Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses.

Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Human vaccine and immunisation research in South Africa: A bibliometric study.

BACKGROUND: Research in human vaccines and immunisation plays a crucial role in shaping national, regional and global health policies aimed at controlling vaccine preventable diseases (VPDs). To our knowledge, the landscape of human vaccine and immunisation research in South Africa (SA) is not well characterised. OBJECTIVES: To characterise research in human vaccines and immunisation in SA. METHODS: We conducted a bibliometric study. Seven electronic databases (PubMed; Scopus; Web of Science; Cochrane; CINAHL; Africa-Wide Information; and MEDLINE (via EBSCOhost)) were searched for eligible studies published in English between 1 January 2007 and 31 March 2020. Selected primary studies needed to be on human vaccine and immunisation research conducted in SA. All types of reviews were excluded. For the included studies, outcomes of interest included publication journals, publication trends, types of studies and VPDs, targeted populations, as well as author affiliations. RESULTS: A total of 9 212 studies was retrieved. After screening for eligibility, 366 studies met the inclusion criteria. The key findings were as follows: (i) a total of 54 (14.8 %) articles on human vaccine and immunisation research in SA appeared in local journals, while 312 (85.2%) were in non-SA (international) journals; (ii) the number of publications on human vaccine and immunisation research in SA increased from 13 in 2007 to 47 in 2015; (iii) there were 189 (51.7%) operational studies and 177 (48.3%) clinical studies, with 88 (49.7%) of the latter being clinical trials; (iv) human vaccine and immunisation research in SA is conducted across all age groups, with a focus on children; and (v) authors of the identified research outputs and those mostly represented were from the universities of Cape Town and the Witwatersrand, Johannesburg. CONCLUSIONS: The landscape of human vaccine and immunisation research in SA is growing and adapting to the emerging trends in vaccinology, with a focus on the duo epidemic of HIV and TB, as well as Expanded Programme on Immunisation (EPI)-related vaccinations. This research contributes to locally relevant evidence that can be used to inform future vaccine and EPI-related research.

Lessons learnt during the national introduction of human papillomavirus (HPV) vaccination programmes in 6 African countries: Stakeholders' perspectives.

BACKGROUND: Infection with human papillomavirus (HPV) significantly increases the risk of developing cervical cancer later in life. Therefore, globally, HPV vaccines targeted to pre-adolescent and adolescent girls have been on the rise since the licensure in 2006. However, the introduction of HPV vaccines has been relatively slow in Africa. At the end of 2016, only 8 of the 54 countries in Africa were reported to have introduced HPV vaccination at a national level. By 2019, the number of countries had grown marginally to 11. OBJECTIVES: To investigate stakeholders' perspectives on the experiences, challenges and lessons learnt during national HPV vaccine introduction in Africa. METHODS: A questionnaire was administered to selected participants from 8 African countries. These countries had successfully introduced HPV vaccination at a national level by the end of 2016. We used in-depth interviews and self-administered online questionnaires for data collection and analysis. Data are presented without naming the country or participants; therefore, readers will not be able to identify the results that are specific to individual countries. Narrative and thematic reporting were used to describe the results. RESULTS: We obtained results from 6 of the 8 targeted countries. The challenges reported during HPV vaccination programmes were: identifying the target population, using a school-based vaccine-delivery strategy, obtaining political support, the need to integrate HPV vaccination with existing school health programmes and engaging multiple stakeholders. These challenges were similar in all 6 countries. The lessons learnt were that a school-based delivery strategy is a successful approach for national HPV vaccination, and that identifying girls for vaccination at schools was less challenging if implemented through a class-based instead of an age-based approach. CONCLUSIONS: Most African countries do not have established platforms to deliver vaccines to pre-adolescent and adolescent populations. The successful introduction of the HPV vaccine through school-based vaccination strategies in African countries may have created a platform to deliver other adolescent vaccines. The similarity of the study findings across the 6 participating countries further strengthens the need to document and disseminate the challenges and lessons learnt during HPV vaccine introduction in Africa. Documentation and dissemination of the challenges and lessons learnt are useful to other countries in Africa that plan to introduce an HPV vaccination programme, and possibly other adolescent vaccines.

Hepatitis A seroprevalence in Western Cape Province, South Africa: Are we in epidemiological transition?

BACKGROUND: Hepatitis A virus (HAV) is the most common cause of viral hepatitis worldwide. Hepatitis A vaccine is not included in the Expanded Programme on Immunisation in South Africa (EPI-SA), as the country is considered to be highly endemic for hepatitis A. OBJECTIVES: To determine the seroprevalence of hepatitis A infection in Western Cape Province (WCP), South Africa. METHODS: We conducted a cross-sectional seroprevalence study in the 1 - 7-year age group in WCP. Our samples (N=482) were blood specimens left over after laboratory testing obtained from referral hospitals between August and October 2015. A Siemens enzyme immunoassay was used to test for total hepatitis A antibodies. We also analysed hepatitis A immunoglobulin G antibody results from the National Health Laboratory Service (NHLS) Disa*Lab database at Groote Schuur Hospital from 2009 to 2014, and included 2009 - 2014 acute hepatitis A (immunoglobulin M-positive) surveillance data from the National Institute for Communicable Diseases to look at trends in notified acute infections over the same period. RESULTS: Our cross-sectional study showed 44.1% seroprevalence in the 1 - 7-year age group. Hepatitis A data from the NHLS database indicated a seroprevalence of <90% up to age 10 years, indicating intermediate endemicity. The surveillance data showed that a substantial number of symptomatic hepatitis A infections occurred in the 7 - 40-year age group, suggesting that an increasing proportion of the population is susceptible to HAV infection. CONCLUSIONS: These results suggest an urgent need for detailed evidence-based considerations to introduce hepatitis A vaccine into the EPI-SA.

Coagulation parameters following equine herpesvirus type 1 infection in horses.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) is the cause of respiratory disease, abortion storms, and outbreaks of herpesvirus myeloencephalopathy (EHM). Infection of the spinal cord is characterised by multifocal regions of virally infected vascular endothelium, associated with vasculitis, thrombosis and haemorrhage that result in ischaemia and organ dysfunction. However, the mechanism of thrombosis in affected horses is unknown. OBJECTIVES: To evaluate tissue factor (TF) procoagulant activity and thrombin-antithrombin complex (TAT) levels in horses following infection with EHV-1. STUDY DESIGN: In vitro and in vivo studies following experimental EHV-1 infection. METHODS: Horses were infected with EHV-1 and levels of peripheral blood mononuclear cell (PBMC)-associated TF activity; plasma and cerebrospinal fluid (CSF)-derived microvesicle (MV)-associated TF activity and TAT complexes in plasma were examined. RESULTS: EHV-1 infection increased PBMC TF procoagulant activity in vitro and in vivo. In infected horses, this increase was observed during the acute infection and was most marked at the onset and end of viraemia. However, no significant differences were observed between the horses that showed signs of EHM and the horses that did not develop EHM. Significant changes in MV-associated TF procoagulant activity and TAT complexes were not observed in infected horses. MAIN LIMITATIONS: A small number of horses typically exhibit clinical EHM following experimental infection. CONCLUSIONS: The results indicate that EHV-1 infection increases PBMC-associated TF procoagulant activity in vivo and in vitro. Additional in vivo studies are needed to better understand the role of TF-dependent coagulation during EHM pathogenesis in horses.

Anti-inflammatory drugs decrease infection of brain endothelial cells with EHV-1 in vitro.

BACKGROUND: Equine herpesvirus-associated myeloencephalopathy is the result of endothelial cell infection of the spinal cord vasculature with equine herpesvirus-1 (EHV-1) during cell-associated viraemia. Endothelial cell infection requires contact between infected peripheral blood mononuclear and endothelial cells. Inflammation generated during viraemia likely upregulates adhesion molecule expression on both cell types increasing contact and facilitating endothelial cell infection. OBJECTIVES: Evaluating the role of anti-inflammatory drugs in decreasing endothelial cell infection with EHV-1. STUDY DESIGN: In vitro assay, crossover design, multiple drug testing. METHODS: In vitro modified infectious centre assay using immortalised carotid artery endothelial cells or primary brain endothelial cells with plaque counts per well as outcome. Cells were either anti-inflammatory drug treated or left untreated. RESULTS: Significant reduction of plaque count when cells were treated compared with untreated cells. No dose-dependent effect when drug concentrations were increased to 10× dose. Treatment of both peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) is required for significant plaque count reduction. MAIN LIMITATIONS: In vitro study. CONCLUSIONS: Anti-inflammatory drugs decrease infection of endothelial cells likely by reducing contact between EHV-1 infected PBMC and endothelial cells in vitro. The role of adhesion molecules in this process needs further investigation. In vitro results suggest anti-inflammatory drug therapy during EHV-1 infection and viraemia in horses could be clinically relevant.

Translational regulation of inhibin ßA by TGFß via the RNA-binding protein hnRNP E1 enhances the invasiveness of epithelial-to-mesenchymal transitioned cells.

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and has an important role in the process of metastasis. The transforming growth factor (TGF)-ß signaling pathway is a key inducer of EMT and we have elucidated a posttranscriptional mechanism by which TGFß modulates expression of select transcripts via the RNA-binding protein hnRNP E1 during EMT. One such transcript inhibin ßA is a member of the TGFß superfamily. Here, we show by polysome profiling that inhibin ßA is translationally regulated by TGFß via hnRNP E1. TGFß treatment or knockdown of hnRNP E1 relieves silencing of the inhibin ßA transcript, resulting in increased protein expression and secreted levels of the inhibin ßA homodimer, activin A. Our data indicate that the translational upregulation of inhibin ßA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

A controlled trial of sputum induction and routine collection methods for TB diagnosis in a South African community.

The diagnostic yield of pulmonary tuberculosis (TB) by sputum induction (SI) at the first point of contact with health services, conducted in all patients with suspected TB regardless of the ability to expectorate spontaneously, has not been evaluated. We compared the diagnostic yield of SI to routine sputum collection in a South African community setting. Ambulatory patients with suspected TB provided a 'spot' expectorated sputum sample, an SI sample by hypertonic (5 %) saline nebulization, and early morning expectorated sputum sample. The diagnostic yield of sputum smear microscopy and liquid culture (denominator all subjects with any positive Mycobacterium tuberculosis culture), and time-to-positivity of culture were compared between SI and expectorated samples. A total of 555 subjects completed the SI procedure, of whom 132 (24 %) were human immunodeficiency virus (HIV)-infected. One hundred and twenty-nine samples (129, 23 %) were M. tuberculosis culture-positive. The time-to-positivity of Mycobacteria Growth Indicator Tube (MGIT) culture was shorter for SI (median difference 2 days, p = 0.63) and for early morning expectorated sputum (median difference 2 days, p = 0.02) compared to spot expectorated sputum. However, there was no difference in the culture-positive diagnostic yield between SI and spot expectorated sputum [difference +0.7 %; confidence interval (CI) -7.0 to +8.5 %, p = 0.82] or SI and early morning expectorated sputum (difference +4.7 %; CI -3.2 to +12.5 %, p = 0.20) for all subjects or for HIV-infected subjects. SI reduces the time to positive M. tuberculosis culture, but does not increase the rate of positive culture compared to routine expectorated sputum collection. SI cannot be recommended as the routine collection method at first contact among ambulatory patients with suspected TB in high-burden communities.

Plasma D-dimer concentrations during experimental EHV-1 infection of horses.

BACKGROUND: Central nervous system blood vessel thrombosis is a part of the pathogenesis of equid herpesvirus-associated myeloencephalopathy (EHM). D-dimers (DD) are stable breakdown products of cross-linked fibrin, and increased DD-plasma concentrations could reflect the degree of systemic coagulation during EHV-1 infection. HYPOTHESIS: We hypothesized that blood DD concentrations will be increased during periods of EHV-1 fever and viremia, reflecting an activated coagulation cascade with fibrinolysis. ANIMALS: Twenty-eight equids were infected with EHV-1 in 3 experimental infection studies. Three (uninfected) horses were included in a separate study to evaluate methodology for DD concentration measurements. METHODS: Clinical data and quantitative viremia were evaluated, and DD concentrations were measured in blood samples on the day before the infection and during days 1-12 postchallenge. Uninfected horses were sampled every 3 hours for 48 hours. Logistic and linear regression was used to investigate the potential association between the fever and viremia with the presence or absence of DD concentrations in peripheral blood. RESULTS: DD concentrations were increased for 1-8 days in the majority of infected animals. Both viremia (odds ratio [OR] 6.3; 95% confidence interval [CI] 3.4-11.8; P = .0013) and fever (OR 4.9; CI 2.3-10.1; P = .001) were strongly associated with the likelihood of detecting DD in peripheral blood. CONCLUSIONS AND CLINICAL IMPORTANCE: EHV-1 viremia is associated with increases in DD concentration in horses and ponies. This indicates that EHV-1 viremia can lead to an activation of coagulation and fibrinolysis.

Vaccine trials in the developing world: operational lessons learnt from a phase IV poliomyelitis vaccine trial in South Africa.

BACKGROUND: Conducting vaccine trials in developing nations is necessary but operationally complex. We describe operational lessons learnt from a phase IV poliomyelitis vaccine trial in a semi-rural region of South Africa. METHODS: We reviewed operational data collected over the duration of the trial with respect to staff recruitment and training, participant recruitment and retention, and cold chain maintenance. RESULTS-LESSONS LEARNT: The recruitment model we used that relied on the 24h physical presence of a team member in the birthing unit was expensive and challenging to manage. Forecasting of enrolment rates was complicated by incomplete baseline data and by the linear nature of forecasts that do not take into account changing variables. We found that analyzing key operational data to monitor progress of the trial enabled us to identify problem areas timeously, and to facilitate a collegial problem-solving process by the extended trial team. Pro-actively nurturing a working relationship with the public sector health care system and the community was critical to our success. Despite the wide geographical area and lack of fixed addresses, we maintained an excellent retention rate through community assistance and the use of descriptive residential information. Training needs of team members were ongoing and dynamic and we discovered that these needs that were best met by an in-house, targeted and systemized training programme. The use of vaccine refrigerators instead of standard frost-free refrigerators is cost-effective and necessary to maintain the cold-chain. CONCLUSION: Operational challenges of a vaccine trial in developing world populations include inexperienced staff, the close liaison required between researchers and public health care services, impoverished participants that require complex recruitment and retention strategies, and challenges of distance and access. These challenges can be overcome by innovative strategies that allow for the unique characteristics of the setting, trial population, and trial team.

Health activism in Cape Town: a case study of the Health Workers Society.

The Health Workers Society (HWS), founded in 1980, was one of several progressive health organisations that fought for a democratic health system in South Africa. We document the sociopolitical context within which it operated and some of its achievements. HWS, many of whose members were staff and students of the University of Cape Town (UCT), provided a forum for debate on health-related issues, politics and society, and worked closely with other organisations to oppose the apartheid state's health policies and practices. They assisted with the formation of the first dedicated trade union for all healthcare workers and were one of the first to pioneer the primary healthcare approach in an informal settlement in Cape Town.

Tuberculosis case finding for vaccine trials in young children in high-incidence settings: a randomised trial.

SETTING: A high tuberculosis (TB) burden rural area in South Africa. OBJECTIVE: To compare TB case yield and disease profile among bacille Calmette-Guérin (BCG) vaccinated children using two case-finding strategies from birth until 2 years of age. DESIGN: BCG-vaccinated infants were enrolled within 2 weeks of birth and randomised to 3-monthly home visits for questionnaire-based TB screening plus record surveillance of TB registers, hospital admission and X-ray lists at health facilities for TB suspects and cases (Group 1), or record surveillance (as above) only (Group 2). Both groups received a close-out visit after 2 years. Participants were evaluated for suspected TB disease using standardised investigations. RESULTS: A total of 4786 infants were enrolled: 2392 were randomised to Group 1 and 2394 to Group 2. The case-finding rate was significantly greater in Group 1 (2.2/100 py) than in Group 2 (0.8/100 py), with a case-finding rate ratio of 2.6 (95%CI 1.8-4.0, P < 0.001). Although the proportion of cases with bacteriological confirmation was lower in Group 1, this difference did not reach statistical significance. There was also no significant difference in the proportions with TB symptoms and signs. CONCLUSION: Home visits combined with record surveillance detected significantly more cases than record surveillance with a single study-end visit. The TB case profile did not differ significantly between the two groups.

Sputum induction for the diagnosis of pulmonary tuberculosis: a systematic review and meta-analysis.

Sputum induction (SI) has been proposed as the optimal sample collection method for patients with paucibacillary tuberculosis (TB). Studies reporting the culture of Mycobacterium tuberculosis from SI were reviewed. A random-effects meta-analysis of diagnostic yield (numerator M. tuberculosis SI culture-positive cases; denominator all culture-positive cases) was conducted. Diagnostic yields (95% confidence intervals, CIs) were displayed as Forest plots. Heterogeneity was evaluated using Chi-squared and I-squared tests and meta-regression analysis. Ninety publications were screened, 28 full-text papers reviewed, and 17 analyzed. Collectively, n=627 SI culture-positive cases among n=975 culture-confirmed TB cases were reported. The diagnostic yield of SI ranged from 35 to 95%. The pooled diagnostic yield was 74% (CI 65-81%), with significant heterogeneity (p<0.0001, I2=86%). There were no statistically significant differences in the yield between sub-groups defined by human immunodeficiency virus (HIV) prevalence or age. Univariate analysis demonstrated that the use of fiberoptic bronchoscopy (FOB) as the comparator method was associated with a 22% reduction (CI 2-42%) in the diagnostic yield of SI. However, after adjustment for confounding, the meta-regression analysis showed that FOB usage (p=0.21) and saline concentration (p=0.31) were not independently associated with the diagnostic yield. SI will detect approximately three-quarters of M. tuberculosis culture-positive cases under study conditions. Significant heterogeneity in the diagnostic yield was not explained by HIV prevalence, age, or the use of FOB as the comparator method. The use of a particular nebulized saline concentration for SI cannot be recommended on the basis of this meta-regression analysis.

Safety and tolerability of sputum induction in adolescents and adults with suspected pulmonary tuberculosis.

Sputum induction by the inhalation of hypertonic saline may increase the yield of microbiological diagnosis of pulmonary tuberculosis (TB). This is particularly relevant in paucibacillary TB, such as in children or human immunodeficiency virus (HIV)-infected patients. Sputum induction must be shown to be safe and tolerable in community settings where invasive diagnostic methods are unavailable. The objective of this study was to describe the changes in physiological parameters and adverse events occurring during sputum induction in ambulatory adult and adolescent TB suspects recruited in community clinics. Sputum induction was performed in HIV-infected (n = 35) and HIV-uninfected (n = 67) TB suspects (n = 102). Oxygen saturation (%), blood pressure (mm Hg), heart rate (/minute), respiratory rate (/minute), and adverse events were monitored at baseline, continuously during the salbutamol pre-treatment and saline nebulization phases, and for 30 min afterwards. During nebulization, there was a statistically significant increase in oxygen saturation (1%, p < 0.0001), systolic BP (7 mm Hg, p < 0.0001), and diastolic BP (2 mm Hg, p = 0.008). Post-nebulization decrease in the systolic BP occurred (4 mm Hg, p = 0.016). These changes were not considered to be clinically significant. Eight minor, transitory, self-resolving adverse events occurred (labored breathing, n = 2; chest pain, n = 2; paroxysmal coughing, n = 1; elevated heart rate, n = 1; vomiting, n = 1; hypotension, n = 1), leading to procedure termination in four participants. No serious adverse events occurred. Induced sputum is safe, tolerable, and feasible in adult and adolescent TB suspects in a community healthcare setting.

Tuberculin skin test and QuantiFERON® assay in young children investigated for tuberculosis in South Africa.

SETTING: Although the literature on interferon-gamma release assays on tuberculosis (TB) in children has increased, data pertaining to young children remain relatively limited. OBJECTIVE: To compare results from the tuberculin skin test (TST) and the QuantiFERON®-TB Gold In-Tube assay (QFT) in children aged <3 years investigated for TB disease. DESIGN: TB suspects were evaluated by medical history and examination, TST, QFT, chest radiography, induced sputum and gastric washings for smear and culture for Mycobacterium tuberculosis. RESULTS: A total of 400 children were enrolled. Among 397 children with both test results, 68 (17%) were QFT-positive and 72 (18%) were TST-positive (≥10 mm). Agreement between the tests was excellent (94%, κ = 0.79, 95%CI 0.69-0.89). TB disease was diagnosed in 52/397 (13%) participants: 3 definite, 35 probable and 14 possible TB. QFT sensitivity and specificity for TB disease were respectively 38% and 81%. TST sensitivity and specificity were respectively 35% and 84%. CONCLUSION: While TST and QFT had excellent concordance in this population, both tests had much lower sensitivity for TB disease than has been reported for other age groups. Our results suggested equivalent performance of QFT and TST in the diagnosis of TB disease in young children in a high-burden setting.

Predictive factors for latent tuberculosis infection among adolescents in a high-burden area in South Africa.

SETTING: A high tuberculosis (TB) burden area in South Africa (notification rate for all TB cases 1400 per 100 000 population). OBJECTIVE: To determine the prevalence of and predictive factors associated with latent TB infection in adolescents. DESIGN: Adolescents aged 12-18 years were recruited from high schools, clinical and demographic data were collected, and a tuberculin skin test (TST) and a QuantiFERON®-TB Gold In-Tube (QFT) assay performed. RESULTS: A total of 6363 (58.2%) of 10 942 adolescents at the schools were enrolled. After exclusions, of 5244 participants, 55.2% (95%CI 53.8-56.5) had TST ≥ 5 mm, while 50.9% (49.5-52.2) were QFT-positive. On multivariate analysis, Black/mixed race racial groups, male sex, older age, household TB contact, low income and low education level were predictive factors for both TST- and QFT-positive results. CONCLUSION: About half of the adolescents were found to be latently infected with TB in a high TB burden area with demographic and poverty-related socio-economic factors predicting the risk of TB infection. Adolescents from deprived communities should be considered an important target group for educational interventions by TB control programmes in high-burden settings.

Infection of central nervous system endothelial cells by cell-associated EHV-1.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for ß-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.

Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced interleukin-6 secretion.

Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens. The influence of human TLR6 polymorphisms on susceptibility to infection is only partially understood. Most microbes contain lipopeptides recognized by TLR2/1 or TLR2/6 heterodimers. Our aim was to determine whether single nucleotide polymorphisms in TLR6 are associated with altered immune responses to lipopeptides and whole mycobacteria. We sequenced the TLR6 coding region in 100 healthy South African adults to assess genetic variation and determined associations between polymorphisms and lipopeptide- and mycobacteria-induced interleukin (IL)-6 production in whole blood. We found two polymorphisms, C745T and G1083C, that were associated with altered IL-6 secretion. G1083C was associated with altered IL-6 levels in response to lipopeptides, Mycobacterium tuberculosis lysate (Mtb lysate, P=0.018) and Bacille Calmette-Guerin (BCG P=0.039). The 745T allele was also associated with lower NF-κB signaling in response to di-acylated lipopeptide, PAM2 (P=0.019) or Mtb (P=0.026) in an HEK293 cell line reconstitution assay, compared with the 745C allele. We conclude that TLR6 polymorphisms may be associated with altered lipopeptide-induced cytokine responses and recognition of Mtb. These studies provide new insight into the role of TLR6 variation and the innate immune response to human infection.