Selective ablation of 3' RNA ends and processive RTs facilitate direct cDNA sequencing of full-length host cell and viral transcripts.

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3' RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

Investigation of the immunogenicity of Zika glycan loop.

BACKGROUND: Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus. Previous studies have identified neutralizing antibodies from Zika patients that bind to quaternary epitopes across neighboring envelope (E) proteins, called E dimer epitopes (EDE). An asparagine-linked glycan on the "glycan loop" (GL) of the ZIKV envelope protein protects the functionally important "fusion loop" on the opposite E subunit in the dimer, and EDE antibodies have been shown to bind to both of these loops. Human EDE antibodies have been divided into two subclasses based on how they bind to the glycan loop region: EDE1 antibodies do not require glycosylation for binding, while EDE2 antibodies strongly rely on the glycan for binding. METHODS: ZIKV GL was expressed on tobacco mosaic virus nanoparticles. Mice were immunized with GL or full-length monomeric E and the immune response was analyzed by testing the ability of sera and monoclonal antibodies to bind to GL and to neutralize ZIKV in in vitro cellular assay. RESULTS: We report here the existence of ZIKV moderately neutralizing antibodies that bind to E monomers through epitopes that include the glycan loop. We show that sera from human Zika patients contain antibodies capable of binding to the unglycosylated glycan loop in the absence of the rest of the envelope protein. Furthermore, mice were inoculated with recombinant E monomers and produced neutralizing antibodies that either recognize unglycosylated glycan loop or require glycan for their binding to monomeric E. We demonstrate that both types of antibodies neutralize ZIKV to some extent in a cellular virus neutralization assay. CONCLUSIONS: Analogous to the existing EDE antibody nomenclature, we propose a new classification for antibodies that bind to E monomer epitopes (EME): EME1 and EME2 for those that do not require and those that do require glycan for binding to E, respectively.

Intra thyroid thymic carcinoma: A case report and literature review.

INTRODUCTION AND IMPORTANCE: Intrathyroid thymic carcinoma (ITC) is a malignant epithelial tumor with thymic differentiation within the thyroid gland. Its frequency is up to 0.15 % of all malignant thyroid tumors. It is frequently a low-grade tumor. The clinical status is often misleading to other more advanced tumors like cervical lymph node metastasis of nonkeratinizing squamous cell carcinoma, undifferentiated variant, dedifferentiated carcinoma, and medullary carcinoma of the thyroid. CASE PREPARATION: The patient came to us with the diagnosis of cervical lymph node metastasis of undifferentiated carcinoma. This patient was first diagnosed with cervical lymph node metastasis in the previous hospital. After having an ITC diagnosis, the patient was operated on the rennet of thyroid glands and had a low dose of radio-chemotherapy for recurrent prevention purposes. It is the first case of such a disease diagnosed at our hospital and also the first case reported in Vietnam. CLINICAL DISCUSSION: ITC is rare and appears similar to all thymic carcinoma variants. The most popular type is squamous carcinoma. Immunohistochemical stains are typical for thymic origin tumors with CD5, CD117 positive. ITC is often negative for monoclonal PAX8 but positive in this case (MRQ-50 clone, Sigma-Aldrich). This finding is an exciting one that should considered. CONCLUSION: Reporting the case increases the awareness of the disease, especially among Vietnam Doctors and patients.

Widespread distribution of phthalic acid esters in indoor and ambient air samples collected from Hanoi, Vietnam.

In the present study, distribution characteristics of ten typical phthalic acid esters (PAEs) were investigated in 90 air samples collected from the urban areas in Hanoi, Vietnam from May to August 2022. The total concentrations of PAEs in indoor and ambient air samples were in the range of 320-4770 ng/m3 and 35.9-133 ng/m3, respectively. Total concentrations of PAEs in indoor air were about one order of magnitude higher than those in ambient air. Among PAEs studied, di-(2-ethyl)hexyl phthalate (DEHP) was measured at the highest levels in all air samples, followed by di-n-octyl phthalate (DnOP) and di-n-butyl phthalate (DnBP). The PAEs concentrations in air samples collected from laboratories at nighttime were significantly higher than those during daytime (p < 0.05). Meanwhile, the distributions of PAEs in various micro-environments in the same house are no statistically significant difference. The median exposure doses of PAEs through inhalation for adults and children were 248 and 725 ng/kg-bw/d, respectively. These exposure levels were still lower than the respective reference doses (RfD) proposed by the US EPA for selected compounds such as diethyl phthalate (DEP), DnBP, and DEHP.

The first newborn patient with SARS-CoV-2 variant B.1.1.7 identified in Viet Nam: treatment and care practices.

SARS-CoV-2 variant B.1.1.7, first detected in September 2020 in the United Kingdom of Great Britain and Northern Ireland, has spread quickly to many countries around the world. While some publications have described the clinical features of adult patients with the B.1.1.7 variant, little information is available on newborn patients. We report the clinical characteristics, treatment and care practices for a 21-day-old newborn patient who was confirmed to be infected with SARS-CoV-2 variant B.1.1.7 in Viet Nam during contact tracing after her father was confirmed to be infected with SARS-CoV-2. The patient displayed no symptoms of COVID-19 on admission but 3 days later developed diarrhoea, vomiting, a runny nose and a productive cough. These symptoms lasted for 3 days before becoming milder for 1 day and then stopping until discharge. During treatment, the patient received Vietnamese traditional herbal peppermint extracts for cough and digestive probiotics for diarrhoeal symptoms. A saltwater solution (Sterimar 0.9%) was used to clean the patient's sinuses. The patient was cared for and fed breastmilk by her mother, who was provided with personal protective equipment, including sterilized infant equipment, medical masks and hand sanitizer, during hospitalization. The patient's mother tested negative for SARS-CoV-2 throughout hospitalization. In conclusion, we found no severely abnormal clinical symptoms in a newborn infected with SARS-CoV-2 variant B.1.1.7 during treatment. Our case suggests that newborn patients with the B.1.1.7 variant can receive exclusive breastmilk feeding if sufficient preventive measures are provided for both mother and child.

Profiles of phthalic acid esters (PAEs) in bottled water, tap water, lake water, and wastewater samples collected from Hanoi, Vietnam.

Contamination levels and distribution patterns of ten typical phthalic acid esters (PAEs) were investigated in various types of water samples collected from Hanoi metropolitan area in Vietnam. Concentrations of 10 PAEs in bottled water, tap water, lake water, and wastewater samples were measured in the ranges of 1640-15,700 ng/L (mean/median: 6400/5820 ng/L), 2100-18,000 ng/L (mean/median: 11,200/9270 ng/L), 19,600-127,000 ng/L (mean/median: 51,800/49,300 ng/L), and 20,700-405,000 ng/L (mean/median: 121,000/115,000 ng/L), respectively. Among PAEs, di-(2-ethylhexyl) phthalate (DEHP) accounted for a major proportion of total concentrations (45%) in wastewater, followed by diisobutyl phthalate (DiBP, 10.3%), and dibutyl phthalate (DBP, 9.53%). Concentrations of PAEs in wastewater decreased significantly with distance from the wastewater treatment plants (WWTPs). Concentrations of PAEs in surface water samples did not vary greatly between locations. PAEs were found in bottled water in the following order: DBP (22.4%), DiBP (22.3%), benzylbutyl phthalate (BzBP, 20.1%), and DEHP (15.5%). The estimated mean exposure doses of 10 PAEs through consumption of drinking water for adults and children in Vietnam were 254 and 256 ng/kg-bw/day, respectively. Capsule: Highest concentrations of PAEs were measured in wastewater, followed by lake water, tap water, and bottled water.

Whole Genome Sequencing of Dengue Virus Serotype 2 from Two Clinical Isolates and Serological Profile of Dengue in the 2015-2016 Nepal Outbreak.

Dengue virus (DENV) is the cause of one of the most prevalent neglected tropical diseases, and up to half of the world's population is at risk for infection. Recent results from clinical trials have shown that DENV vaccination can induce the development of severe dengue disease and/or prolong hospitalization after natural infection in certain naive populations. Thus, it is crucial that vaccine development takes into account the history of DENV exposure in the targeted population. In Nepal, DENV infection was first documented in 2004, and despite the increasing prevalence of DENV infection, the population remains relatively naive. However, it is not known which of the four DENV serotypes circulate in Nepal or whether there is evidence of repeated exposure to DENV in the Nepali population. To address this, we studied 112 patients who presented with symptomology suspicious for DENV infection at clinics throughout Nepal during late 2015 and early 2016. Of the 112 patients examined, 39 showed serological and/or genetic evidence of primary or secondary DENV infection: 30 were positive for DENV exposure by IgM/IgG ELISA, two by real-time reverse-transcription PCR (RT-PCR), and seven by both methods. Dengue virus 1-3, but not DENV4, serotypes were detected by RT-PCR. Whole genome sequencing of two DENV2 strains isolated from patients with primary and secondary infections suggests that DENV was introduced into Nepal through India, with which it shares a porous border. Further study is needed to better define the DENV epidemic in Nepal, a country with limited scientific resources and infrastructure.

Sensitive detection of influenza a virus based on a CdSe/CdS/ZnS quantum dot-linked rapid fluorescent immunochromatographic test.

Prevention is the most effective management strategy for influenza A infection in humans. In this study, we developed a CdSe/CdS/ZnS quantum dot (QD) fluorescent dye for rapid and sensitive detection of two common subtypes (H1N1 and H3N2) of influenza A virus, and examined its utility. CdSe/CdS/ZnS QD was conjugated with antibody (Ab) after conjugation with latex, making QD conjugate of QD + Latex + Ab. A stable photoluminescence of QD conjugate and advantage of CdSe/CdS/ZnS QD used was characterized in this study. The performance of a rapid fluorescent immunochromatographic test (FICT) employing QD conjugate (QD-FICT) in detecting influenza A/H1N1 was 8-fold and 64-fold higher than that of a europium nanoparticle-based FICT and a rapid diagnostic test (RDT; Standard Diagnostics BIOLINE Influenza A/B), respectively. For influenza A/H3N2, QD-FICT showed 8-fold and 128-fold higher performance than europium nanoparticle-based FICT and RDT, respectively. In clinical evaluations, QD-FICT showed 93.75% clinical sensitivity [45/48; 95% confidence interval (95% CI): 82.80-98.69], 100% clinical specificity (117/117; 95% CI: 96.90-100.00), and strong correlation (kappa; 0.98) with rRT-PCR (20 ≤ Ct ≤ 40). Europium nanoparticle-linked FICT showed 79.17% clinical sensitivity (38/48; 95% CI: 65.01-89.53) and 100% clinical specificity (117/117; 95% CI: 96.90-100.00), whereas RDT showed 77.08% sensitivity (37/48; 95% CI: 62.69-87.97), 100% specificity (117/117; 95% CI: 96.90-100.00), and reasonably good correlation with rRT-PCR (kappa; 0.93). Water-soluble QDs can therefore be used as an effective material for developing fluorescent diagnostic systems for rapid detection of human influenza A virus in clinical specimens.

Thoracoscopic surgery approach to mediastinal mature teratomas: a single-center experience.

BACKGROUND: Mediastinal mature teratomas are rare tumors with diverse surgical approaches. The aim of this study is to review our experience of thoracoscopic surgery management in patients with teratomas. METHODS: We retrospectively reviewed 28 consecutive patients with mediastinal mature teratomas who underwent thoracoscopic surgery at Viet Duc University Hospital from January 2008 to August2018. Patients were divided into 2 groups with 2 types of thoracoscopic surgery, closed thoracoscopic surgery (CTS) group and video-assisted thoracoscopic surgery (VATS) group. The selection of sugical approach was based on sizes, locations and characteristics of tumors. Post-operative outcomes were assessed and compared between these 2 groups. RESULTS: There were 14 female and 14 male patients with a median age of 41.2 ± 13.8 years. A total of 22 teratomas were located on the right side of the chest cavity and 6 on the left side. We performed CTS in 21 patients (75%) and VATS in 7 patients (25%) for tumor resection. There were 3 cases (10.7%) required conversion to minithoracotomy (5 cm in incision length). Skin appendages accounted for the highest rate (96.4%) in pathology. There was no record of mortality or tumor recurrence detected by computerized tomography. CONCLUSION: A thoracoscopic surgery for a mediastinal mature teratoma was a feasible choice. Challenging factors such as large tumors, intraoperative bleeding and strong tumor cell adhesion were considered handling by conversion to mini-thoracotomy that could ensure safety procedures and complete removal of tumors. Extraction of tumor contents might be performed for patients with large mature cystic teratomas to facilitate thoracoscopic surgery.

CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.

Human Polyclonal Antibodies Prevent Lethal Zika Virus Infection in Mice.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that represents a major threat to global health. ZIKV infections in adults are generally asymptomatic or present with mild symptoms. However, recent outbreaks of ZIKV have revealed that it can cause Congenital Zika Syndrome in neonates and Guillain-Barré syndrome in adults. Currently, no ZIKV-specific vaccines or antiviral treatments are available. In this study, we tested the efficacy of convalescent plasma IgG hyperimmune product (ZIKV-IG) isolated from individuals with high neutralizing anti-ZIKV titers as a therapeutic candidate against ZIKV infection using a model of ZIKV infection in Ifnar1-/- mice. ZIKV-IG successfully protected mice from lethal ZIKV challenge. In particular, ZIKV-IG treatment at 24 hours after lethal ZIKV infection improved survival by reducing weight loss and tissue viral burden and improving clinical score. Additionally, ZIKV-IG eliminated ZIKV-induced tissue damage and inflammation in the brain and liver. These results indicate that ZIKV-IG is efficacious against ZIKV, suggesting this human polyclonal antibody is a viable candidate for further development as a treatment against human ZIKV infection.

Synergism between the tyrosine kinase inhibitor sunitinib and Anti-TNF antibody protects against lethal dengue infection.

Dengue virus (DENV) currently circulates in more than 100 countries and causes an estimated 390 million infections per year. While most cases manifest as a self-resolving fever, ∼1.5% of infections develop into a more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which causes ∼20,000 deaths annually. The underlying pathological feature of DHF/DSS, also known as Severe Dengue, is an acute increase in vascular permeability leading to hypovolemia and shock. Angiogenic factors and cytokines, such as vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF), have been implicated in the increased vascular permeability, suggesting a potential therapeutic strategy for Severe Dengue. Here, we employed a mouse model of antibody-dependent enhancement of DENV infection, which recapitulates the fatal capillary leakage and shock of human Severe Dengue, to investigate the effects of approved VEGF- and TNF-targeting drugs. DENV infection caused a significant increase in serum VEGF levels within 2 days and resulted in ∼80% mortality within 8 days of infection. Treatment of mice with sunitinib, a VEGF receptor tyrosine kinase inhibitor, once (day 2) or twice (days 1 and 2) post-infection reduced mortality by 50-80% compared with untreated mice. Notably, sunitinib treatment decreased serum TNF levels, white blood cell counts, and hematocrit levels relative to untreated mice, but had only marginal effects on tissue viral burden. Combination therapy with anti-TNF antibody and sunitinib significantly reduced vascular leakage and synergized to provide superior protection from lethal DENV infection compared with either agent alone. These data suggest that a two-pronged anti-angiogenic and anti-inflammatory approach may be useful for the rapid treatment of DHF/DSS.

Cross-reactive Dengue virus-specific CD8+ T cells protect against Zika virus during pregnancy.

As Zika virus (ZIKV) emerges into Dengue virus (DENV)-endemic areas, cases of ZIKV infection in DENV-immune pregnant women may rise. Here we show that prior DENV immunity affects maternal and fetal ZIKV infection in pregnancy using sequential DENV and ZIKV infection models. Fetuses in ZIKV-infected DENV-immune dams were normal sized, whereas fetal demise occurred in non-immune dams. Moreover, reduced ZIKV RNA is present in the placenta and fetuses of ZIKV-infected DENV-immune dams. DENV cross-reactive CD8+ T cells expand in the maternal spleen and decidua of ZIKV-infected dams, their depletion increases ZIKV infection in the placenta and fetus, and results in fetal demise. The inducement of cross-reactive CD8+ T cells via peptide immunization or adoptive transfer results in decreased ZIKV infection in the placenta. Prior DENV immunity can protect against ZIKV infection during pregnancy in mice, and CD8+ T cells are sufficient for this cross-protection. This has implications for understanding the natural history of ZIKV in DENV-endemic areas and the development of optimal ZIKV vaccines.