Impact of yellow fever virus envelope protein on wild-type and vaccine epitopes and tissue tropism.

The envelope (E) protein of flaviviruses is functionally associated with viral tissue tropism and pathogenicity. For yellow fever virus (YFV), viscerotropic disease primarily involving the liver is pathognomonic for wild-type (WT) infection. In contrast, the live-attenuated vaccine (LAV) strain 17D does not cause viscerotropic disease and reversion to virulence is associated with neurotropic disease. The relationship between structure-function of the E protein for WT strain Asibi and its LAV derivative 17D strain is poorly understood; however, changes to WT and vaccine epitopes have been associated with changes in virulence. Here, a panel of Asibi and 17D infectious clone mutants were generated with single-site mutations at the one membrane residue and each of the eight E protein amino acid substitutions that distinguish the two strains. The mutants were characterized with respect to WT-specific and vaccine-specific monoclonal antibodies (mAbs) binding to virus plus binding of virus to brain, liver, and lung membrane receptor preparations (MRPs) generated from AG129 mice. This approach shows that amino acids in the YFV E protein domains (ED) I and II contain the WT E protein epitope, which overlap with those that mediate YFV binding to mouse liver. Furthermore, amino acids in EDIII associated with the vaccine epitope overlap with those that facilitate YFV binding mouse brain MRPs. Taken together, these data suggest that the YFV E protein is a key determinant in the phenotype of WT and 17D vaccine strains of YFV.

Japanese encephalitis virus live attenuated vaccine strains display altered immunogenicity, virulence and genetic diversity.

Japanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE). The most commonly used vaccine used to prevent JE is the live-attenuated strain SA14-14-2, which was generated by serial passage of the wild-type (WT) JEV strain SA14. Two other vaccine candidates, SA14-5-3 and SA14-2-8 were derived from SA14. Both were shown to be attenuated but lacked sufficient immunogenicity to be considered effective vaccines. To better contrast the SA14-14-2 vaccine with its less-immunogenic counterparts, genetic diversity, ribavirin sensitivity, mouse virulence and mouse immunogenicity of the three vaccines were investigated. Next generation sequencing demonstrated that SA14-14-2 was significantly more diverse than both SA14-5-3 and SA14-2-8, and was slightly less diverse than WT SA14. Notably, WT SA14 had unpredictable levels of diversity across its genome whereas SA14-14-2 is highly diverse, but genetic diversity is not random, rather the virus only tolerates variability at certain residues. Using Ribavirin sensitivity in vitro, it was found that SA14-14-2 has a lower fidelity replication complex compared to SA14-5-3 and SA14-2-8. Mouse virulence studies showed that SA14-2-8 was the most virulent of the three vaccine strains while SA14-14-2 had the most favorable combination of safety (virulence) and immunogenicity for all vaccines tested. SA14-14-2 contains genetic diversity and sensitivity to the antiviral Ribavirin similar to WT parent SA14, and this genetic diversity likely explains the (1) differences in genomic sequences reported for SA14-14-2 and (2) the encoding of major attenuation determinants by the viral E protein.

Attenuation of Zika Virus by Passage in Human HeLa Cells.

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Development of disease and immunity at the genital epithelium following intrarectal inoculation of male guinea pigs with herpes simplex virus type 2.

Most analyses of genital immunity to herpes simplex virus type 2 (HSV-2) have been performed in females, consequently immune protection of the male genital epithelium is incompletely understood. We developed a model of male genital HSV-2 infection resulting from intrarectal inoculation of guinea pigs. Vesicular lesions developed transiently on the perineum and foreskin concurrent with acute virus shedding. Virus shedding and recurrent genital lesions were also detected after establishment of a latent infection. Analysis of perineum and foreskin RNA detected transcripts for IFNγ, proinflammatory and regulatory cytokines, and for genes involved in migration and regulation of leukocytes. HSV-specific T cells were detected in lymphoid and genital tissues after resolution of the primary infection whereas virus-specific antibody secreting cells were detected only in lymphoid tissue. Taken together, the ability to quantify pathogenesis and local immunity in this guinea pig model represent an important advance towards understanding immunity to HSV-2 in males.

Spectrum of activity testing for therapeutics against all four dengue virus serotypes in AG129 mouse models: Proof-of-concept studies with the adenosine nucleoside inhibitor NITD-008.

Dengue is a mosquito-borne disease of global public health importance caused by four genetically and serologically related viruses (DENV-1 to DENV-4). Efforts to develop effective vaccines and therapeutics for dengue have been slowed by the paucity of preclinical models that mimic human disease. DENV-2 models in interferon receptor deficient AG129 mice were an important advance but only allowed testing against a single DENV serotype. We have developed complementary AG129 mouse models of severe disseminated dengue infection using strains of the other three DENV serotypes. Here we used the adenosine nucleoside inhibitor NITD-008 to show that these models provide the ability to perform comparative preclinical efficacy testing of candidate antivirals in vivo against the full-spectrum of DENV serotypes. Although NITD-008 was effective in modulating disease caused by all DENV serotypes, the variability in protection among DENV serotypes was greater than expected from differences in activity in in vitro testing studies emphasizing the need to undertake spectrum of activity testing to help in prioritization of candidate compounds for further development.

Characterization of a murine model of non-lethal, symptomatic dengue virus infection.

The mosquito-borne disease dengue is caused by four serologically- and genetically-related viruses, termed DENV-1 to DENV-4. Historical setbacks due to lack of human-like mouse models of dengue were partially remedied with characterization of lethal DENV-2 infection in immunocompromised AG129 mice (deficient in IFN-α/ß/γ receptors). Recently, our group established lethal AG129 mouse infection models of DENV-1, DENV-3, and DENV-4 using human isolates. Here we compare a non-lethal, disseminated model of DENV-3 infection using strain D83-144 to that of the lethal outcome following infection by strain C0360/94. Both strains belong to DENV-3 genotype II and differ by only 13 amino acids. Intraperitoneal inoculation of AG129 mice with strain D83-144 led to clinical signs of dengue infection, such as cytokine induction, thrombocytopenia, and systemic infection. However, C0360/94 infection led to features of severe human dengue, including coagulopathy and lethal outcome, whereas D83-144 infection does not. This study is the first to investigate a low passage, non-mouse lethal strain in AG129 mice and demonstrates that D83-144 infection induces milder features of human dengue than those induced by lethal C0360/94 infection. The results suggest that the AG129 mouse model has applications to investigate factors associated with mild or severe disease.

Cultivated Human Vaginal Microbiome Communities Impact Zika and Herpes Simplex Virus Replication in ex vivo Vaginal Mucosal Cultures.

The human vaginal microbiome (VMB) is a complex bacterial community that interacts closely with vaginal epithelial cells (VECs) impacting the mucosal phenotype and its responses to pathogenic insults. The VMB and VEC relationship includes nutrient exchange and regulation of signaling molecules that controls numerous host functions and defends against invading pathogens. To better understand infection and replication of sexually transmitted viral pathogens in the human vaginal mucosa we used our ex vivo VEC multilayer culture system. We tested the hypothesis that selected VMB communities could be identified that alter the replication of sexually transmitted viruses consistent with reported clinical associations. Sterile VEC multilayer cultures or those colonized with VMB dominated by specific Lactobacillus spp., or VMB lacking lactobacilli, were infected with Zika virus, (ZIKV) a single stranded RNA virus, or Herpes Simplex Virus type 2 (HSV-2), a double stranded DNA virus. The virus was added to the apical surface of the cultured VEC multilayer to model transmission during vaginal intercourse. Viral replication was measured 48 h later by qPCR. The results indicated that VEC cultures colonized by VMB containing Staphylococcus spp., previously reported as inflammatory, significantly reduced the quantity of viral genomes produced by ZIKV. HSV-2 titers were decreased by nearly every VMB tested relative to the sterile control, although Lactobacillus spp.-dominated VMBs caused the greatest reduction in HSV-2 titer consistent with clinical observations. To explore the mechanism for reduced ZIKV titers, we investigated inflammation created by ZIKV infection, VMB colonization or pre-exposure to selected TLR agonists. Finally, expression levels of human beta defensins 1-3 were quantified in cultures infected by ZIKV and those colonized by VMBs that impacted ZIKV titers. Human beta defensins 1-3 produced by the VEC showed no association with ZIKV titers. The data presented expands the utility of this ex vivo model system providing controlled and reproducible methods to study the VMB impact on STIs and indicated an association between viral replication and specific bacterial species within the VMB.

A lethal model of disseminated dengue virus type 1 infection in AG129 mice.

The mosquito-borne disease dengue is caused by four serologically and genetically related flaviviruses termed DENV-1 to DENV-4. Dengue is a global public health concern, with both the geographical range and burden of disease increasing rapidly. Clinically, dengue ranges from a relatively mild self-limiting illness to a severe life-threatening and sometimes fatal disease. Infection with one DENV serotype produces life-long homotypic immunity, but incomplete and short-term heterotypic protection. The development of small-animal models that recapitulate the characteristics of the disseminated disease seen clinically has been difficult, slowing the development of vaccines and therapeutics. The AG129 mouse (deficient in interferon alpha/beta and gamma receptor signalling) has proven to be valuable for this purpose, with the development of models of disseminated DENV-2,-3 and -4 disease. Recently, a DENV-1 AG129 model was described, but it requires antibody-dependent enhancement (ADE) to produce lethality. Here we describe a new AG129 model utilizing a non-mouse-adapted DENV-1 strain, West Pacific 74, that does not require ADE to induce lethal disease. Following high-titre intraperitoneal challenge, animals experience a virus infection with dissemination to multiple visceral tissues, including the liver, spleen and intestine. The animals also become thrombocytopenic, but vascular leakage is less prominent than in AG129 models with other DENV serotypes. Taken together, our studies demonstrate that this model is an important addition to dengue research, particularly for understanding the pathological basis of the disease between DENV serotypes and allowing the full spectrum of activity to test comparisons for putative vaccines and antivirals.

A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease.

UNLABELLED: The mosquito-borne disease dengue (DEN) is caused by four serologically and genetically related viruses, termed DENV-1 to DENV-4. Infection with one DENV usually leads to acute illness and results in lifelong homotypic immunity, but individuals remain susceptible to infection by the other three DENVs. The lack of a small-animal model that mimics systemic DEN disease without neurovirulence has been an obstacle, but DENV-2 models that resemble human disease have been recently developed in AG129 mice (deficient in interferon alpha/beta and interferon gamma receptor signaling). However, comparable DENV-1, -3, and -4 models have not been developed. We utilized a non-mouse-adapted DENV-3 Thai human isolate to develop a lethal infection model in AG129 mice. Intraperitoneal inoculation of six to eight-week-old animals with strain C0360/94 led to rapid, fatal disease. Lethal C0360/94 infection resulted in physical signs of illness, high viral loads in the spleen, liver, and large intestine, histological changes in the liver and spleen tissues, and increased serum cytokine levels. Importantly, the animals developed vascular leakage, thrombocytopenia, and leukopenia. Overall, we have developed a lethal DENV-3 murine infection model, with no evidence of neurotropic disease based on a non-mouse-adapted human isolate, which can be used to investigate DEN pathogenesis and to evaluate candidate vaccines and antivirals. This suggests that murine models utilizing non-mouse-adapted isolates can be obtained for all four DENVs. IMPORTANCE: Dengue (DEN) is a mosquito-borne disease caused by four DENV serotypes (DENV-1, -2, -3, and -4) that have no treatments or vaccines. Primary infection with one DENV usually leads to acute illness followed by lifelong homotypic immunity, but susceptibility to infection by the other three DENVs remains. Therefore, a vaccine needs to protect from all four DENVs simultaneously. To date a suitable animal model to mimic systemic human illness exists only for DENV-2 in immunocompromised mice using passaged viruses; however, models are still needed for the remaining serotypes. This study describes establishment of a lethal systemic DENV-3 infection model with a human isolate in immunocompromised mice and is the first report of lethal infection by a nonadapted clinical DENV isolate without evidence of neurological disease. Our DENV-3 model provides a relevant platform to test DEN vaccines and antivirals.

Hypothalamic progesterone receptor-A mediates gonadotropin surges, self priming and receptivity in estrogen-primed female mice.

Ovarian progesterone (Prog) is an essential steroid hormone for the secretion of GnRH and reproductive behavior. It exerts primary effects through the progesterone receptor (PR). When analyzed separately in vitro, PR isoforms (PR-A, PR-B) display striking differences in transcriptional activity. The present study was undertaken to determine the in vivo impact of each isoform on hypothalamic function in female mice with ablation of a single isoform, either PR-A or PR-B. To this end, we used single-cell RNA analyses, reverse transcriptase real-time (q)PCR mRNA analyses of punched-out tissue, immunohistochemistry, and reproductive behavior. We provide evidence for the requirement of PR-A in individual ventrolateral ventromedial nucleus (vlVMN) neurons for Prog-facilitated proceptive and receptive behaviors in estrogen benzoate (EB)-primed females and the reciprocal male interactions. We clarify histological and molecular mechanisms of PR isoform activity by showing that (1) PR-A is predominant in individual vlVMN neurons controlling female lordosis circuitry, whilst (2) PR-B is predominant in those VMN subdivisions that provide for amplification of PR-A activity. We go on to demonstrate that PR-A is dominant in the anteroventral periventricular nucleus but not the arcuate nucleus that feed fibers into and around the VMN. In the medial preoptic area, high levels of GnRH RNA in EB-primed PR-A-expressing mice were seen coincident with increased plasma LH levels. Two consecutive GnRH pulses enhanced LH only in primed PR-A-expressing females. In all, the findings are consistent with the hypothesis that hypothalamic PR-A-mediated genomic activities result in reproductive behavior coordinated with ovulation.