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.

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.

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.

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.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains.

Chemokine receptor Ccr2 is critical for monocyte accumulation and survival in West Nile virus encephalitis.

West Nile virus (WNV) is a re-emerging pathogen responsible for outbreaks of fatal meningoencephalitis in humans. Previous studies have suggested a protective role for monocytes in a mouse model of WNV infection, but the molecular mechanisms have remained unclear. In this study, we show that genetic deficiency in Ccr2, a chemokine receptor on Ly6c(hi) inflammatory monocytes and other leukocyte subtypes, markedly increases mortality due to WNV encephalitis in C57BL/6 mice; this was associated with a large and selective reduction of Ly6c(hi) monocyte accumulation in the brain. WNV infection in Ccr2(+/+) mice induced a strong and highly selective monocytosis in peripheral blood that was absent in Ccr2(-/-) mice, which in contrast showed sustained monocytopenia. When a 1:1 mixture of Ccr2(+/+) and Ccr2(-/-) donor monocytes was transferred by vein into WNV-infected Ccr2(-/-) recipient mice, monocyte accumulation in the CNS was not skewed toward either component of the mixture, indicating that Ccr2 is not required for trafficking of monocytes from blood to brain. We conclude that Ccr2 mediates highly selective peripheral blood monocytosis during WNV infection of mice and that this is critical for accumulation of monocytes in the brain.

Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype.

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome.

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E(315)) and NS5 (NS5(654,655)) proteins, and into the 3' non-coding region (Delta30) of TBEV/DEN4. The variant that contained all three mutations (vDelta30/E(315)/NS5(654,655)) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vDelta30/E(315)/NS5(654,655) should be further evaluated as a TBEV vaccine.

Cellular inflammatory response to flaviviruses in the central nervous system of a primate host.

Flaviviruses such as tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, and St. Louis encephalitis virus are important neurotropic human pathogens, typically causing a devastating and often fatal neuroinfection. Flaviviruses induce neuroinflammation with typical features of viral encephalitides, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. Development of safe and effective live-virus vaccines against neurotropic flavivirus infections demands a detailed knowledge of their neuropathogenesis in a primate host that is evolutionarily close to humans. Here, we used computerized morphometric analysis to quantitatively assess the cellular inflammatory responses in the central nervous system (CNS) of rhesus monkeys infected with three antigenically divergent attenuated flaviviruses. The kinetics, spatial pattern, and magnitude of microglial activation, trafficking of T and B cells, and changes in T cell subsets within the CNS define unique phenotypic signatures for each of the three viruses. Our results provide a benchmark for investigation of cellular inflammatory responses induced by attenuated flaviviruses in the CNS of primate hosts and provide insight into the neuropathogenesis of flavivirus encephalitis that might guide the development of safe and effective live-virus vaccines.

Attenuated West Nile viruses bearing 3'SL and envelope gene substitution mutations.

Four viable West Nile (WN) 3'SL-mutant viruses were evaluated for neuroinvasiveness and neurovirulence in mice. All mutants were highly attenuated for neuroinvasiveness. However, only one of these four (WNmutE virus) was significantly attenuated for neurovirulence. To attenuate WNmutE virus further, we introduced five substitution mutations into the envelope (env) gene segment in wild-type (wt) WN and WNmutE genomes, based on differences in the env gene sequence between the live Japanese encephalitis vaccine (SA14-14-2) and its virulent parent. The env gene mutations had an attenuating effect in the context of the wt WNV genome but only a marginal enhancing effect on the attenuation of WNmutE virus.

Chimeric West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity.

A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4Delta30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against WN challenge. Here, we report the generation of a clinical lot of WN/DEN4Delta30 virus and its further preclinical evaluation for safety and immunogenicity in mice, geese and monkeys. The vaccine candidate had lost neuroinvasiveness in highly sensitive immunodeficient mice inoculated intraperitoneally and had greatly reduced neurovirulence in suckling mice inoculated intracerebrally (IC). Compared to the wild-type WN parent, the chimeric virus was highly restricted in replication in both murine and human neuroblastoma cells as well as in brains of suckling mice. The WN/DEN4Delta30 virus failed to infect geese, indicating that chimerization of WN with DEN4 completely attenuated WN for this avian host. This observation suggests that the WN/DEN4 chimeric viruses would be restricted in their ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4Delta30 vaccine candidate was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4Delta30 vaccine virus was safe and readily induced neutralizing antibodies against WN in monkeys immune to each of the four serotypes of dengue virus. These studies confirm the attenuation of WN/DEN4Delta30 for non-human primates, including dengue-immune monkeys, and demonstrate both a highly restricted replication (>10(8)-fold decrease) in the brain of mice inoculated IC and an absence of infectivity for birds, findings that indicate this vaccine should be safe for both the recipient and the environment.

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice.

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.

Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys.

Three antigenic chimeric live attenuated tick-borne encephalitis virus (TBEV) vaccine candidates were compared for level of replication in murine and human neuroblastoma cells, for neurovirulence and neuroinvasiveness in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. Two chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue type 4 virus (DEN4) with the corresponding genes of a Far Eastern TBEV, Sofjin strain, in the presence (TBEV/DEN4Delta30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome. A third chimeric TBEV vaccine candidate was based on the antigenically distant, but naturally attenuated Langat virus (LGT). Chimerization of LGT with DEN4 resulted in decreased neurovirulence and neuroinvasiveness in mice and highly restricted viremia in rhesus monkeys. Also, the LGT/DEN4 chimera was highly restricted in replication in both murine and human neuroblastoma cells. In contrast, TBEV/DEN4 and TBEV/DEN4Delta30 were neither attenuated for neurovirulence in the mice nor restricted in replication in the neuroblastoma cells. However, both were highly attenuated for neuroinvasiveness in mice. TBEV/DEN4 replicated to moderately high titer in rhesus monkeys (mean peak viremia=10(3.1)PFU/ml) indicating that the TBEV/DEN4 chimerization had only a modest, if any, attenuating effect in monkeys. However, the addition of the Delta30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for rhesus monkeys (mean peak viremia=10(0.7)PFU/ml) and induced a higher level of antibody against the TBEV than did LGT/DEN4. A single dose of either highly attenuated TBEV/DEN4Delta30 or LGT/DEN4 vaccine candidate or three doses of an inactivated TBEV vaccine were efficacious in monkeys against wild-type LGT challenge. These results indicate that both TBEV/DEN4Delta30 and LGT/DEN4 are safe and efficacious in rhesus monkeys and should be further evaluated as vaccine candidates for use in humans.

Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus.

Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta 30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Delta 30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Delta 30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Delta 30, will be evaluated first in our initial clinical studies.

Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis.

The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.

West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy.

A candidate live attenuated vaccine strain was constructed for West Nile virus (WN), a neurotropic flavivirus that has recently emerged in the U.S. Considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (DEN4). The genes for the structural premembrane and envelope proteins of DEN4 present in an infectious cDNA clone were replaced by the corresponding genes of WN strain NY99. Two of 18 cDNA clones of a WN/DEN4 chimera yielded full-length RNA transcripts that were infectious when transfected into susceptible cells. The two infectious clones shared a motif in the transmembrane signal domain located immediately downstream of the NS2B-NS3 protease cleavage site that separates the DEN4 capsid protein and the WN premembrane protein of the chimera. This motif, Asp and Thr at a position 3 and 6 amino acids downstream of the cleavage site, respectively, was not present in the 16 noninfectious cDNA clones. The WN/DEN4 chimera was highly attenuated in mice compared with its WN parent; the chimera was at least 28,500 times less neurovirulent in suckling mice inoculated intracerebrally and at least 10,000 times less virulent in adult mice inoculated intraperitoneally. Nonetheless, the WN/DEN4 chimera and a deletion mutant derived from it were immunogenic and provided complete protection against lethal WN challenge. These observations provide the basis for pursuing the development of a live attenuated WN vaccine.