Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

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.

Combination of Cystatins 9 and C Modulates Serum Biomarkers Associated with Inflammation and Provides Prophylactic as Well as Long-Term Protection against Multidrug-Resistant Klebsiella pneumoniae.

We have identified recombinant human cystatins 9 (rCST9) and C (rCSTC) as a combination immunotherapeutic treatment against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae We evaluated the lasting protection of rCST9/rCSTC treatment against MDR NDM-1 K. pneumoniae pneumonia. Results showed that rCST9/rCSTC treatment modulated endogenous serum biomarkers, cystatins 9 and C and amyloid A, associated with poor patient outcomes and provided prophylactic and long-term protection in a murine model of pneumonia.

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.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice.

UNLABELLED: The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. IMPORTANCE: The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

In Vivo Rectal Mucosal Barrier Function Imaging in a Large-Animal Model by Using Confocal Endomicroscopy: Implications for Injury Assessment and Use in HIV Prevention Studies.

Injury occurring on the surface of the rectal mucosal lining that causes defects in barrier function may result in increased risk for transmission of infection by HIV and other pathogens. Such injury could occur from microbicidal or other topical agents, mechanical trauma during consensual or nonconsensual intercourse, or inflammatory conditions. Tools for evaluation of rectal mucosal barrier function for assessing the mucosa under these conditions are lacking, particularly those that can provide in vivo structural and functional barrier integrity assessment and are adaptable to longitudinal imaging. We investigated confocal endomicroscopy (CE) as a means for in vivo imaging of the rectal epithelial barrier in the ovine model following spatially confined injury to the surface at a controlled site using a topical application of the microbicide test agent benzalkonium chloride. Topical and intravenous (i.v.) fluorescent probes were used with CE to provide subcellular resolution imaging of the mucosal surface and assessment of barrier function loss. A 3-point CE grading system based on cellular structure integrity and leakage of dye through the mucosa showed significant differences in score between untreated (1.19 ± 0.53) and treated (2.55 ± 0.75) tissue (P < 0.0001). Histological grading confirmed findings of barrier compromise. The results indicate that CE is an effective means for detecting epithelial injury and barrier loss following localized trauma in a large-animal model. CE is promising for real-time rectal mucosal evaluation after injury or trauma or topical application of emerging biomedical prevention strategies designed to combat HIV.

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.

Characterization of lethal dengue virus type 4 (DENV-4) TVP-376 infection in mice lacking both IFN-α/ß and IFN-γ receptors (AG129) and comparison with the DENV-2 AG129 mouse model.

Dengue is a mosquito-borne disease caused by four related but distinct dengue viruses, DENV-1 to DENV-4. Dengue is endemic in most tropical countries, and over a third of the world's population is at risk of being infected. Although the global burden is high, no vaccine or antiviral is licensed to combat this disease. An obstacle complicating dengue research is the lack of animal challenge models that mimic human disease. Advances in immunocompromised murine infection models resulted in development of lethal DENV-2, DENV-3 and DENV-4 models in AG129 mice, which are deficient in both the IFN-α/ß receptor (IFN-α/ßR) and the IFN-γ receptor (IFN-γR). These models mimic features of dengue disease in humans. Here, we characterized lethal infection of AG129 mice by DENV-4 strain TVP-376 and found that AG129 mice developed clinical signs of illness and high viral loads in multiple tissues and succumbed 5 days after infection. Moreover, the splenic and hepatic histopathology of TVP-376-infected mice demonstrated the presence of cell activation and destruction of tissue architecture. Furthermore, infected mice had heightened levels of circulating cytokines. Comparison of the virulence phenotypes of DENV-4 strain TVP-376 and DENV-2 strain D2S10 revealed that TVP-376-induced mortality occurred in the absence of both IFN-α/ßR and IFN-γR signalling, but not with intact signalling from the IFN-γR, whereas D2S10 required the absence of IFN-α/ßR signalling only, indicating that it is more virulent than TVP-376. In conclusion, TVP-376 is lethal in AG129 mice, and this model provides a useful platform to investigate vaccine candidates and antivirals against DENV-4.

TLR3- and MyD88-dependent signaling differentially influences the development of West Nile virus-specific B cell responses in mice following immunization with RepliVAX WN, a single-cycle flavivirus vaccine candidate.

Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.

Dynamic species distribution models from categorical survey data.

1. Species distribution models are static models for the distribution of a species, based on Hutchinson's niche concept. They make probabilistic predictions about the distribution of a species, but do not have a temporal interpretation. In contrast, density-structured models based on categorical abundance data make it possible to incorporate population dynamics into species distribution modelling. 2. Using dynamic species distribution models, temporal aspects of a species' distribution can be investigated, including the predictability of future abundance categories and the expected persistence times of local populations, and how these may respond to environmental or anthropogenic drivers. 3. We built density-structured models for two intertidal marine invertebrates, the Lusitanian trochid gastropods Phorcus lineatus and Gibbula umbilicalis, based on 9 years of field data from around the United Kingdom. Abundances were recorded on a categorical scale, and stochastic models for year-to-year changes in abundance category were constructed with winter mean sea surface temperature (SST) and wave fetch (a measure of the exposure of a shore) as explanatory variables. 4. Both species were more likely to be present at sites with high SST, but differed in their responses to wave fetch. Phorcus lineatus had more predictable future abundance and longer expected persistence times than G. umbilicalis. This is consistent with the longer lifespan of P. lineatus. 5. Where data from multiple time points are available, dynamic species distribution models of the kind described here have many applications in population and conservation biology. These include allowing for changes over time when combining historical and contemporary data, and predicting how climate change might alter future abundance conditional on current distributions.

Influence of methamphetamine on genital herpes simplex virus type 2 infection in a mouse model.

BACKGROUND: Use of the stimulant methamphetamine (METH) is increasingly common, with >35 million users worldwide. There is a known association between stimulant use and an increased incidence of HIV and other sexually transmitted infections (STIs). METH is known to have immune modulatory properties. However, the impact of METH on normal immune responses and disease pathogenesis with STIs has not been fully examined. METHODS: We used a well-characterized murine model to investigate the impact of METH use on genital herpes simplex virus type 2 infection. Plaque assay and quantitative real-time polymerase chain reaction were used to measure viral replication. Cytokine bead array and enzyme-linked immunosorbent assay were used to determine levels of cytokines during host innate immune response. RESULTS: METH treatment altered behavior, onset of clinical signs, and disease progression. METH-treated mice also had a thinned vaginal epithelium and an increase in virus present in the sensory ganglia. In addition, METH produced a local dysregulation of cytokine secretion that contrasts with its minimal impact on systemic cytokine secretion. CONCLUSIONS: Results suggest that the METH alterations of the host immune response partially contribute to enhanced genital herpes disease progression. These findings will improve understanding of METH use on host immune responses and susceptibility to disease.

Baseline mapping of Oropouche virology, epidemiology, therapeutics, and vaccine research and development.

Oropouche virus (OROV) is an arthropod-borne orthobunyavirus found in South America and causes Oropouche fever, a febrile infection similar to dengue. It is the second most prevalent arthropod-borne viral disease in South America after dengue. Over 500,000 cases have been diagnosed since the virus was first discovered in 1955; however, this is likely a significant underestimate given the limited availability of diagnostics. No fatalities have been reported to date, however, up to 60% of cases have a recurrent phase of disease within one month of recovery from the primary disease course. The main arthropod vector is the biting midge Culicoides paraensis, which has a geographic range as far north as the United States and demonstrates the potential for OROV to geographically expand. The transmission cycle is incompletely understood and vertebrate hosts include both non-human primates and birds further supporting the potential ability of the virus to spread. A number of candidate antivirals have been evaluated against OROV in vitro but none showed antiviral activity. Surprisingly, there is only one report in the literature on candidate vaccines. We suggest that OROV is an undervalued pathogen much like chikungunya, Schmallenberg, and Zika viruses were before they emerged. Overall, OROV is an important emerging disease that has been under-investigated and has the potential to cause large epidemics in the future. Further research, in particular candidate vaccines, is needed for this important pathogen.

Characterization of the Ovine Vaginal Microbiome and Inflammation Patterns as an Improved Testing Model of Human Vaginal Irritation.

The development of therapies targeted to improve the health of women has utilized direct vaginal delivery as a more effective and less toxic method of protection from HIV and other pathogens. Vaginal applicants and delivery devices that provide sustained effects have been met with increasing acceptability, but the efficacy and toxicity outcomes have not been successfully predicted by preclinical in vitro studies and animal modeling. We have explored the utilization of sheep as a model for testing the safety of vaginal applicants and devices based on spatial and structural similarities to the human vagina. As recently noted by the FDA, an additional safety measure is an impact on the vaginal microbiome (VMB) that is known to contribute to vaginal health and influence pathogen susceptibility and drug metabolism. To advance the utility of the sheep vaginal model, we completed a thorough molecular characterization of the ovine VMB utilizing both next-generation sequencing (NGS) and PCR methods. The process also created a custom PCR array to quantify ovine VMB community profiles in an affordable, higher throughput fashion. The results from vaginal swabs (>475 samples) collected from non-pregnant crossbred Dorset and Merino ewes treated with selected vaginal applicants or collected as sham samples established 16 VMB community types (VMB CTs). To associate VMB CTs with eubiosis or dysbiosis, we also completed custom ELISAs for six cytokines identifying IL1B, IL8, TNFa, and CXCL10 as useful markers to support the characterization of ovine vaginal inflammation. The results indicated that Pasteurella, Actinobacillus, Pseudomonas, Bacteroides, Leptotrichia, and E. coli were common markers of eubiosis (low inflammatory marker expression), and that Haemophilus, Ureaplasma, and Corynebacterium were associated with dysbiosis (high cytokine levels). Utilizing the optimized workflow, we also confirmed the utility of three commonly used vaginal applicants for impact on the VMB and inflammatory state, producing a dataset that supports the recommendation for the use of sheep for testing of vaginal applicants and devices as part of preclinical pipelines.

Differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2.

We previously reported that transgenic (Tg) mice expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), were highly susceptible to SARS-CoV infection, which resulted in the development of disease of various severity and even death in some lineages. In this study, we further characterized and compared the pathogeneses of SARS-CoV infection in two of the most stable Tg lineages, AC70 and AC22, representing those susceptible and resistant to the lethal SARS-CoV infection, respectively. The kinetics of virus replication and the inflammatory responses within the lungs and brains, as well as the clinical and pathological outcomes, were assessed in each lineage. In addition, we generated information on lymphocyte subsets and mitogen-mediated proliferation of splenocytes. We found that while both lineages were permissive to SARS-CoV infection, causing elevated secretion of many inflammatory mediators within the lungs and brains, viral infection appeared to be more intense in AC70 than in AC22 mice, especially in the brain. Moreover, such infection was accompanied by a more profound immune suppression in the former, as evidenced by the extensive loss of T cells, compromised responses to concanavalin A stimulation, and absence of inflammatory infiltrates within the brain. We also found that CD8(+) T cells were partially effective in attenuating the pathogenesis of SARS-CoV infection in lethality-resistant AC22 mice. Collectively, our data revealed a more intense viral infection and immunosuppression in AC70 mice than in AC22 mice, thereby providing us with an immunopathogenic basis for the fatal outcome of SARS-CoV infection in the AC70 mice.

Benzalkonium chloride causes colposcopic changes and increased susceptibility to genital herpes infection in mice.

BACKGROUND: Colposcopy is widely used in clinical microbicide safety testing but not in preclinical small animal studies. Endoscopic colposcopy could be employed in small animals allowing colposcopy to be used as one component in a multifactorial safety testing paradigm. STUDY DESIGN: We conducted dose-response studies in mice using 2%, 0.2%, or 0.02% benzalkonium chloride (BZK) as the test compound, and using multiple safety end points that included endoscopic colposcopy, susceptibility to vaginal HSV-2 infection, histology, and entry of inflammatory cells into the vagina. RESULTS: Animals treated with 0.2% or higher BZK experienced vaginal toxicities detectable by all tests used including colposcopy. In contrast, 0.02% BZK produced no significant changes except by histology in which a significant thinning of the vaginal epithelium was seen. CONCLUSION: Endoscopic colposcopy detected microbicide-elicited changes in the mouse vagina with similar sensitivity to the other endpoints used in these studies and would appear to be useful as part of a multifactorial microbicide safety testing paradigm in mice.

3,4-Methylenedioxymethamphetamine increases susceptibility to genital herpes simplex virus infection in mice.

Abused by >1.2 million Americans, 3,4-methylenedioxymethamphetamine (MDMA) (commonly referred to as ecstasy) is popular in the dance club, rave, and circuit party scenes. MDMA and other similar drugs are reportedly associated with increased incidence of sexually transmitted infectious diseases, such as AIDS and genital herpes, and may have immunological effects. In the present study, we demonstrate that MDMA causes increased susceptibility to herpes simplex virus type 2 infection in mice and earlier onset of genital herpes. We also demonstrate that MDMA has an effect on the cytokines of the innate immune system-both systemically and, for the first time, in the genital tract. These data suggest that MDMA may play an important biological role in infection.

Baseline mapping of severe fever with thrombocytopenia syndrome virology, epidemiology and vaccine research and development.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emergent tick-borne bunyavirus first discovered in 2009 in China. SFTSV is a growing public health problem that may become more prominent owing to multiple competent tick-vectors and the expansion of human populations in areas where the vectors are found. Although tick-vectors of SFTSV are found in a wide geographic area, SFTS cases have only been reported from China, South Korea, Vietnam, and Japan. Patients with SFTS often present with high fever, leukopenia, and thrombocytopenia, and in some cases, symptoms can progress to severe outcomes, including hemorrhagic disease. Reported SFTSV case fatality rates range from ~5 to >30% depending on the region surveyed, with more severe disease reported in older individuals. Currently, treatment options for this viral infection remain mostly supportive as there are no licensed vaccines available and research is in the discovery stage. Animal models for SFTSV appear to recapitulate many facets of human disease, although none of the models mirror all clinical manifestations. There are insufficient data available on basic immunologic responses, the immune correlate(s) of protection, and the determinants of severe disease by SFTSV and related viruses. Many aspects of SFTSV virology and epidemiology are not fully understood, including a detailed understanding of the annual numbers of cases and the vertebrate host of the virus, so additional research on this disease is essential towards the development of vaccines and therapeutics.

Effector CD4+ T-cell involvement in clearance of infectious herpes simplex virus type 1 from sensory ganglia and spinal cords.

In primary infection, CD8(+) T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4(+) T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4(+) T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4(+) T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8(+) T-cell-deficient and CD8(+) T-cell-depleted mice, suggesting that CD4(+) T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4(+) T cells resolved neural infection, CD8(+) T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4(+) T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.

T lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection.

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4(+) and CD8(+) T cells cooperate synergistically in hMPV eradication during primary infection, but CD4(+) more than CD8(+) T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4(+) and CD8(+) T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4(+) T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8(+) T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.