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Neurological symptoms associated with COVID-19, acute and long-term, suggest SARS-CoV-2 affects both central and peripheral nervous systems. Although studies have shown olfactory and hematogenous entry into the brain and neuroinflammation, little attention has been paid to the susceptibility of the peripheral nervous system to infection or to alternative routes of CNS invasion. We show that neurons in the central and peripheral nervous system are susceptible to productive infection with SARS-CoV-2. Infection of K18-hACE2 mice, wild-type mice, golden Syrian hamsters, and primary neuronal cultures demonstrate viral RNA, protein, and infectious virus in peripheral nervous system neurons and satellite glial cells, spinal cord, and specific brain regions. Moreover, neuropilin-1 facilitates SARS-CoV-2 neuronal infection. Our data show that SARS-CoV-2 rapidly invades and establishes a productive infection in the peripheral and central nervous system via direct invasion of neurons prior to viremia, which may underlie some cognitive and sensory symptoms associated with COVID-19.

Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label-free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes' drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 μm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated-electrode insulator-based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.

Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons. Published version

Felis catus, the domestic cat, is the definitive host for parasites that may result in adverse health outcomes in humans. Prevalence data of zoonotic parasites in feral cats, which are free-roaming domestic cats that are born and live in the wild, are limited. The objective of this study was to assess seroprevalence of Toxoplasma gondii antibodies and copro-prevalence of potentially zoonotic parasites in feral cats and to evaluate risk factors for seropositivity and faecal excretion of parasites. In this cross-sectional survey, 275 feral cats at Trap-Neuter-Release clinics in Central Virginia were tested for parasites via faecal flotation, direct immunofluorescence assay (faeces) and modified agglutination testing (serum). Toxoplasma gondii seroprevalence was 22.35% (95% CI: 17.47-27.86). Faecal prevalence of T. gondii-like oocysts was 1.04% (95% CI: 0.13-3.71), Toxocara cati 58.85% (95% CI: 51.54-65.89), Ancylostoma spp. 18.75% (95% CI: 13.49-25.00), Giardia duodenalis 5.73% (95% CI: 2.89-10.02) and Cryptosporidium spp. 3.33% (95% CI: 1.37-7.24). Female cats were more likely than males to excrete faecal Ancylostoma spp. eggs (OR 2.88; 95% CI 1.34-6.17). Adults were more likely than immature cats to be seropositive (OR 2.10; 95% CI: 1.11-3.97) and to excrete faecal Ancylostoma spp. eggs (OR 2.57; 95% CI: 1.10-5.99). However, immature cats were more likely than adults to excrete T. cati eggs (OR 6.79; 95% CI: 3.31-13.90) and to excrete one or more potentially zoonotic species (OR 4.67; 95% CI: 2.28-9.55) in faeces. Results of this study have implications for human and animal health and highlight the importance of collaboration between public health, medical and veterinary communities in preventive efforts.

Alzheimer’s Disease (AD) is the sixth leading cause of death in the United States. Recent studies have established a potential link between herpes simplex virus 1 (HSV-1) infection and the development of AD. HSV-1 DNA has been detected in AD amyloid plaques in human brains, and treatment with the antiviral acyclovir (ACV) was reported to block the accumulation of the AD-associated proteins beta-amyloid (Aβ) and hyper-phosphorylated tau (p-tau) in Vero and glioblastoma cells. Our goal was to determine whether the accumulation of AD-related proteins is attributable to acute and/or latent HSV-1 infection in mature hippocampal neurons, a region of the brain severely impacted by AD. Primary adult murine hippocampal neuronal cultures infected with HSV-1, with or without antivirals, were assessed for Aβ and p-tau expression over 7 days postinfection. P-tau expression was transiently elevated in HSV-1-infected neurons, as well as in the presence of antivirals alone. Infected neurons, as well as uninfected neurons treated with antivirals, had a greater accumulation of Aβ(42) than uninfected untreated neurons. Furthermore, Aβ(42) colocalized with HSV-1 latency-associated transcript (LAT) expression. These studies suggest that p-tau potentially acts as an acute response to any perceived danger-associated molecular pattern (DAMP) in primary adult hippocampal neurons, while Aβ aggregation is a long-term response to persistent threats, including HSV-1 infection. IMPORTANCE Growing evidence supports a link between HSV-1 infection and Alzheimer’s disease (AD). Although AD is clearly a complex multifactorial disorder, an infectious disease etiology provides alternative therapy opportunities for this devastating disease. Understanding the impact that HSV-1 has on mature neurons and the proteins most strongly associated with AD pathology may identify specific mechanisms that could be manipulated to prevent progression of neurodegeneration and dementia.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis. IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

A common characteristic among schizophrenia and bipolar disorder patients is cognitive dysfunction, especially for memory and attention. Recent evidence has suggested that cognitive impairment in schizophrenia and bipolar disorder patients could be associated with herpes simplex virus 1 (HSV-1) infection, due to the ability of HSV-1 to infect neurons in the temporal lobe, which plays a key role in the formation of memory and processing of sensory input. The objective of this review is to analyze the aggregate neuropsychological testing data from previous studies regarding the impact of HSV-1 infection on cognitive function in schizophrenia and bipolar disorder. A systematic literature search generated a total of 379 articles; 12 full-text case control and cross-sectional studies met the eligibility criteria to be included in the review. Pooled effects assessed the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) total scores and the three index scores for immediate memory, delayed memory, and attention in a random effects model. The overall effect for RBANS total score was in favor of the HSV-1 positive group (z = 3.10, p = 0.002). A statistically significant overall effect of cognitive impairment for memory and attention indices was in favor of HSV positive schizophrenia patients (z = 5.95 p 0.00001). The findings from the meta-analysis suggest that serological evidence of HSV-1 infection has a significant impact on cognitive function with small to moderate effect sizes (-0.23 to -0.49), particularly affecting memory and attention, in schizophrenia and bipolar patients.

Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection.

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease. IMPORTANCE Herpes simplex virus 1 (HSV-1) and HSV-2 establish latent infections, from which the viruses reactivate to cause recurrent disease throughout the life of the host. However, the viruses exhibit different manifestations and frequencies of recurrent disease. HSV-1 and HSV-2 establish latency in both sensory and autonomic ganglia. Autonomic ganglia are more responsive than sensory ganglia to stimuli associated with recurrent disease in humans, such as stress and hormone fluctuations, suggesting that autonomic ganglia may play an important role in recurrent disease. We show that HSV-1 can reactivate from autonomic ganglia, independently from sensory ganglia, to cause recurrent ocular disease. We found no evidence that HSV-2 could reactivate from autonomic ganglia independently from sensory ganglia after ocular infection, but HSV-2 did replicate in both ganglia simultaneously to cause persistent disease. Thus, viral replication and reactivation in autonomic ganglia contribute to different clinical disease manifestations of HSV-1 and HSV-2 after ocular infection.

A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor., Supported by National Institutes of Health Grants R01 EY 01869, R01 EY023179, T32 EY007120, R21 EY025435, P30 EY002162, EY022697, and F32 EY02114. Additional support was provided by That Man May See (San Francisco), Benign Essential Blepharospasm Research Foundation and Unrestricted Grants from Research to Prevent Blindness (UCSF, UW).

Zika virus (ZIKV) has recently surged in human populations, causing an increase in congenital and Guillain-Barré syndromes. While sexual transmission and presence of ZIKV in urine, semen, vaginal secretions, and saliva have been established, the origin of persistent virus shedding into biological secretions is not clear. Using a primary adult murine neuronal culture model, we have determined that ZIKV persistently and productively infects sensory neurons of the trigeminal and dorsal root ganglia, which innervate glands and mucosa of the face and the genitourinary tract, respectively, without apparent injury. Autonomic neurons that innervate these regions are not permissive for infection. However, productive ZIKV infection of satellite glial cells that surround and support sensory and autonomic neurons in peripheral ganglia results in their destruction. Persistent infection of sensory neurons, without affecting their viability, provides a potential reservoir for viral shedding in biological secretions for extended periods of time after infection. Furthermore, viral destruction of satellite glial cells may contribute to the development of Guillain-Barré Syndrome via an alternative mechanism to the established autoimmune response. Published version

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons. IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become infected by HSV-1 and HSV-2 express stress hormone receptors and are responsive to hormone fluctuations. Our results show that autonomic neurons are more responsive to epinephrine and corticosterone than are sensory neurons, demonstrating that the autonomic nervous system plays a substantial role in HSV pathogenesis. Furthermore, these results suggest that stress responses have the potential to differentially impact HSV-1 and HSV-2 so as to produce divergent outcomes of infection.

Feral cats raise public health concerns due to their large population numbers and ability to harbour pathogens that cause disease in people, pets, and wildlife. Information regarding the potential for feral cats to intersect with areas frequented by humans is lacking. This study examined the potential for feral cats and human territories to overlap in the Richmond metropolitan area of Central Virginia. Feral cats (n = 275) were trapped for monthly trap-neuter-release (TNR) clinics from July to November 2016. A geographic information system (GIS) was used to map feral cat trapping locations, elementary and preschools, public parks, and community gardens, and to evaluate the potential for cat interaction with these areas, presuming a maximum habitat radius of 0.44 miles. We found that 8.0% of feral cats in the Richmond metropolitan area had potential to range onto public elementary or preschool property, and 81.5% of feral cats trapped in Richmond City had potential to roam into one or more places of interest, including elementary and preschool grounds, public parks, and community gardens. This provides public health, veterinary, and human health professionals with important information that can be used to focus resources in an effort to reduce zoonosis associated with feral cat populations.

Due to the recent epidemic of Zika virus (ZIKV) infection and resulting sequelae, as well as concerns about both the sexual and vertical transmission of the virus, renewed attention has been paid to the pathogenesis of this unique arbovirus. Numerous small animal models have been used in various ZIKV pathogenicity studies, however, they are often performed using immunodeficient or immunosuppressed animals, which may impact disease progression in a manner not relevant to immunocompetent humans. The use of immunocompetent animal models, such as macaques, is constrained by small sample sizes and the need for specialized equipment/staff. Here we report the establishment of ZIKV infection in an immunocompetent small animal model, the guinea pig, using both subcutaneous and vaginal routes of infection to mimic mosquito-borne and sexual transmission. Guinea pigs developed clinical signs consistent with mostly asymptomatic and mild disease observed in humans. We demonstrate that the route of infection does not significantly alter viral tissue tropism but does impact mucosal shedding mechanics. We also demonstrate persistent infection in sensory and autonomic ganglia, identifying a previously unrecognized niche of viral persistence that could contribute to viral shedding in secretions. We conclude that the guinea pig represents a useful and relevant model for ZIKV pathogenesis. Published version

Herpes simplex viruses (HSV1 and HSV2) establish latency in peripheral ganglia after ocular or genital infection, and can reactivate to produce different patterns and frequencies of recurrent disease. Previous studies showed that nerve growth factor (NGF) maintains HSV1 latency in embryonic sympathetic and sensory neurons. However, adult sensory neurons are no longer dependent on NGF for survival, some populations cease expression of NGF receptors postnatally, and the viruses preferentially establish latency in different populations of sensory neurons responsive to other neurotrophic factors (NTFs). Thus, NGF may not maintain latency in adult sensory neurons. To identify NTFs important for maintaining HSV1 and HSV2 latency in adult neurons, we investigated acute and latently-infected primary adult sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) after NTF removal. NGF and glial cell line-derived neurotrophic factor (GDNF) deprivation induced HSV1 reactivation in adult sympathetic neurons. In adult sensory neurons, however, neurturin (NTN) and GDNF deprivation induced HSV1 and HSV2 reactivation, respectively, while NGF deprivation had no effects. Furthermore, HSV1 and HSV2 preferentially reactivated from neurons expressing GFRα2 and GFRα1, the high affinity receptors for NTN and GDNF, respectively. Thus, NTN and GDNF play a critical role in selective maintenance of HSV1 and HSV2 latency in primary adult sensory neurons.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

The herpes simplex virus 2 (HSV-2) viral microRNA (miRNA) designated miR-H6 is located upstream of the latency-associated transcript (LAT) promoter region on the strand opposite the LAT. Deletion of the LAT promoter and part of LAT exon 1 abolished HSV-2 miR-H6 expression in acutely and latently infected guinea pig dorsal root ganglia (DRG), suggesting that this region is needed both for the expression of LAT-encoded miRNAs and for miR-H6 expression in vivo . Relative to cells infected with a viral rescuant, miR-H6 expression was significantly reduced in cells infected with a mutant HSV-2 virus, NotPolyA, with an insertion of a simian virus (SV40) polyadenylation signal sequence between the LAT promoter and miR-H6 sequences. In addition, expression of miR-H6, but not LAT or viral DNA, was significantly reduced in both mouse trigeminal ganglia (TG) and guinea pig DRG latently infected with the NotPolyA mutant. Guinea pigs infected with NotPolyA experienced reduced neurological complications of acute infection relative to those infected with the rescuant, but the recurrence phenotype of the NotPolyA mutant was similar to those of its rescuant and wild-type HSV-2, indicating that reduction of miR-H6 expression is not by itself able to alter the establishment of latency for the wild-type virus or the recurrence phenotype. Furthermore, the mutation in NotPolyA did not affect the propensity of wild-type HSV-2 to establish latency in neurons positive for subtype marker KH10. In contrast to published reports regarding its HSV-1 homolog, HSV-2 miR-H6 did not affect ICP4 expression in transfected or infected cells. We hypothesize that viral miRNAs associated with LAT expression are likely to work collectively, contributing to the phenotype attributed to the LAT.

Background: This study explores how the Roma in Romania, the EU’s most concentrated population, are faring in terms of a number of quality of life indicators, including poverty levels, healthcare, education, water, sanitation, and hygiene. It further explores the role of synthetic populations and modelling in identifying at-risk populations and delivering targeted aid.Methods: 135 surveys were conducted across five geographically diverse Romanian communities. Household participants were selected through a comprehensive random walk method. Analyses were conducted on all data using Pandas for Python. Combining land scan data, time-use survey analyses, interview data, and ArcGIS, the resulting synthetic population was analysed via classification and regression tree (CART) analysis to identify hot-spots of need, both ethnically and geographically.Results: These data indicate that the Roma in Romania face significant disparities in education, with Roma students less likely to progress beyond 8 th grade. In addition, the Roma population remains significantly disadvantaged with regard to safe and secure housing, poverty, and healthcare status, particularly in connection to diarrheal disease. In contrast, however, both Roma and non-Roma in rural areas face difficulties regarding full-time employment, sanitation, and water, sanitation, and hygiene infrastructure. In addition, the use of a synthetic population can generate information about ‘hot spots’ of need, based on geography, ethnicity, and type of aid required.Conclusions: These data demonstrate the challenges that remain to the Roma population in Romania, and also point to the myriad of ways in which all rural Romanians, regardless of ethnicity, are encountering hardship. This study highlights an approach that combines traditional survey data with more wide-reaching geographically based data and CART analysis to determine ‘hot spot’ areas of need in a given population. With the appropriate inputs, this tool can be extrapolated to any population in any country.

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in different neuronal subtypes (A5+ and KH10+) in murine trigeminal ganglia, results which correlate with restricted productive infection in these neurons in vitro . HSV-2 latency-associated transcript (LAT) contains a cis -acting regulatory element near the transcription start site that promotes productive infection in A5+ neurons and a second element in exon 1 that inhibits productive infection in KH10+ neurons. HSV-1 contains no such regulatory sequences, demonstrating different mechanisms for regulating productive HSV infection in neurons.

PURPOSE How herpes simplex virus (HSV) is transported from the infected neuron cell body to the axon terminal is poorly understood. Several viral proteins are candidates for regulating the process, but the evidence is controversial. We compared the results of Us9 deletions in two HSV strains (F and NS) using a novel quantitative assay to test the hypothesis that the viral protein Us9 regulates the delivery of viral DNA to the distal axon of retinal ganglion cells in vivo. We also deleted a nine-amino acid motif in the Us9 protein of F strain (Us9-30) to define the role of this domain in DNA delivery. METHODS The vitreous chambers of murine eyes were infected with equivalent amounts of F or NS strains of HSV. At 3, 4, or 5 days post infection (dpi), both optic tracts (OT) were dissected and viral genome was quantified by qPCR. RESULTS At 3 dpi, the F strain Us9- and Us9-30 mutants delivered less than 10% and 1%, respectively, of the viral DNA delivered after infection with the Us9R (control) strain. By 4 and 5 dpi, delivery of viral DNA had only partially recovered. Deletion of Us9 in NS-infected mice has a less obvious effect on delivery of new viral DNA to the distal OT. By 3 dpi the NS Us9-strain delivered 22% of the DNA that was delivered by the NS wt, and by 4 and 5 dpi the amount of Us9-viral DNA was 96% and 81%, respectively. CONCLUSIONS A highly conserved acidic cluster within the Us9 protein plays a critical role for genome transport to the distal axon. The transport is less dependent on Us9 expression in the NS than in the F strain virus. This assay can be used to compare transport efficiency in other neurotropic viral strains.

After HSV infection, some trigeminal ganglion neurons support productive cycle gene expression, while in other neurons the virus establishes a latent infection. We previously demonstrated that HSV-1 and HSV-2 preferentially establish latent infection in A5+ and KH10+ sensory neurons, respectively, and that exchanging the latency-associated transcript (LAT) between HSV-1 and HSV-2 also exchanges the neuronal preference. Since many viral genes besides the LAT are functionally interchangeable between HSV-1 and HSV-2, we co-infected HSV-1 and HSV-2, both in vivo and in vitro, to determine if trans-acting viral factors regulate whether HSV infection follows a productive or latent pattern of gene expression in sensory neurons. The pattern of HSV-1 and HSV-2 latent infection in trigeminal neurons was no different following co-infection than with either virus alone, consistent with the hypothesis that a trans-acting viral factor is not responsible for the different patterns of latent infection of HSV-1 and HSV-2 in A5+ and KH10+ neurons. Since exchanging the LAT regions between the viruses also exchanges neuronal preferences, we infected transgenic mice that constitutively express 2.8 kb of the LAT region with the heterologous viral serotype. Endogenous expression of LAT did not alter the pattern of latent infection after inoculation with the heterologous serotype virus, demonstrating that the LAT region does not act in trans to direct preferential establishment of latency of HSV-1 and HSV-2. Using HSV1-RFP and HSV2-GFP in adult trigeminal ganglion neurons in vitro, we determined that HSV-1 and HSV-2 do not exert trans-acting effects during acute infection to regulate neuron specificity. Although some neurons were productively infected with both HSV-1 and HSV-2, no A5+ or KH10+ neurons were productively infected with both viruses. Thus, trans-acting viral factors do not regulate preferential permissiveness of A5+ and KH10+ neurons for productive HSV infection and preferential establishment of latent infection.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency and express the latency-associated transcript (LAT) preferentially in different murine sensory neuron populations, with most HSV-1 LAT expression in A5 neurons and most HSV-2 LAT expression in KH10 neurons. To study the mechanisms regulating the establishment of HSV latency in specific subtypes of neurons, cultured dissociated adult murine trigeminal ganglion (TG) neurons were assessed for relative permissiveness for productive infection. In contrast to that for neonatal TG, the relative distribution of A5 and KH10 neurons in cultured adult TG was similar to that seen in vivo. Productive infection with HSV was restricted, and only 45% of cultured neurons could be productively infected with either HSV-1 or HSV-2. A5 neurons supported productive infection with HSV-2 but were selectively nonpermissive for productive infection with HSV-1, a phenomenon that was not due to restricted viral entry or DNA uncoating, since HSV-1 expressing -galactosidase under the control of the neurofilament promoter was detected in 90% of cultured neurons, with no preference for any neuronal subtype. Infection with HSV-1 reporter viruses expressing enhanced green fluorescent protein (EGFP) from immediate early (IE), early, and late gene promoters indicated that the block to productive infection occurred before IE gene expression. Trichostatin A treatment of quiescently infected neurons induced productive infection preferentially from non-A5 neurons, demonstrating that the nonpermissive neuronal subtype is also nonpermissive for reactivation. Thus, HSV-1 is capable of entering the majority of sensory neurons in vitro; productive infection occurs within a subset of these neurons; and this differential distribution of productive infection is determined at or before the expression of the viral IE genes. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) replication at the periphery is accompanied by infection of neuronal axons and subsequent retrograde axonal transport to cell bodies of primary sensory neurons, where infection may follow either a productive or a latent pathway. For some neurons, lytic gene expression and progeny virus production are thought to result in cell death, while in other neurons, the productive cycle fails and the virus establishes a latent infection. The factors that determine whether HSV progresses through a productive cycle or establishes latency are not clear. It is suspected that different neuronal subtypes and/or the presence or absence of certain host factors may be critical in determining the outcome

Levels of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in dorsal root ganglia (DRG) and spinal cord (SC) were quantified after inoculation of guinea pig genitals and footpads. In genital infection, viral DNA reached SC and DRG simultaneously (at 2 to 3 days after inoculation) but was more abundant in SC than in DRG. After inoculation of footpads, which lack parasympathetic innervation, the viruses spread more efficiently to DRG than to SC. These results show important differences between genital and footpad infections, including independence of spread to DRG and SC, and imply that autonomic neurons may play an important role in the pathogenesis of viral latency after genital inoculation.

Herpes simplex virus 1 (HSV-1) and HSV-2 cause similar acute infections but differ in their abilities to reactivate from trigeminal and lumbosacral dorsal root ganglia. During latency, HSV-1 and HSV-2 also preferentially express their latency-associated transcripts (LATs) in different sensory neuronal subtypes that are positive for A5 and KH10 markers, respectively. Chimeric virus studies showed that LAT region sequences influence both of these viral species-specific phenotypes. To further map the LAT region sequences responsible for these phenotypes, we constructed the chimeric virus HSV2-LAT-E1, in which exon 1 (from the LAT TATA to the intron splice site) was replaced by the corresponding sequence from HSV-1 LAT. In intravaginally infected guinea pigs, HSV2-LAT-E1 reactivated inefficiently relative to the efficiency of its rescuant and wild-type HSV-2, but it yielded similar levels of viral DNA, LAT, and ICP0 during acute and latent infection. HSV2-LAT-E1 preferentially expressed the LAT in A5+ neurons (as does HSV-1), while the chimeric viruses HSV2-LAT-P1 (LAT promoter swap) and HSV2-LAT-S1 (LAT sequence swap downstream of the promoter) exhibited neuron subtype-specific latent LAT expression phenotypes more similar to that of HSV-2 than that of HSV-1. Rescuant viruses displayed the wild-type HSV-2 phenotypes of efficient reactivation in the guinea pig genital model and a tendency to express LAT in KH10+ neurons. The region that is critical for HSV species-specific differences in latency and reactivation thus lies between the LAT TATA and the intron splice site, and minor differences in the 5′ ends of chimeric sequences in HSV2-LAT-E1 and HSV2-LAT-S1 point to sequences immediately downstream of the LAT TATA.

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo . miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are similar viruses with several notable differences. While both viruses establish latency in sensory ganglia and reactivate to cause recurrent disease, HSV-1 reactivates more efficiently from trigeminal ganglia to cause cold sores or keratitis and HSV-2 reactivates more efficiently from lumbosacral dorsal root ganglia (DRG) to cause genital herpes. Both viruses are capable of causing central nervous system (CNS) disease, but HSV-1 CNS infections typically manifest as severe necrotizing encephalitis while HSV-2 is more commonly associated with relatively benign meningitis. Potential mechanisms for type-specific differences between HSV-1 and HSV-2 have not been closely examined, although the latency associated transcript (LAT) of HSV plays a critical role in the establishment of latency and site-specific reactivation and may have regulatory influence over viral replication and spread