Search

To evaluate the clinical accuracy of rapid diagnostic tests for the detection of Ebola virus.We searched MEDLINE®, Embase® and Web of Science for articles published between 1976 and October 2021 reporting on clinical studies assessing the performance of Ebola virus rapid diagnostic tests compared with reverse transcription polymerase chain reaction (RT-PCR). We assessed study quality using the QUADAS-2 criteria. To estimate the pooled sensitivity and specificity of these rapid diagnostic tests, we used a bivariate random-effects meta-analysis.Our search identified 113 unique studies, of which nine met the inclusion criteria. The studies were conducted in the Democratic Republic of the Congo, Guinea, Liberia and Sierra Leone and they evaluated 12 rapid diagnostic tests. We included eight studies in the meta-analysis. The pooled sensitivity and specificity of the rapid tests were 86% (95% confidence interval, CI: 80-91) and 95% (95% CI: 91-97), respectively. However, pooled sensitivity decreased to 83% (95% CI: 77-88) after removing outliers. Pooled sensitivity increased to 90% (95% CI: 82-94) when analysis was restricted to studies using the RT-PCR from altona Diagnostics as gold standard. Pooled sensitivity increased to 99% (95% CI: 67-100) when the analysis was restricted to studies using whole or capillary blood specimens.The included rapid diagnostic tests did not detect all the Ebola virus disease cases. While the sensitivity and specificity of these tests are moderate, they are still valuable tools, especially useful for triage and detecting Ebola virus in remote areas.Évaluer la précision clinique des tests de diagnostic rapide pour dépister le virus Ebola.Nous avons exploré les bases de données MEDLINE®, Embase® et Web of Science à la recherche d'articles, publiés entre 1976 et octobre 2021, qui évoquaient des études cliniques mesurant les performances des tests de diagnostic rapide du virus Ebola comparés à une réaction en chaîne par polymérase après transcription inverse (RT-PCR). Nous avons déterminé la qualité de ces études à l'aide des critères QUADAS-2. Enfin, pour estimer la sensibilité et la spécificité regroupées de ces tests de diagnostic rapide, nous avons eu recours à une méta-analyse bivariée à effets aléatoires.Nos recherches nous ont permis d'identifier 113 études uniques, dont neuf correspondaient aux critères d'inclusion. Ces études avaient été menées en Guinée, au Libéria, en République démocratique du Congo et en Sierra Leone, et portaient sur douze tests de diagnostic rapide. Nous avons inclus huit études dans notre méta-analyse. La sensibilité et la spécificité combinées des tests rapides s'élevaient respectivement à 86% (intervalle de confiance de 95%, IC: 80–91) et 95% (IC de 95%: 91–97). Néanmoins, la sensibilité combinée baissait à 83% (IC de 95%: 77–88) après retrait des valeurs aberrantes. Lorsque l'analyse se limitait aux études utilisant la RT-PCR d'altona Diagnostics en guise de référence, la sensibilité combinée augmentait jusqu'à 90% (IC de 95%: 82–94). Elle atteignait même 99% (IC de 95%: 67–100) quand l'analyse se limitait aux études basées sur des échantillons de sang total ou capillaire.Les tests de diagnostic rapide pris en compte ne détectaient pas tous les cas de maladie à virus Ebola. Néanmoins, en dépit d'une sensibilité et d'une spécificité modérées, ils demeurent de précieux outils, en particulier pour le triage et le dépistage du virus Ebola dans les zones reculées.Evaluar la precisión clínica de las pruebas diagnósticas rápidas para la detección del virus del Ébola.Se realizaron búsquedas en MEDLINE®, Embase® y Web of Science de artículos publicados entre 1976 y octubre de 2021 que informaran sobre estudios clínicos en los que se evaluara el rendimiento de las pruebas diagnósticas rápidas para detectar el virus del Ébola en comparación con la reacción en cadena de la polimerasa con retrotranscripción (RT-PCR). Se evaluó la calidad de los estudios mediante los criterios QUADAS-2. Para estimar la sensibilidad y la especificidad combinadas de estas pruebas diagnósticas rápidas, se utilizó un metanálisis bivariante de efectos aleatorios.La búsqueda identificó 113 estudios singulares, de los que nueve cumplían los criterios de inclusión. Los estudios se realizaron en la República Democrática del Congo, Guinea, Liberia y Sierra Leona y evaluaron doce pruebas diagnósticas rápidas. Se incluyeron ocho estudios en el metanálisis. La sensibilidad y la especificidad agrupadas de las pruebas rápidas fueron del 86 % (intervalo de confianza del 95 %, IC: 80-91) y del 95 % (IC del 95 %: 91-97), respectivamente. Sin embargo, la sensibilidad agrupada disminuyó al 83 % (IC del 95 %: 77-88) tras eliminar los valores atípicos. La sensibilidad agrupada aumentó al 90 % (IC del 95 %: 82-94) cuando el análisis se restringió a los estudios que utilizaban la RT-PCR de altona Diagnostics como criterio de referencia. La sensibilidad agrupada aumentó al 99 % (IC del 95 %: 67-100) cuando el análisis se restringió a los estudios que utilizaban muestras de sangre total o capilar.Las pruebas diagnósticas rápidas incluidas no detectaron todos los casos de enfermedad por el virus del Ébola. Aunque la sensibilidad y la especificidad de estas pruebas son moderadas, siguen siendo herramientas valiosas, sobre todo útiles para el triaje y la detección del virus del Ébola en zonas remotas.تقييم الدقة السريرية لاختبارات التشخيص السريع للكشف عن فيروس الإيبولا.قمنا بالبحث فيحدد بحثنا 113 دراسة فريدة، تسعة منها حققت معايير الاشتمال. تم إجراء الدراسات في جمهورية الكونغو الديمقراطية وغينيا وليبيريا وسيراليون، وقامت بتقييم اثني عشر اختبارًا تشخيصيًا سريعًا. قمنا بتضمين ثماني دراسات في التحليل التلوي. كانت الحساسية والخصوصية المجمعة للاختبارات السريعة 86% (بفاصل ثقة مقداره 95%: 80 إلى 91) و95% (بفاصل ثقة مقداره 95%: 91 إلى 97)، على الترتيب. ومع ذلك، انخفضت الحساسية المجمعة إلى 83% (بفاصل ثقة مقداره 95%: 77 إلى 88) بعد إزالة القيم المتطرفة. انخفضت الحساسية المجمعة إلى 90% (بفاصل ثقة مقداره 95%: 82 إلى 94) عندما اقتصر التحليل على الدراسات التي تستخدملم تكتشف الاختبارات التشخيصية السريعة المُضمّنة جميع حالات الإصابة بمرض فيروس الإيبولا. في حين أن حساسية وخصوصية هذه الاختبارات معتدلة، إلا أنها لا تزال أدوات ذات قيمة كبيرة، ومفيدة بشكل خاص لفرز واكتشاف فيروس الإيبولا في المناطق النائية.评估埃博拉病毒快速诊断检测法的临床准确性。.我们搜索了 MEDLINE®、Embase® 和 Web of Science 上于 1976 年至 2021 年 10 月期间发表的文章，这些文章报道了埃博拉病毒快速诊断检测与实时逆转录聚合酶链反应 (RT–PCR) 相比的效果评估。我们采用 QUADAS-2 标准评估了研究的质量，并且采用双变量随机效应元分析来评估这些快速诊断检测的综合敏感性和特异性。.我们搜索了 113 项研究，其中有九项符合纳入标准。这些研究是在刚果民主共和国、几内亚、利比里亚和塞拉利昂开展的，研究评估了十二种快速诊断检测方法。我们在元分析中纳入了八项研究。快速检测的综合敏感性和特异性分别为 86%（95% 置信区间，CI: 80–91）和 95% (95% CI: 91–97)。但是，在去除离群值之后，综合敏感性降至 83% (95% CI: 77–88)。当分析限定于将采用来自 altona Diagnostics 公司的 RT–PCR 检测作为黄金标准的研究时，综合敏感性增至 90% (95% CI: 82–94)。当分析限定于采用全血标本或末梢采血标本的研究时，综合敏感性增至 99% (95% CI: 67–100)。.纳入的快速诊断检测并未检测出所有埃博拉病毒病例。尽管这些检测在敏感性和特异性方面表现一般，但仍然是有价值的工具，尤其适用于偏远地区的埃博拉病毒分诊和检测。.Оценить клиническую точность экспресс-тестов для диагностики вируса Эбола.Авторы провели поиск в базах данных MEDLINE®, Embase® и Web of Science статей, опубликованных в период с 1976 г. по октябрь 2021 г., в которых сообщалось о клинических исследованиях, оценивающих эффективность экспресс-тестов для диагностики вируса Эбола по сравнению с полимеразной цепной реакцией с обратной транскрипцией (ОТ-ПЦР). Авторы оценивали качество исследования с использованием критериев QUADAS-2. Для оценки совокупной чувствительности и специфичности диагностических экспресс-тестов авторы использовали двумерный метаанализ случайных эффектов.Поиск выявил 113 уникальных исследований, девять из которых соответствовали критериям включения. Исследования проводились в Гвинее, Демократической Республике Конго, Либерии и Сьерра-Леоне, в которых оценивались двенадцать диагностических экспресс-тестов. Авторы включили восемь исследований в метаанализ. Совокупная чувствительность и специфичность экспресс-тестов составила 86% (95%-й ДИ: 80–91) и 95% (95%-й ДИ: 91–97) соответственно. Однако совокупная чувствительность снизилась до 83% (95%-й ДИ: 77–88) после удаления выбросов. Совокупная чувствительность увеличилась до 90% (95%-й ДИ: 82–94), когда анализ был ограничен исследованиями с использованием ОТ-ПЦР от altona Diagnostics в качестве главного стандарта. Совокупная чувствительность увеличилась до 99% (95%-й ДИ: 67–100), когда анализ был ограничен исследованиями с использованием образцов цельной или капиллярной крови.Включенные диагностические экспресс-тесты не выявили всех случаев болезни, вызванной вирусом Эбола. Хотя чувствительность и специфичность этих тестов умеренные, они по-прежнему являются ценными инструментами, особенно полезными для сортировки больных и диагностики вируса Эбола в удаленных районах.

The aim of this study was to evaluate the microbiota of normal milk in dairy cows and their relationship with host factors, such as the age of the cow (Age), somatic cell counts in milk (SCCs), and days in milk (DIM). We investigated 48 milk samples from 22 cows with no systemic or local clinical signs using MinIONTM nanopore sequencing for a 16S rRNA gene amplicon. Bacterial richness was positively correlated with the DIM (P=0.043), and both the Shannon-Wiener Index and Simpson's Index, which are metrics of alpha-diversity, were also significantly positively correlated with the SCC (P

Approximately 2.9 million people worldwide suffer from cholera each year, many of whom are destitute. However, understanding of immunity against cholera is still limited. Several studies have reported the duration of antibodies following cholera; however, systematic reviews including a quantitative synthesis are lacking.To meta-analyze cohort studies that have evaluated vibriocidal, cholera toxin B subunit (CTB), and lipopolysaccharide (LPS) antibody levels following a clinical cholera case.Design: Systematic review and meta-analysis. We searched PubMed and Web of science for studies assessing antibodies againstNine studies met our inclusion criteria for systematic review and seven for meta-analysis. The pooled mean of vibriocidal antibody titers in adults and older children (aged ≥ 6 years) was 123 on day 2 post-symptom onset, which sharply increased on day 7 (pooled mean = 6956) and gradually waned to 2247 on day 30, 578 on day 90, and 177 on day 360. Anti-CTB IgA antibodies also peaked on day 7 (pooled mean = 49), followed by a rapid decrease on day 30 (pooled mean = 21), and further declined on day 90 (pooled mean = 10), after which it plateaued from day 180 (pooled mean = 8) to 360 (pooled mean = 6). Similarly, anti-CTB IgG antibodies peaked in early convalescence between days 7 (pooled mean = 65) and 30 (pooled mean = 69), then gradually waned on days 90 (pooled mean = 42) and 180 (pooled mean = 30) and returned to baseline on day 360 (pooled mean = 24). Anti-LPS IgA antibodies peaked on day 7 (pooled mean = 124), gradually declined on day 30 (pooled mean = 44), which persisted until day 360 (pooled mean = 10). Anti LPS IgG antibodies peaked on day 7 (pooled mean = 94). Thereafter, they decreased on day 30 (pooled mean = 85), and dropped further on days 90 (pooled mean = 51) and 180 (pooled mean = 47), and returned to baseline on day 360 (pooled mean = 32). Sensitivity analysis including data from young children (aged 2-5 years) showed very similar findings as in the primary analysis.This study confirms that serological antibody (vibriocidal, CTB, and LPS) titers return to baseline levels within 1 year following clinical cholera, i.e., before the protective immunity against subsequent cholera wanes. However, this decay should not be interpreted as waning immunity because immunity conferred by cholera against subsequent disease lasts 3-10 years. Our study provides evidence for surveillance strategies and future research on vaccines and also demonstrates the need for further studies to improve our understanding of immunity against cholera.

The purpose of this study was to determine the concentrations of antimicrobial components (immunoglobulin A (IgA), lactoferrin (LF), lingual antimicrobial peptide (LAP), and S100A7) in normal milk and their relation to host factors (Age, somatic cell count (SCC), days in milk, richness, and alpha diversity of the milk microbiota) in dairy cows using multivariate regression tree analyses, and to clarify how the milk microbiota is related to the obtained results. Thirty normal milk samples were collected from a commercial dairy farm in June 2020. The thresholds that predicted the concentration of each antimicrobial component in milk were obtained by regression tree analysis, and the beta-diversity of the milk microbiota composition between groups divided according to each threshold was compared by an analysis of similarities test. The IgA and LF concentrations were mainly predicted by the SCC (177,500 and 70,000 cells/ml, respectively), and the LAP and S100A7 concentrations were predicted by Age (29.667 and 40.3 months, respectively). No relationship was observed between the concentration of IgA, LAP, or S100A7 and the milk microbiota composition between the groups divided by the threshold for prediction, but the milk microbiota composition was significantly different between the groups divided by the threshold for predicting the LF concentration. Our results indicated that the LF concentration in normal milk may be associated with the milk microbiota composition.

The rapid diagnosis of cholera contributes to adequate outbreak management. This meta-analysis assesses the diagnostic accuracy of cholera rapid tests (RDTs) to detect Vibrio cholerae O1. Methods: Systematic review and meta-analysis. We searched four databases (Medline, EMBASE, Google Scholar, and Web of Science up to 8 September 2021) for studies that evaluated cholera RDTs for the detection of V. cholerae O1 compared with either stool culture or polymerase chain reaction (PCR). We assessed the studies’ quality using the QUADAS-2 criteria. In addition, in this update, GRADE approach was used to rate the overall certainty of the evidence. We performed a bivariate random-effects meta-analysis to calculate the pooled sensitivity and specificity of cholera RDTs. Results: Overall, 20 studies were included in this meta-analysis. Studies were from Africa (n = 11), Asia (n = 7), and America (Haiti; n = 2). They evaluated eight RDTs (Crystal VC-O1, Crystal VC, Cholkit, Institut Pasteur cholera dipstick, SD Bioline, Artron, Cholera Smart O1, and Smart II Cholera O1). Using direct specimen testing, sensitivity and specificity of RDTs were 90% (95% CI, 86 to 93) and 86% (95% CI, 81 to 90), respectively. Cholera Sensitivity was higher in studies conducted in Africa [92% (95% CI, 89 to 94)] compared with Asia [82% (95% CI, 77 to 87)]. However, specificity [83% (95% CI, 71 to 91)] was lower in Africa compared with Asia [90% (95% CI, 84 to 94)]. GRADE quality of evidence was estimated as moderate. Conclusions: Against culture or PCR, current cholera RDTs have moderate sensitivity and specificity for detecting Vibrio cholerae O1.

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

Fecal contamination of water sources and open defecation have been linked to cholera outbreaks in India. However, a systematic review on the drivers responsible for these outbreaks has yet to be published. Here, we systematically review the published literature on cholera outbreaks in India between 2011 and 2020. We searched studies in English in three databases (MEDLINE, EMBASE, and Web of Science) and the Integrated Disease Surveillance Program that tracks cholera outbreaks throughout India. Two authors independently extracted data and assessed the quality of the included studies. Quantitative data on the modes of transmission reviewed in this study were assessed for any change over time between 2011–2015 and 2016–2020. Our search retrieved 10823 records initially, out of which 81 full-text studies were assessed for eligibility. Among these 81 studies, 20 were eligible for inclusion in this review. There were 565 reported outbreaks between 2011 and 2020 that led to 45,759 cases and 263 deaths. Outbreaks occurred throughout the year; however, they exploded with monsoons (June through September). In Tamil Nadu, a typical peak of cholera outbreaks was observed from December to January. Seventy-two percent (33,089/45,759) of outbreak-related cases were reported in five states, namely Maharashtra, West Bengal, Punjab, Karnataka, and Madhya Pradesh. Analysis of these outbreaks highlighted the main drivers of cholera including contaminated drinking water and food, inadequate sanitation and hygiene (including open defecation), and direct contact between households. The comparison between 2011–2015 and 2016–2020 showed a decreasing trend in the outbreaks that arose due to damaged water pipelines. Many Indians still struggle with open defecation, sanitation, and clean water access. These issues should be addressed critically. In addition, it is essential to interrupt cholera short-cycle transmission (mediated by households, stored drinking water and foodstuffs) during an outbreak. As cholera is associated with deprivation, socio-economic development is the only long-term solution.

Air pollution caused by fine particulate matter (PM2.5), volatile organic compounds, and bioaerosols is a major environmental risk to health. We developed a photocatalytic air cleaner for reducing the pollution levels of indoor air; we improved the photocatalytic system by using UV-LED for the removal of acetaldehyde and PM2.5 and by reducing the weight and size of the system. The efficiency of photocatalysis depends on the surface area and materials. Therefore, we prepared a nanosized titanium dioxide (TiO2)-coated aluminum plate irradiated by UV-LED lamps (wavelength: 375 nm) as a photocatalytic air cleaner. Passing air continuously through a TiO2-coated aluminum plate (5 × 10 × 1 cm) under black light for 200 min decomposed 90% of 5 ppm acetaldehyde (12.4 µmol h–1) and generated two carbon dioxide molecules (25.43 µmol h–1) at a molar ratio of 1:2, indicating complete decomposition of acetaldehyde with high efficiency. This photocatalytic air cleaner was applied to the decomposition of acetaldehyde and inactivation and removal of aerosol-associated influenza virus. Acetaldehyde (20 ppm) in a 1-m3 cubic space was eliminated in 60 min at a half-life of 8 min. The aerosol-associated infectivity and the RNA genome of influenza virus A/PR/8/1934 (H1N1) produced by a nebulizer in a 779-L cubic space were eliminated within 7 min; however, they were detectable for up to 28 minutes when the functional photocatalytic air cleaner was not used. The presence of intermediate breakdown products of influenza virus indicated that the virus was broken down by photocatalysis. Thus, the photocatalytic air cleaner efficiently decomposed and eliminated organic chemicals, acetaldehyde, and aerosol-associated influenza virus infectivity and viral RNA, indicating that it can clean and detoxify the indoor air in a closed space for maintaining a safer environment.

Short tandem repeats (STRs) are repetitive DNA sequences that are highly polymorphic and widely used for personal identification in the field of forensic medicine. The standard method for determining the repeat number of STRs is capillary electrophoresis of PCR products; however, the use of DNA sequencing has increased because it can identify same-sized alleles with nucleotide substitutions (iso-alleles). In this study, we performed human STR genotyping using a portable nanopore-based DNA sequencer, the MinION, and evaluated its performance. Because the sequence quality obtained by MinION is considerably lower than those obtained with other DNA sequencers, we developed an original scoring scheme for judging the genotypes from MinION reads. Analysis of seven human samples for 21-45 STR loci yielded an average of 857 thousand reads per sample, and the accuracy of genotyping and iso-allele identification reached 75.7% and 82%, respectively. Although the accuracy is higher than that reported previously, further improvements are required before this method can be practically applied.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T-lymphotropic virus. Bovine major histocompatibility complex (BoLAs) are used extensively as markers of disease and immunological traits in cattle. For BLV diagnosis, proviral load is a major diagnosis index for the determination of disease progression and transmission risk. Therefore, we investigated the frequency of BoLA-DRB3 alleles, BoLA-DQA1 alleles, and haplotypes of BoLA class II isolated from the heads of 910 BLV-infected cows out of 1290 cows assessed from BLV-positive farms, in a nationwide survey from 2011 to 2014 in Japan. Our aim was to identify BoLA class II polymorphisms associated with the BLV proviral load in the Holstein cow. The study examined 569 cows with a high proviral load and 341 cows with a low proviral load. Using the highest odds ratio (OR) as a comparison index, we confirmed that BoLA-DRB3 was the best marker for determining which cow spread the BLV (OR 13.9 for BoLA-DRB3, OR 11.5 for BoLA-DQA1, and OR 6.2 for BoLA class II haplotype). In addition, DRB3*002:01, *009:02, *012:01, *014:01, and *015:01 were determined as BLV provirus associated alleles. BoLA-DRB3*002:01, *009:02, and *014:01 were determined as resistant alleles (OR > 1), and BoLA-DRB3*012:01 and *015:01 were determined as susceptible alleles (OR

Objectives We have developed a portable system for the rapid determination of bacterial composition for the diagnosis of infectious diseases. Our system comprises of a nanopore technology‐based sequencer, MinION, and two laptop computers. To examine the accuracy and time efficiency of our system, we provided a proof‐of‐concept for the detection of the causative bacteria of 11 meningitis patients in Zambia. Methods We extracted DNA from cerebrospinal fluid samples of each patient and amplified the 16S rRNA gene regions. The sequencing library was prepared, and the sequenced reads were simultaneously processed for bacterial composition determination using the minimap2 software and the representative prokaryote genomes. Results The sequencing results of four of the six culture‐positive samples were consistent with those of conventional culture‐based methods. The dominant bacterial species in each of these samples were identified from the sequencing data within only 3 min. Although the major bacterial species were also detected from the other two culture‐positive samples and five culture‐negative samples, their presence could not be confirmed. Moreover, as a whole, although the number of sequencing reads obtained within a short sequencing run was small, there was no change in the major bacterial species over time with prolonged sequencing. In addition, the processing time strongly correlated with the number of sequencing reads used for the analysis. Conclusion Our results suggest that time‐effective analysis could be achieved by determining the number of sequencing reads required for the rapid diagnosis of infectious bacterial species depending on the complexity of bacterial species in a sample., This study used a portable system for rapid bacterial composition determination. The right PC connected to the MinION DNA sequencer obtains the DNA sequence, while the left PC analyzed the DNA sequence simultaneously.

We previously reported that oral low-dose acyclovir (200 mg/day) for the prevention of herpes simplex virus (HSV) infections after allogenic hematopoietic stem cell transplantation (HSCT) is effective without the emergence of acyclovir-resistant HSV infections. However, HSV infections are of significant concern because the number of allogeneic HSCT with T-cell depletion, which is a risk factor of the emergence of drug-resistant HSV infections, has been increasing. We experienced a 25-year-old female who received allogenic HSCT from an unrelated donor with 1-antigen mismatch using anti-thymocyte globulin. Despite acyclovir prophylaxis (200 mg/day), she developed the right palatal ulcer that was positive for HSV-1 specific antigen by fluorescent antibody on day 20 and developed new hypoglossal and tongue ulcers on day 33. Replacement of acyclovir with foscarnet improved her ulcers. We isolated 2 acyclovir-resistant and foscarnet-sensitive strains from the right palatal and hypoglossal ulcers, which had the same frame shift mutation in the thymidine kinase genes. The rate of proliferation of the isolate from the hypoglossal ulcer was faster than that from the right palatal ulcer in the plaque reduction assay. HSV strains that acquired acyclovir-resistant mutations at the right palatal ulcer with larger plaque might spread to the hypoglossal ulcer as the secondary site of infection because of better growth property. Second-line antiviral agents should be considered when we suspect treatment failure of HSV infection, especially in HSCT with T-cell depletion. Further studies are required whether low-dose acyclovir prophylaxis leads to the emergence of virological resistance.

The bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL) which is the most common neoplastic disease of cattle. Recent studies show bovine leukocyte antigen (BoLA)-DRB3 gene plays a direct role in controlling the number of proviral load in cattle and the homozygote BoLA-DRB3*1601 is associated with high proviral load. The other hand, the CD8+ cytotoxic T cell (CTL) response is an important defense against viral invasion. To find the candidate of vaccine for BLV infection we performed CD8+ T cell epitopes mapping in BLV-infected cattle with different BoLA-DRB3 alleles. In this study, 20 amino acids in length of 115 synthetic peptides were made from the sequence of Gag, Env and Tax protein of BLV, overlapping by 10 amino acids. Then total 11 CD8+ T cell epitopes were found out in BLV-infected four Japanese Black cattle with different BoLA-DRB3 alleles that are both of DRB3 *1601/1601, and DRB3 *1501/2703 and DRB3 *1501/0503 allele by WST assay. Different CD8+ T cell epitopes were recognized by cattle with different BoLA-DRB3 alleles and then dose-dependent cytotoxicity activity were measured by non-radioactive cytotoxicity assay. All of four cattle responded to gp51N11, but it has higher variability values by Wu-Kabat variability index analysis. By contrast, CD8+ T cell epitopes were determined from all of four cattle on gp30 region and they have high conservatives. gp30N16 among 4 epitopes was detected in each low proviral load cattle from susceptibility and neutrality groups. Those results were suggested that gp30 region maybe the best candidate for vaccine that can induce cell-mediated immunity against this disease.

Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (380 copies/1×10(5) cells) than that in the peripheral blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. However, there are no comprehensive studies on the distribution of BLV in the Philippines, and the genetic characteristics of Philippine BLV strains are unknown. Therefore, the aim of this study was to detect BLV infections in the Philippines and determined their genetic variability. Blood samples were obtained from 1116 cattle from different farms on five Philippine islands, and BLV provirus was detected by BLV-CoCoMo-qPCR-2 and nested PCR targeting BLV long terminal repeats. Out of 1116 samples, 108 (9.7 %) and 54 (4.8 %) were positive for BLV provirus, as determined by BLV-CoCoMo-qPCR-2 and nested PCR, respectively. Of the five islands, Luzon Island showed the highest prevalence of BLV infection (23.1 %). Partial env gp51 genes from 43 samples, which were positive for BLV provirus by both methods, were sequenced for phylogenetic analysis. Phylogenetic analysis based on a 423-bp fragment of the env gene revealed that Philippine BLV strains clustered into either genotype 1 or genotype 6. Substitutions were mainly found in antigenic determinants, such as the CD4(+) T-cell epitope, the CD8(+) T-cell epitope, the second neutralizing domain, B and E epitopes, and these substitutions varied according to genotype. This study provides comprehensive information regarding BLV infection levels in the Philippines and documents the presence of two BLV genotypes, genotypes 1 and 6, in this population.

Background Human respiratory syncytial virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and young children. However, molecular characteristic of HRSV is still unknown in the Philippines. Objective To describe the molecular epidemiology of circulating HRSV detected in the Philippines. Study design From May 2008 to April 2012, nasopharyngeal swabs were collected from infants and children aged between 7 days and 14 years who were hospitalized with severe pneumonia. HRSV was detected by nested PCR targeting M2 gene, and C-terminus of the G gene was sequenced for phylogenetic analysis. Result Out of total 2150 samples, 19.3% (n = 415) were positive for HRSV, and 65.0% of them (n = 270) were identified as HRSV-A and 35.0% (n = 145) as HRSV-B. There were two major HRSV outbreaks: between June 2008 and February 2009, and between June and March 2012. Majority of HRSV strains detected during the former outbreak were HRSV-A (97.5%, 203/208) whereas during the later outbreak, both HRSV-A (54/158, 34.2%) and HRSV-B (104/158, 65.8%) were detected. All HRSV-A strains were classified as genotype NA1 and all HRSV-B as genotype BA, which had 60-nucleotide duplication in secondary hypervariable region of the G gene. Among HRSV-B positive samples, there were 2 distinct clusters with unique amino acid changes and low homology in compared to other strains in BA, suggesting emergence of new variant of HRSV-B. Conclusion The study provides an overview of the genetic variation in circulating HRSV viruses in the Philippines along with identification of possibly a novel variant of HRSV-B.

Analyzing the evolutionary pattern of the influenza A(H1N1)pdm09 strain in different regions is important for understanding its diversification. We therefore conducted this study to elucidate the genetic variability and molecular evolution of the influenza A(H1N1)pdm09 strains that circulated during the 2009–2010 and 2010–2011 influenza seasons in Sendai, Japan. Nasopharyngeal swab specimens were collected from patients with influenza-like illnesses who visited outpatient clinics in Sendai City, Japan, from September 2009 to April 2011. A total of 75 isolates were selected from September 2009 to April 2011 to analyze the genetic changes in the entire hemagglutinin 1 (HA1) segment of the HA gene and the neuraminidase (NA) gene based on sequence analysis. Bayesian coalescent Markov chain Monte Carlo analyses of HA1 and NA gene sequences were performed for further analysis. High sequence identities were observed for HA1 and NA in influenza A(H1N1)pdm09, displaying 99.06 and 99.33 % nucleotide identities, respectively, with the A(H1N1)pdm09 vaccine strain A/California/07/2009. The substitution rates of nucleotides for HA1 in the 2009–2010 and 2010–2011 were 1.5 × 10−3 and 1.6 × 10−3 substitutions per site per year, respectively. Phylogenetic tree analysis demonstrated that Sendai isolates were clustered into global clade 7, which is characterized by an S203T mutation in the HA1 gene. Moreover, two distinct circulation clusters were present in the 2010–2011 season. Mutations were present in antigenic or receptor-binding domains of the HA1 segment, including A141V, S143G, S183P, S185T, and S203T. The Bayesian skyline plot model illustrated a steady rate for the maintenance of genetic diversity, followed by a slight increase in the later part of the 2010–2011 season. Selection analysis revealed that the HA1 (position 197) and NA (position 46) sites were under positive selection; however, no known mutation conferring resistance to NA inhibitors such as H275Y was observed. The effect on control of the influenza A(H1N1)pdm09 virus, including vaccine strain selection, requires continuous monitoring of the strain by genetic surveillance. Electronic supplementary material The online version of this article (doi:10.1007/s11262-013-0980-5) contains supplementary material, which is available to authorized users.

Bovine leukemia virus (BLV) is causative virus of enzootic bovine leukosis (EBL) that is malignant B lymphoma. BLV infected cattle to be asymptomatic carrier, persistent lymphocytosis (PL), and bovine leukosis. BLV is spreading over the world and causes serious problems. After infection, BLV genome integrated and can be amplified during latency periods. We designed degenerated primers by Coordination of Common Motifs (CoCoMo) algorism to establish a new quantitative real-time PCR method (BLV-CoCoMo-qPCR) that measure the proviral load of known and novel BLV variants. In present study, to analyze correlation between proviral load and risk factors such as breeds, numbers of lymphocytes, age and reproductive history, we collected blood and serum from 1,010 cattle in 44 BLV positive farms of the 22 prefectures in Japan, and measured BLV proviral load and detected anti-BLV antibody. The 682 of 1,010 samples (67.5%) were positive by BLV-CoCoMo-qPCR and ELISA, 22 of 1,010 samples (2.2%) were positive by only BLV-CoCoMo-qPCR, and 13 of 1,010 samples (1.3%) were positive by only ELISA. We detected BLV provirus from 22 samples that showed BLV negative by serological test, and average of proviral load was 31,533 copies. Therefore, BLV-CoCoMo-qPCR seems useful methods to determine BLV infection. It was not correlation between age and proviral load. Meanwhile, positive correlation showed between lymphocytes and proviral load. Therefore, we classified BLV provirus positive samples in three groups by disease progression of lymphocytosis (asymptomatic carrier, false positive and lymphocytosis) by age and numbers of lymphocytes, and compared proviral load between these groups. In fact, proviral load were increased according disease progression of lymphocytosis. Thus, the measurement of proviral load seems to be available for pathologic diagnosis of lymphocytosis.