Skin tropism during Usutu virus and West Nile virus infection: an amplifying and immunological role.

Usutu virus (USUV) and West Nile virus (WNV) are closely related emerging arboviruses belonging to the Flavivirus genus and posing global public health concerns. Although human infection by these viruses is mainly asymptomatic, both have been associated with neurological disorders such as encephalitis and meningoencephalitis. Since USUV and WNV are transmitted through the bite of an infected mosquito, the skin represents the initial site of virus inoculation and provides the first line of host defense. Although some data on the early stages of WNV skin infection are available, very little is known about USUV. Herein, USUV-skin resident cell interactions were characterized. Using primary human keratinocytes and fibroblasts, an early replication of USUV during the first 24 hours was shown in both skin cells. In human skin explants, a high viral tropism for keratinocytes was observed. USUV infection of these models induced type I and III interferon responses associated with upregulated expression of various interferon-stimulated genes as well as pro-inflammatory cytokine and chemokine genes. Among the four USUV lineages studied, the Europe 2 strain replicated more efficiently in skin cells and induced a higher innate immune response. In vivo, USUV and WNV disseminated quickly from the inoculation site to distal cutaneous tissues. In addition, viral replication and persistence in skin cells were associated with an antiviral response. Taken together, these results provide a better understanding of the pathophysiology of the early steps of USUV infection and suggest that the skin constitutes a major amplifying organ for USUV and WNV infection.IMPORTANCEUsutu virus (USUV) and West Nile virus (WNV) are closely related emerging Flaviviruses transmitted through the bite of an infected mosquito. Since they are directly inoculated within the upper skin layers, the interactions between the virus and skin cells are critical in the pathophysiology of USUV and WNV infection. Here, during the early steps of infection, we showed that USUV can efficiently infect two human resident skin cell types at the inoculation site: the epidermal keratinocytes and the dermal fibroblasts, leading to the induction of an antiviral innate immune response. Moreover, following cutaneous inoculation, we demonstrated that both viruses can rapidly spread, replicate, and persist in all distal cutaneous tissues in mice, a phenomenon associated with a generalized skin inflammatory response. These results highlight the key amplifying and immunological role of the skin during USUV and WNV infection.

Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

American alligators are capable of West Nile virus amplification, mosquito infection and transmission.

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

Mosquito population structure, pathogen surveillance and insecticide resistance monitoring in urban regions of Crete, Greece.

BACKGROUND: In Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece's major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority. METHODOLOGY: Under the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018-2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species. PRINCIPAL FINDINGS: Important disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25-46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV.

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes' ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

Accounting for spatial sampling patterns in Bayesian phylogeography.

Statistical phylogeography provides useful tools to characterize and quantify the spread of organisms during the course of evolution. Analyzing georeferenced genetic data often relies on the assumption that samples are preferentially collected in densely populated areas of the habitat. Deviation from this assumption negatively impacts the inference of the spatial and demographic dynamics. This issue is pervasive in phylogeography. It affects analyses that approximate the habitat as a set of discrete demes as well as those that treat it as a continuum. The present study introduces a Bayesian modeling approach that explicitly accommodates for spatial sampling strategies. An original inference technique, based on recent advances in statistical computing, is then described that is most suited to modeling data where sequences are preferentially collected at certain locations, independently of the outcome of the evolutionary process. The analysis of georeferenced genetic sequences from the West Nile virus in North America along with simulated data shows how assumptions about spatial sampling may impact our understanding of the forces shaping biodiversity across time and space.

First Evidence of West Nile Virus Overwintering in Mosquitoes in Germany.

Mosquitoes collected from mid-December 2020 to early March 2021 from hibernacula in northeastern Germany, a region of West Nile virus (WNV) activity since 2018, were examined for WNV-RNA. Among the 6101 mosquitoes tested in 722 pools of up to 12 specimens, one pool of 10 Culex pipiens complex mosquitoes collected in early March 2021 in the cellar of a medieval castle in Rosslau, federal state of Saxony-Anhalt, tested positive. Subsequent mosquito DNA analysis produced Culex pipiens biotype pipiens. The pool homogenate remaining after nucleic acid extraction failed to grow the virus on Vero and C6/36 cells. Sequencing of the viral NS2B-NS3 coding region, however, demonstrated high homology with virus strains previously collected in Germany, e.g., from humans, birds, and mosquitoes, which have been designated the East German WNV clade. The finding confirms the expectation that WNV can overwinter in mosquitoes in Germany, facilitating an early start to the natural transmission season in the subsequent year. On the other hand, the calculated low infection prevalence of 0.016-0.20%, depending on whether one or twelve of the mosquitoes in the positive pool was/were infected, indicates a slow epidemic progress and mirrors the still-hypoendemic situation in Germany. In any case, local overwintering of the virus in mosquitoes suggests its long-term persistence and an enduring public health issue.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

The envelope protein of Usutu virus attenuates West Nile virus virulence in immunocompetent mice.

West Nile virus (WNV) and Usutu virus (USUV) are the two most widespread mosquito-borne flaviviruses in Europe causing severe neuroinvasive disease in humans. Here, following standardization of the murine model with wild type (wt) viruses, we engineered WNV and USUV genome by reverse genetics. A recombinant virus carrying the 5' UTR of WNV within the USUV genome backbone (r-USUV5'-UTR WNV) was rescued; when administered to mice this virus did not cause signs or disease as wt USUV suggesting that 5' UTR of a marked neurotropic parental WNV was not per se a virulence factor. Interestingly, a chimeric virus carrying the envelope (E) protein of USUV in the WNV genome backbone (r-WNVE-USUV) showed an attenuated profile in mice compared to wt WNV but significantly more virulent than wt USUV. Moreover, except when tested against serum samples originating from a live WNV infection, r-WNVE-USUV showed an identical antigenic profile to wt USUV confirming that E is also the major immunodominant protein of USUV.

Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth's average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector-virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

West Nile virus seroprevalence and associated risk factors among horses in Egypt.

Determination of the seroprevalence and risk factors that are associated with West Nile virus (WNV) in horses is essential for adoption of effective prevention strategies. Our objective in this study, therefore, was to determine the seroprevalence and to identify the risk factors associated with WNV infection in the most densely horse-populated governorates in Egypt. A cross-sectional study was conducted in 2018 on 930 horses, which were distributed over five governorates in the Nile delta of Egypt. The horses, which were randomly selected, were serologically tested through use of an ID screen West Nile competition enzyme-linked immunosorbent assay (ELISA) to detect anti-WNV immunoglobulin G (IgG) and plaque reduction neutralization tests (PRNT; gold standard) to confirm the seropositive status of animals and to avoid cross reaction with other flavi-viruses. Four variables (geographical location, breed, sex and age) were considered in the risk analysis. Univariable and stepwise forward multivariable logistic regression methods were used for risk-factor analysis. The odds ratio (OR) was used as an approximate measure of relative risk. A total of 156 (16.8%; 95% confidence interval (CI) 14.4-19.2; P < 0.001) serum samples were found to be serologically positive for WNV. The highest seroprevalence rate was detected in horses of age ≥ 15 years (68.1%; 95% CI 49.8-72.4), stallions (26.4%; 95% CI 22.7-30.4), and those of mixed breed (21.5%; 95% CI 17.7-27.5). Horses older than 15 years were found to be at increased risk of WNV infection with OR = 4.3 (95% CI 3.0-6.2, P < 0.001) compared with horses aged under 2.5 years. Also, when all the risk factors were considered, stallions were more likely than mares to be WNV seropositive (OR = 2.4, 95% CI 1.6-3.7, P < 0.001), and of the breeds, mixed-breed (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) and Arabian horses (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) were more likely to be seropositive. Geographical location seemed to have no impact on the seroprevalence of exposure to WNV among these horses. Due to these findings, we strongly recommend intensive surveillance and implementation of effective control and prevention strategies against WNV, especially in stallion, mixed-breed horses with ages ≥ 15 years.

Influence of Biological Model on the Formation of the Pathogenic Properties of the West Nile Virus Isolate.

Various biological models are used to isolate West Nile virus, but their role as a selection factor that facilitates selection of isolates with certain properties is usually not evaluated. We compared pathogenic properties of three strains of the West Nile virus obtained from one sample of virus-containing material using different models: WNV Volgograd 900m/18 (on the model of suckling mice), WNV Volgograd 900a/18 (on C6/36 cells) and WNV Volgograd 900v/18 (on Vero cells). WNV Volgograd 900m/18 strain demonstrated virulent (LD50 5×103±0.005×104 PFU, p≤0.05) and neuroinvasive properties, induced viremia and pathomorphological changes in the liver, lymph nodes, and brain of nonlinear white mice. WNV Volgograd 900v/18 strain had similar characteristics except for neuroinvasiveness. WNV Volgograd 900a/18 variant demonstrated minimum virulence (LD50 5×104±0.005×104 PFU, p≤0.05), did not cause neurological symptoms, and was not isolated from the blood of infected animals.

Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens.

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

Tick-Borne Encephalitis Virus (TBEV) Infection in Two Horses.

A final diagnosis in a horse with clinical signs of encephalopathy can be challenging despite the use of extensive diagnostics. Clinical signs are often not pathognomonic and need to be interpreted in combination with (specific) laboratory results and epidemiological data of the geographical region of the origin of the case(s). Here we describe the diagnostic pathway of tick-borne encephalitis virus infection in two horses using established molecular diagnostic methods and a novel in situ hybridization technique to differentiate between regionally important/emerging diseases for central Europe: (i) hepatoencephalopathy, (ii) Borna disease virus, and (iii) West Nile virus infections.

The Effect of Fluctuating Incubation Temperatures on West Nile Virus Infection in Culex Mosquitoes.

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.

Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.

Mother-to-Child Transmission of Arboviruses during Breastfeeding: From Epidemiology to Cellular Mechanisms.

Most viruses use several entry sites and modes of transmission to infect their host (parenteral, sexual, respiratory, oro-fecal, transplacental, transcutaneous, etc.). Some of them are known to be essentially transmitted via arthropod bites (mosquitoes, ticks, phlebotomes, sandflies, etc.), and are thus named arthropod-borne viruses, or arboviruses. During the last decades, several arboviruses have emerged or re-emerged in different countries in the form of notable outbreaks, resulting in a growing interest from scientific and medical communities as well as an increase in epidemiological studies. These studies have highlighted the existence of other modes of transmission. Among them, mother-to-child transmission (MTCT) during breastfeeding was highlighted for the vaccine strain of yellow fever virus (YFV) and Zika virus (ZIKV), and suggested for other arboviruses such as Chikungunya virus (CHIKV), dengue virus (DENV), and West Nile virus (WNV). In this review, we summarize all epidemiological and clinical clues that suggest the existence of breastfeeding as a neglected route for MTCT of arboviruses and we decipher some of the mechanisms that chronologically occur during MTCT via breastfeeding by focusing on ZIKV transmission process.

Surveillance of West Nile virus infection in Kashgar Region, Xinjiang, China, 2013-2016.

West Nile virus (WNV) was first isolated in mainland China from mosquitoes in Jiashi County, Kashgar Region, Xinjiang in 2011, following local outbreaks of viral meningitis and encephalitis caused by WNV. To elaborate the epidemiological characteristics of the WNV, surveillance of WNV infection in Kashgar Region, Xinjiang from 2013 to 2016 were carried out. Blood and CSF samples from surveillance human cases, blood of domestic chicken, cattle, sheep and mosquitoes in Kashgar Region were collected and detected. There were human 65 WNV Immunoglobulin M (IgM) antibody positive cases by ELISA screening, 6 confirmed WNV cases by the plaque reduction neutralization test (PRNT) screening. These cases occurred mainly concentrated in August to September of each year, and most of them were males. WNV-neutralizing antibodies were detected in both chickens and sheep, and the positive rates of neutralizing antibodies were 15.5% and 1.78%, respectively. A total of 15,637 mosquitoes were collected in 2013-2016, with Culex pipiens as the dominant mosquito species. Four and 1 WNV-positive mosquito pools were detected by RT-qPCR in 2013 and 2016 respectively. From these data, we can confirm that Jiashi County may be a natural epidemic foci of WNV disease, the trend highlights the routine virology surveillance in WNV surveillance cases, mosquitoes and avian should be maintained and enhanced to provide to prediction and early warning of outbreak an epidemic of WNV in China.

High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease.

BACKGROUND: Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a 'microglia-like' phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. METHODS: Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells during WNV-infection. Validating our strategy, we (1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, (2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and (3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. RESULTS: Using this gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12hi CD86-, P2RY12hi CD86+ and P2RY12lo CD86- P2RY12lo CD86+. During infection, 2 further populations were identified as 'inflammatory' and 'microglia-like' macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12hi CD86- subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. CONCLUSIONS: Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells.