Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Role of vaccination and anti-SARS-CoV-2 antibodies in the clinical outcome of hospitalized COVID-19 patients.

Background: Although vaccination has considerably reduced the risk of hospitalization and death from COVID19, the impact of vaccination and anti-SARS-CoV-2 antibody status on the outcome of patients who required hospitalization has been poorly investigated. Material and methods: A prospective observational study in 232 patients hospitalized for COVID19 was carried out from October 2021 to January 2022 to evaluate the role on patient outcome of their vaccination and anti-SARS-CoV-2 antibody status and titer, comorbidities, analytical determinations, clinical presentation at admission, treatments and requirements for respiratory support. Cox regression and survival analyzes were performed. The SPSS and "R" programs were used. Results: Patients with complete vaccination schedule had higher S-protein antibody titers (log10 3.73 [2.83-4.6] UI/ml vs 1.6 [2.99-2.61] UI/ml; p < 0.001), lower probability of radiographic worsening (21.6% vs. 35.4%; p = 0.005), less likely required high doses of dexamethasone (28.4% vs. 45.4%; p = 0.012), high-flow oxygen (20.6% vs. 35.4%; p = 0.02), ventilation (13.7% vs, 33.8%; p = 0.001) and intensive care admissions (10.8% vs. 32.6%; p < 0.001). Remdesivir (HR = 0.38; p < 0.001) and complete vaccination schedule (HR = 0.34; p = 0.008) were protective factors. No differences in antibody status were detected between groups (HR = 0.58; p = 0.219). Conclusions: SARS-CoV-2 vaccination was associated with higher S-protein antibody titers and lower probability of radiological progression, immunomodulators requirement and respiratory support or death. However, vaccination but not antibody titters protected from adverse events pointing a role of immune-protective mechanisms in addition to humoral response.

Antecedentes: Aunque la vacunación ha reducido considerablemente el riesgo de hospitalización y muerte por COVID-19, se ha investigado poco el impacto de la vacunación y el estado de los anticuerpos anti-SARS-CoV-2 en la evolución de los pacientes que requieren hospitalización. Material y métodos: Se realizó un estudio observacional prospectivo en 232 pacientes hospitalizados por COVID-19 desde octubre del 2021 hasta enero del 2022 para evaluar el impacto en la evolución clínica del estado vacunal, el título de anticuerpos anti-SARS-CoV-2, la presencia de comorbilidades, analítica, la clínica al ingreso, tratamientos y soporte respiratorio. Se realizaron análisis de supervivencia y regresión de Cox. Se utilizaron los programas SPSS y «R¼. Resultados: Los pacientes con esquema de vacunación completo presentaron títulos de anticuerpos contra la proteína S más elevados (log10 3,73 [2,83-4,6] UI/mL vs. 1,6 [2,99-2,61] UI/mL; p < 0,001), menor probabilidad de empeoramiento radiográfico (21,6 vs. 35,4%; p = 0,005), requirieron con menor probabilidad dosis elevadas de dexametasona (28,4 vs. 45,4%; p = 0,012), oxígeno de alto flujo (20,6 vs. 35,4%; p = 0,02), ventilación (13,7 vs. 33,8%; p = 0,001) e ingresos en cuidados intensivos (10,8 vs. 32,6%; p < 0,001). El remdesivir (HR = 0,38; p < 0,001) y el esquema completo de vacunación (HR = 0,34; p = 0,008) fueron factores protectores de mala evolución. No se detectaron diferencias en el estado de los anticuerpos entre los grupos (HR = 0,58; p = 0,219). Conclusiones: La vacunación contra el SARS-CoV-2 se asoció con mayores títulos de anticuerpos contra la proteína S y menor probabilidad de progresión radiológica, requerimiento de inmunomoduladores y soporte respiratorio o muerte. Sin embargo, la vacunación, pero no los títulos de anticuerpos, protegió de los eventos adversos, lo que indica un papel de los mecanismos de protección inmunológica además de la respuesta humoral.

Remdesivir plus dexamethasone is associated to improvement in the clinical outcome of COVID-19 hospitalized patients regardless of their vaccination status.

INTRODUCTION: Remdesivir seems to reduce the risk of hospitalization and improve clinical outcome in hospitalized patients with COVID-19. OBJECTIVES: To compare the clinical outcome of COVID-19 hospitalized patients treated with remdesivir plus dexamethasone versus dexamethasone alone, according to their vaccination status. MATERIAL AND METHODS: A retrospective observational study was carried out in 165 patients hospitalized for COVID-19 from October 2021 to January 2022. Multivariate logistic regression, Kaplan-Meier and the log-rank tests were used to evaluate the event (need for ventilation or death). RESULTS: Patients treated with remdesivir plus dexamethasone (n=87) compared with dexamethasone alone (n=78) showed similar age (60±16, 47-70 vs. 62±37, 51-74 years) and number of comorbidities: 1 (0-2) versus 1.5 (1-3). Among 73 fully vaccinated patients, 42 (47.1%) were in remdesivir plus dexamethasone and 31 (41%) in dexamethasone alone. Patients treated with remdesivir plus dexamethasone needed intensive care less frequently (17.2% vs. 31%; p=0.002), high-flow oxygen (25.3% vs. 50.0%; p=0.002) and non-invasive mechanical ventilation (16.1% vs. 47.4%; p<0.001). Furthermore, they had less complications during hospitalization (31.0% vs. 52.6%; p=0.008), need of antibiotics (32.2% vs. 59%; p=0.001) and radiologic worsening (21.8% vs. 44.9%; p=0.005). Treatment with remdesivir plus dexamethasone (aHR, 0.26; 95% CI: 0.14-0.48; p<0.001) and vaccination (aHR 0.39; 95% CI: 0.21-0.74) were independent factors associated with lower progression to mechanical ventilation or death. CONCLUSIONS: Remdesivir in combination with dexamethasone and vaccination independently and synergistically protects hospitalized COVID-19 patients requiring oxygen therapy from progression to severe disease or dead.

Whole Sequencing and Detailed Analysis of SARS-CoV-2 Genomes in Southeast Spain: Identification of Recurrent Mutations in the 20E (EU1) Variant with Some Clinical Implications.

During the COVID-19 pandemic caused by SARS-CoV-2, new waves have been associated with new variants and have the potential to escape vaccinations. Therefore, it is useful to conduct retrospective genomic surveillance research. Herein, we present a detailed analysis of 88 SARS-CoV-2 genomes belonging to samples taken from COVID-19 patients from October 2020 to April 2021 at the "Reina Sofía" Hospital (Murcia, Spain) focused to variant appeared later. The results at the mentioned stage show the turning point since the 20E (EU1) variant was still prevalent (71.6%), but Alpha was bursting to 14.8%. Concern mutations have been found in 5 genomes classified as 20E (EU1), which were not characteristic of this still little evolved variant. Most of those mutations are found in the spike protein, namely Δ69-70, E484K, Q675H and P681H. However, a relevant deletion in ORF1a at positions 3675-3677 was also identified. These mutations have been reported in many later SARS-CoV-2 lineages, including Omicron. Taken together, our data suggest that preferential emergence mutations could already be present in the early converging evolution. Aside from this, the molecular information has been contrasted with clinical data. Statistical analyses suggest that the correlation between age and severity criteria is significantly higher in the viral samples with more accumulated changes.

Role of vaccination and anti-SARS-CoV-2 antibodies in the clinical outcome of hospitalized COVID-19 patients.

BACKGROUND: Although vaccination has considerably reduced the risk of hospitalization and death from COVID19, the impact of vaccination and anti-SARS-CoV-2 antibody status on the outcome of patients who required hospitalization has been poorly investigated. MATERIAL AND METHODS: A prospective observational study in 232 patients hospitalized for COVID19 was carried out from October 2021 to January 2022 to evaluate the role on patient outcome of their vaccination and anti-SARS-CoV-2 antibody status and titer, comorbidities, analytical determinations, clinical presentation at admission, treatments and requirements for respiratory support. Cox regression and survival analyzes were performed. The SPSS and "R" programs were used. RESULTS: Patients with complete vaccination schedule had higher S-protein antibody titers (log10 3.73 [2.83-4.6]UI/ml vs 1.6 [2.99-2.61]UI/ml; p<0.001), lower probability of radiographic worsening (21.6% vs. 35.4%; p=0.005), less likely required high doses of dexamethasone (28.4% vs. 45.4%; p=0.012), high-flow oxygen (20.6% vs. 35.4%; p=0.02), ventilation (13.7% vs, 33.8%; p=0.001) and intensive care admissions (10.8% vs. 32.6%; p<0.001). Remdesivir (HR=0.38; p<0.001) and complete vaccination schedule (HR=0.34; p=0.008) were protective factors. No differences in antibody status were detected between groups (HR=0.58; p=0.219). CONCLUSIONS: SARS-CoV-2 vaccination was associated with higher S-protein antibody titers and lower probability of radiological progression, immunomodulators requirement and respiratory support or death. However, vaccination but not antibody titters protected from adverse events pointing a role of immune-protective mechanisms in addition to humoral response.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

Moderate to Intense Physical Activity Is Associated With Improved Clinical, CD4/CD8 Ratio, and Immune Activation Status in HIV-Infected Patients on ART.

BACKGROUND: Physical activity has anti-inflammatory effects and reduces morbidity and mortality in the general population, but its role in the clinical, CD4/CD8 ratio, and immune activation status of HIV-infected patients has been poorly studied. METHODS: A cross-sectional study was carried out in a cohort of 155 HIV-infected patients on stable antiretroviral therapy (ART) to compare clinical, biochemical, CD4/CD8 ratio, and immune activation status according to their physical activity in the last 2 years (sedentary/low vs moderate/intense) assessed by the iPAQ. A binary logistic regression and mixed analysis of variance were performed to evaluate the impact of levels of physical activity on CD4/CD8 ratio. RESULTS: In our series, 77 (49.7%) out of 155 patients were sedentary, and 78 (50.3%) practiced moderate/intense physical activity. Moderate/intense physical activity was associated with better metabolic control (lower body mass index, P = .024; glucose, P = .024; and triglyceride, P = .002) and CDC HIV stage (P = .046), lower CD8+ (P =  .018), CD4+CD8+ (P = .026), CD4+CD86+ (P = .045), CD4+HLA-DR+ (P = .011), CD8+HLA-DR+ (P = .048) T lymphocytes and CD16+HLA-DR+ natural killer cells (P = .026), and higher CD3+CD4+ T lymphocytes (P = .016) and CD4/CD8 ratio (P = .001). Sedentary lifestyle (odds ratio [OR], 2.12; P = .042), CD4 nadir (OR, 1.005; P < .001), and CD8+CD38+ T cells (OR, 1.27; P = .006) were independently associated with low CD4/CD8 ratio (<0.8). Earlier and more intense CD4/CD8 ratio recovery was observed in patients with higher physical activity in the 2-year follow-up with a significant interaction between these variables: F(2, 124) = 3.31; P = .049; partial η2 = 0.042. CONCLUSIONS: Moderate to high physical activity is associated with beneficial health effects, improvement in metabolic profile, and reduction of chronic inflammation in patients with HIV. Although more studies and clinical trials are needed to confirm these findings, a healthy lifestyle including at least moderate physical activity should be recommended to HIV patients on stable ART.

Subclinical atherosclerosis and immune activation in young HIV-infected patients with telomere shortening.

BACKGROUND: To date, available data on premature aging in young HIV-infected adults are scarce and no reports offer comprehensive assessment of telomere shortening (TS) in relation to subclinical atherosclerosis (SCA). In this study, we investigate if telomere shortening and immune activation markers are associated with SCA, which is one of the main degenerative diseases in young HIV-infected adults. METHODS: A descriptive cross-sectional study was carried out in 149 HIV-infected patients on stable antiretroviral regimen (ART). Carotid intima-media thickness (cIMT) was estimated by carotid ultrasound. Quantitative singleplex PCR was performed to evaluate TS. The expression of activation/senescence markers was evaluated by multiparametric flow cytometry. RESULTS: TS was observed in 73 patients (49%). Higher cIMT was observed in patients with TS than those without it (0.86 vs. 0.80 mm; p=0.041). Patients under the age of 50 (defined as young adults) with TS showed higher absolute numbers of activated lymphocyte T cells CD8+CD38+ (3.94 vs. 2.34 cell/µl; p=0.07) and lymphocyte B cells CD19+CD38+ (3.07 vs. 2.10 cell/µl; p=0.004) compared to those without TS. In the multivariate analysis, the only factor independently associated with TS was the absolute number of lymphocyte T cells CD8+CD38+ T cells (OR = 1.18; 95%-CI = 1.00-1.39; p = 0.05). CONCLUSION: Young HIV-infected adults show premature biological aging with accentuated immune activation. Chronic inflammation with excessive T-cells activation could be associated to TS, premature aging, and SCA in young HIV-infected adults.

Bariatric and metabolic endoscopy in the handling of fatty liver disease. A new emerging approach?

BACKGROUND: non-alcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic hepatopathy in our environment. However, the benefits of the bariatric endoscopy in this disease are barely documented. OBJECTIVES: to evaluate changes in NAFLD, via non-invasive methods in obese patients who underwent bariatric restrictive endoscopy. Weight, metabolic changes and the level of technical safety were also analyzed as secondary objectives. METHODS: thirty patients with NAFLD and obesity (mean BMI 38.22 ± 6.55 kg/m2) underwent bariatric restrictive endoscopy; this included 15 one-year intragastric balloons and 15 sutured gastroplasties (ESG-Apollo®). A non-invasive prospective analysis was performed via analytical (hepatic function, insulin-resistance and hepatic steatosis/fibrosis scores) and ultrasonographic parameters. In addition, anthropometric features and the evolution of the main obesity-related comorbidities were evaluated. The follow-up period was one year in all cases. RESULTS: thirty patients were included; 63% were female with a mean age of 46 ± 13.8 years. There was a decrease in FLI, HSI, NAFLD-Fibrosis Score, hepatic ultrasonographic steatosis, subcutaneous fat (p < 0.001), HOMA-IR, insulin and triglycerides (p < 0.05) after 12 months. An average EWL of 44.02% (16.34% TBWL) was obtained after one year, with EWL > 25% in 27/30 patients (TBWL > 10% in 25/30 patients) (p < 0.001). Obesity-related comorbidities were resolved in 17/30 (57%) of cases, 5/8 (62.5%) HTA, 5/12 (41.7%) DLP, 2/4 (50%) T2DM, 2/3 (66.7%) SOAS and 3/3 (100%) arthropathy. An improvement in HbA1c in the ESG-Apollo group (p = 0.017) was the only difference. One migrated and spontaneously expelled balloon was the only technical incidence. CONCLUSIONS: bariatric endoscopy could be proposed during short-term follow-up as an effective and safe alternative in patients with obesity and NAFLD. It stimulates weight loss and improves analytical and ultrasound parameters from hepatic fat, insulin-resistance and hypertriglyceridemia. It also improves associated major comorbidities.

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality.

BACKGROUND: During freezing the selective precipitation of substances in the medium may provoke a pH shift and lead to sperm damage. OBJECTIVE: To study the effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality. METHODS: A total of 15 ejaculates from different boars were obtained and divided into six aliquots prior to a standard straw cryopreservation in freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) with different pH. After thawing, sperm quality (plasma membrane integrity, motility and acrosome status) were assessed at 30 and 90 minutes of post-thaw incubation at 37 degree C. RESULTS: When the boar sperm were frozen in a freezing media with pH basic, and particularly at pH 8, it had higher post-thaw sperm quality. CONCLUSION: The pre-adjustment at pH 8 of the freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) is able to improve the post-thaw boar sperm quality.

Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl ß-cyclodextrin or cholesterol-loaded cyclodextrin.

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-ß-cyclodextrin/120 × 10(6) sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P <0.05), even in semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P <0.05). Thus, CLC-treated sperm showed a higher tolerance to high glycerol concentrations (5%) in terms of sperm viability (59.19% ± 3) than non-treated sperm (45.58% ± 3; P<0.05). Therefore, it could be necessary to modify the freezing extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.

Effect of the holding time at 15 °C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation.

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8h), thawing rate (37 °C for 20s or 70 °C for 8s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing-thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4h post-thawing.

In vivo fertilising ability of frozen-thawed boar sperm treated with cholesterol-loaded cyclodextrins prior to cryopreservation.

The use of frozen-thawed (FT) sperm for the artificial insemination of pigs is rare. Treating boar sperm with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances the penetration of immature oocytes in vitro, and this phenomenon has been positively correlated with in vivo fertilisation ability in pigs. The objective of this study was to compare the in vivo fertilising ability of boar sperm treated with 0 (control) or 1mg CLC/120×10(6) sperm (CLC) prior to freezing. The fertilising ability of the FT sperm was compared in hormonally treated (equine/human chorionic gonadotropin hormones; eCG/hCG) weaned sows inseminated once (cervical insemination) at the following fixed-times after hCG administration: 37h (experiment 1) or 30h (experiment 2). In experiment 1, both treatments exhibited similar fertility rates of 67.7 and 55.9% for the control and CLC, respectively (P>0.05); however, the CLC group had a smaller litter size (11.3±0.9) than the control group (13.6±0.8) (P<0.05). In experiment 2, the pregnancy and farrowing rates were 65.2 and 66.7% for the control and CLC groups, respectively, and the litter size was 12.9±1 and 11.3±1 for the control and CLC groups, respectively, which were similar (P>0.05) for both treatments. These results indicate that the timing of insemination developed for the FT control sperm may not be suitable for CLC-treated sperm and that CLC-treated sperm may benefit from a shorter time interval between the hCG treatment and insemination. Moreover, acceptable results can be obtained with FT boar sperm with a single artificial insemination performed 30h after hCG treatment, which is the time interval recommended for fresh sperm.

Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus.

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation.

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.

Inclusion of seminal plasma in sperm cryopreservation of Iberian pig.

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process.

Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

Treating sperm with cholesterol-loaded cyclodextrins (CLC) improves the cryosurvival of the sperm of different cold-shock sensitive species. However, the response of boar sperm to this treatment is not fully understood. The aim of this study was to determine how CLC and methyl-ß-cyclodextrin (MßCD, not loaded with cholesterol) affect the parameters for boar sperm functionality, including sperm osmotic resistance, and the ability of the sperm to capacitate and to penetrate the sow's immature oocytes in vitro. Samples treated with CLC or MßCD prior to freezing exhibited similar percentages of motile sperm, live sperm and sperm with intact acrosomes as the control samples (P>0.05). In addition, these treatments did not alter the response of the boar sperm to capacitating conditions. However, when compared to the controls and the MßCD-treated samples, the CLC-treated sperm maintained greater percentages of motile sperm and live sperm in a wide range of osmotic solutions including hypo- (50, 75 and 150 mOsm/kg) and hyper-osmotic (600, 800 mOsm/kg) conditions (P<0.05). In addition, the CLC-treated sperm exhibited greater oocyte penetration ability than the control and the MßCD-treated sperm (P<0.0001). In conclusion, the pre-freezing treatment of boar sperm with CLC does not alter the ability of the sperm to respond to capacitating conditions. Despite not increasing the cryosurvival of the sperm, this treatment widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

A globally occurring indel polymorphism in the promoter of the IFNA2 gene is not associated with severity of malaria but with the positivity rate of HCV.

BACKGROUND: Type I Interferons (IFNs) are well known cytokines which exert antiviral activity, antitumor activity and immunomodulatory effects. Single-nucleotide polymorphisms (SNP) and deletions in the gene coding for IFNA2 have been shown to influence the level of expression in vitro. The indel polymorphism -305_-300delAACTTT showed the strongest effect in vitro. To analyse the worldwide distribution of this polymorphism we analyzed five different populations (586 Vietnamese, 199 Central Africans, 265 Brazilians, 108 Kaingang and 98 Guarani). To investigate a possible association with susceptibility to infectious diseases we determined the polymorphism in malaria patients suffering either mild or severe malaria and in a cohort of hepatitis C virus infected individuals. RESULTS: We could detect the indel polymorphism in all populations analysed. There was no association with this polymorphism and the outcome of malaria but we found an increase of this indel polymorphism in hepatitis C virus positive individuals compared to healthy controls (p = 0.014). CONCLUSION: Polymorphisms in genes involved in the interferon pathway have been implicated in the resistance or susceptibility against cerebral malaria and HBV. Here we show that an indel polymorphism, which mediates a disadvantageous effect in HBV patients, may also play a disadvantageous role in HCV infections stressing the importance of a fully functional interferon pathway.

Genetic conservation of the GIL blood group determining aquaporin 3 gene in African and Caucasian populations.

BACKGROUND: The human GIL blood group is encoded by the aquaporin 3 (AQP3), a water-glycerol channel present in human red cell (RBC) membranes. So far no molecular investigation of this gene has been performed in an African population. STUDY DESIGN AND METHODS: To analyze the genetic variability of the AQP3 gene in African and European persons, all exons including the boundaries to introns and the promoter region were sequenced. AQP3 mRNA levels in children affected by uncomplicated and by severe malarial anemia were determined by real-time polymerase chain reaction (RT-PCR). RESULTS: Two mutations in the promoter region and three synonymous mutations in the exons 1, 2, and 4 were found. The promoter polymorphisms, however, were not associated with the expression level of AQP3 measured in whole blood. Significant differences in AQP3 expression were found between malaria diseases severity groups (p = 0.017). CONCLUSION: AQP3 is a highly conserved gene. Although reticulocytes are highly abundant in malarial anemia, low AQP3 expression was observed. This may be a result of an impaired erythropoiesis.