrathalamically inoculated with a wild-type WNV. The localization of WNV within the CNS was mapped to specific neuronal groups and anatomical structures. The neurological functions related to structures containing WNV-labeled neurons were reviewed and summarized. Intraneuronal localization of WNV was investigated by electron microscopy. The known anatomical connectivity of WNV-labeled neurons was used to reconstruct the directionality of WNV spread within the CNS using a connectogram design. Anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belonged to the pathways of motor control. Ultrastructurally, virions were found predominantly within vesicular structures (including autophagosomes) in close vicinity to the axodendritic synapses, either at pre- or post-synaptic positions (axonal terminals and dendritic spines, respectively), strongly indicating transsynaptic spread of the virus between connected neurons. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons. CONCLUSIONS/SIGNIFICANCE: This study offers a new insight into the neuropathogenesis of WNV infection in a primate model that closely mimics WNV encephalomyelitis in humans. We show that within the primate CNS, WNV primarily infects the anatomical structures and pathways responsible for the control of movement. Our findings also suggest that WNV most likely propagates within the CNS transsynaptically, by both, anterograde and retrograde axonal transport.

A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development.

UNLABELLED: An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. IMPORTANCE: The availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell-adapted version of it, which will be used for virus-host interaction studies and vaccine development.

Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell-Mediated Viral Clearance.

Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5(-/-) mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5(-/-) mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control-treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.

Kissing-loop interaction between 5' and 3' ends of tick-borne Langat virus genome 'bridges the gap' between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development.

Insertion of microRNA target sequences into the flavivirus genome results in selective tissue-specific attenuation and host-range restriction of live attenuated vaccine viruses. However, previous strategies for miRNA-targeting did not incorporate a mechanism to prevent target elimination under miRNA-mediated selective pressure, restricting their use in vaccine development. To overcome this limitation, we developed a new approach for miRNA-targeting of tick-borne flavivirus (Langat virus, LGTV) in the duplicated capsid gene region (DCGR). Genetic stability of viruses with DCGR was ensured by the presence of multiple cis-acting elements within the N-terminal capsid coding region, including the stem-loop structure (5'SL6) at the 3' end of the promoter. We found that the 5'SL6 functions as a structural scaffold for the conserved hexanucleotide motif at its tip and engages in a complementary interaction with the region present in the 3' NCR to enhance viral RNA replication. The resulting kissing-loop interaction, common in tick-borne flaviviruses, supports a single pair of cyclization elements (CYC) and functions as a homolog of the second pair of CYC that is present in the majority of mosquito-borne flaviviruses. Placing miRNA targets into the DCGR results in superior attenuation of LGTV in the CNS and does not interfere with development of protective immunity in immunized mice.

MicroRNA-based control of tick-borne flavivirus neuropathogenesis: Challenges and perspectives.

In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenicity of both DNA and RNA viruses. Previously, we reported the successful application of this strategy to control the neurovirulent phenotype of a model chimeric tick-borne encephalitis/dengue type 4 virus (TBEV/DEN4), containing the structural protein genes of a highly virulent TBEV in the genetic backbone of non-neuroinvasive DEN4 virus. In the present study, we investigated the suitability of this approach for the attenuation of the more neurovirulent chimeric virus (TBEV/LGTV), which is based on the genetic backbone of the naturally attenuated member of the TBEV serocomplex, a Langat virus (LGTV). Unlike the TBEV/DEN4, the TBEV/LGTV virus retained the ability of its parental viruses to spread from the peripheral site of inoculation to the CNS. We evaluated ten potential sites in the 3'NCR of the TBEV/LGTV genome for placement of microRNA (miRNA) targets and found that the TBEV/LGTV genome is more restrictive for such genetic manipulations compared to TBEV/DEN4. In addition, unlike TBEV/DEN4 virus, the introduction of multiple miRNA targets into either the 3'NCR or ORF of the TBEV/LGTV genome had only a modest effect on virus attenuation in the developing CNS of highly permissive newborn mice. However, simultaneous miRNA-targeting in the ORF and 3'NCR had synergistic effect on control and silencing of virus replication in the brain and completely abolished the virus neurotropism. Furthermore, neuroinvasiveness of miRNA-targeted TBEV/LGTV viruses in very sensitive immunodeficient SCID mice was significantly limited. Immunocompetent animals immunized with such viruses were completely protected against challenge with the neurovirulent LGTV parent. These findings support the rationale of the miRNA-targeting approach to develop live attenuated virus vaccines against various neurotropic viruses.