Clinical characteristics, treatment, and prognosis of 74 2019 novel coronavirus disease patients in Hefei: A single-center retrospective study.

ABSTRACT: Since December 2019, pneumonia caused by a novel coronavirus (SARS-CoV-2), namely 2019 novel coronavirus disease (COVID-19), has rapidly spread from Wuhan city to other cities across China. The present study was designed to describe the epidemiology, clinical characteristics, treatment, and prognosis of 74 hospitalized patients with COVID-19.Clinical data of 74 COVID-19 patients were collected to analyze the epidemiological, demographic, laboratory, radiological, and treatment data. Thirty-two patients were followed up and tested for the presence of the viral nucleic acid and by pulmonary computed tomography (CT) scan at 7 and 14 days after they were discharged.Among all COVID-19 patients, the median incubation period for patients and the median period from symptom onset to admission was all 6 days; the median length of hospitalization was 13 days. Fever symptoms were presented in 83.78% of the patients, and the second most common symptom was cough (74.32%), followed by fatigue and expectoration (27.03%). Inflammatory indicators, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) of the intensive care unit (ICU) patients were significantly higher than that of the non-ICU patients (P < .05). However, 50.00% of the ICU patients had their the ratio of T helper cells to cytotoxic T cells (CD4/CD8) ratio lower than 1.1, whose proportion is much higher than that in non-ICU patients (P < .01).Compared with patients in Wuhan, COVID-19 patients in Anhui Province seemed to have milder symptoms of infection, suggesting that there may be some regional differences in the transmission of SARS-CoV-2 between different cities.

The impact of recent chemotherapy on immunity in 2 COVID-19 cases with gastrointestinal tumors: A case report.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a rapidly emerging infectious respiratory disease caused by severe acute respiratory syndrome coronavirus 2. Currently, more than 100 million cases of COVID-19 have been confirmed worldwide, with over 2.4 million mortalities. The pandemic affects people of all ages but older individuals and those with severe chronic illnesses, including cancer patients, are at higher risk. PATIENT CONCERNS: The impact of cancer treatment on the progression of COVID-19 is unclear. Therefore, we assessed the effects of chemotherapy on COVID-19 outcomes for 2 cancer patients. On January 24, 2020, a level I response to a major public health emergency was initiated in Hubei Province, China, which includes Enshi Autonomous Prefecture that has a population of 4.026 million people. As of April 30, 2020, 252 confirmed cases of COVID-19 and 11 asymptomatic carriers were identified in Enshi. DIAGNOSIS: Among the confirmed cases and asymptomatic carriers, 2 patients were identified who were previously diagnosed with malignant tumors, including one with hepatocellular carcinoma and the other with cardia carcinoma. INTERVENTIONS: These 2 patients were receiving or just completed chemotherapy at the time of their COVID-19 diagnosis. OUTCOMES: Both patients were followed and presented favorable outcomes. The positive outcomes for these 2 patients could be partially explained by their recent chemotherapy that impacted their immune status. Also, their relatively younger ages and lack of comorbidities were likely factors in their successful recovery from COVID-19. CONCLUSIONS: Anticancer treatment might enhance a patient's ability to respond favorably to COVID-19 infection. However, anticancer treatment is likely to impact immune function differently in different individuals, which can influence disease outcomes.

Detection of Enteric Viruses and Bacterial Indicators in a Sewage Treatment Center and Shallow Water Bay.

The incidence of enteric viruses in treated wastewater and their potential release into the environment or use for agriculture are very critical matters in public health. In our study, PCR (polymerase chain reaction) analysis of enteric viruses was performed on 59 samples of influents and effluents collected from Tubli wastewater treatment plant (Water Pollution Control Center (WPCC)) and Tubli Bay, where the effluents were discharged, in Kingdom of Bahrain during two sampling periods. Four clinically essential waterborne enteric viruses were examined: enterovirus (EV), hepatitis A virus (HAV), astroviruses (AV), and rotaviruses (RV) and compared to standard bacterial and bacteriophages indicators of fecal pollution. Detection rates of EV, AV, HAV, and RV in the influent samples were 100%, 75%, 12.5%, and 12.5%, respectively, while 50% of the effluent samples from Tubli WPCC contained only EV RNA. None of the tested enteric viruses could be detected in any of the samples collected directly from Tubli Bay. Effluent samples from Tubli plant did not show significant seasonal differences. Since detection of enteric viruses genome does not necessarily indicate infectivity, the infectivity of these viruses was evaluated through isolation and growth of indictor bacteria and bacteriophages. High concentration of fecal bacteriological indicators was detected in all effluents samples (100%): 3.20 × 103 cfu/mL for E. coli, 1.32 × 103 cfu/mL for Salmonella spp., and 1.92 × 103 cfu/mL for Shigella spp. E. coli and Salmonella specific bacteriophages were also detected in the effluent samples in high titers. The combined results of PCR and bacterial enumeration point to a probable public health risk via the use of these wastewaters in agriculture or their discharge into the sea. Continuous surveillance of viral and bacterial prevalence and their resistance to sewage disinfection procedures could contribute to a better control of risks associated with the recycling of effluent wastewater and its release into the environment.

Viral Hepatitis and Human Immunodeficiency Virus Testing and Linkage to Care for Individuals Enrolled in an Opioid Treatment Program.

BACKGROUND: In the United States, many opioid treatment programs (OTPs) do not offer viral hepatitis (VH) or human immunodeficiency virus (HIV) testing despite high prevalence among OTP clients. We initiated an opt-out VH and HIV testing and linkage-to-care program within our OTP. METHODS: All OTP intakes are screened for VH and HIV and evaluated for rescreening annually. A patient navigator reviews laboratory results and provides counseling in the OTP clinic. The medical record is queried to identify individuals with previously diagnosed, untreated VH or HIV. Navigation support is provided for linkage or relinkage to VH or HIV care. RESULTS: Between March 2018 and Februrary 2019, 532 individuals were screened for hepatitis C virus (HCV), 180 tested HCV antibody positive (34%), and 108 were HCV-ribonucleic acid (RNA) positive (20%). Sixty individuals were identified with previously diagnosed, untreated HCV. Of all HCV RNA+, 49% reported current injection drug use (82 of 168). Ninety-five individuals were seen by an HCV specialist (57% of HCV RNA+), 72 started treatment (43%), and 69 (41%) completed treatment. Individuals with primary care providers were most likely to start treatment. Four individuals were diagnosed with hepatitis B; 0 were diagnosed with HIV. CONCLUSIONS: The implementation of an OTP-based screening and navigation protocol has enabled significant gains in the identification and treatment of VH in this high prevalence setting.

The Application of Single-Cell RNA Sequencing in Vaccinology.

Single-cell RNA sequencing allows highly detailed profiling of cellular immune responses from limited-volume samples, advancing prospects of a new era of systems immunology. The power of single-cell RNA sequencing offers various opportunities to decipher the immune response to infectious diseases and vaccines. Here, we describe the potential uses of single-cell RNA sequencing methods in prophylactic vaccine development, concentrating on infectious diseases including COVID-19. Using examples from several diseases, we review how single-cell RNA sequencing has been used to evaluate the immunological response to different vaccine platforms and regimens. By highlighting published and unpublished single-cell RNA sequencing studies relevant to vaccinology, we discuss some general considerations how the field could be enriched with the widespread adoption of this technology.

Clinical display, diagnostics and genetic implication of novel Coronavirus (COVID-19) epidemic.

COVID-19 pandemic can cause irreparable damage to the involved society. This study aimed to provide a summary of the up-to-dated clinical display, diagnostics, molecular and genetic implications for COVID-19 infected patients. In this review, 73 research articles published before 25 March 2020 were analyzed to better understand the clinical characteristics of patients and to introduce the available serological, hematology and molecular diagnostic methods. Apart from articles extracted from PubMed and Google Scholar, WHO (https://www.who.int/), NHC (National Health Commission of the People's Republic of China (http://www.nhc.gov.cn/), NICE (National Institute for Health and Clinical Excellence, https://www.nice.org.uk/), CDC (Centers for Disease Control and Prevention, https://www.cdc.gov/), and National Administration of Traditional Chinese Medicine (http://www.satcm.gov.cn/) were also accessed to search for eligible studies. Papers published between January 1, 2020, and 25 March 2020 were searched in English and the terms "2019-nCoV, Covid-19, Clinical Characteristics OR manifestation, method of detection, COVID-19 Genome and molecular test" were used. As the pandemic continues to evolve, there have been reports about the possibility of asymptomatic transmission of this newly emerged pneumonia virus. We highlighted the role of HLA haplotype in virus infection as HLA typing will provide susceptibility information for personalized prevention, diagnosis, and treatment in future studies. All the data in this article will assist researchers and clinicians to develop their clinical views regarding infected patients and to emphasize the origin of SARS-CoV-2 for diagnostics.

A report of clinical diagnosis and treatment of nine cases of coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 has become an important public health issue in the world. More than 118 000 cases were confirmed around the world. The main clinical manifestations were respiratory symptoms and occasional gastrointestinal symptoms. However, there is no unified standard for the diagnosis and treatment of COVID-19. In the retrospective analysis, we report nine cases of COVID-19, describe the history of contact, clinical manifestations, the course of diagnosis and clinical treatment before, during and after treatment.

Activation of the Liver X Receptor Pathway Inhibits HBV Replication in Primary Human Hepatocytes.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is ranked among the top health priorities worldwide. Accumulating evidence suggests that HBV infection and replication are closely associated with liver metabolism. The liver X receptors (LXRs), which belong to the superfamily of nuclear hormone receptors, are important physiological regulators of lipid and cholesterol metabolism. However, the association between the LXR pathway and HBV infection remains largely unclear. APPROACH AND RESULTS: In this study, the antiviral activity of LXR agonists was investigated using multiple HBV cellular models. We observed that in HBV-infected primary human hepatocytes (PHHs), synthetic LXR agonists (T0901317, GW3965, and LXR-623), but not an LXR antagonist (SR9238), potently inhibited HBV replication and gene expression, as demonstrated by substantial reductions in viral RNA, DNA, and antigen production following agonist treatment. However, covalently closed circular DNA (cccDNA) levels were not significantly reduced by the agonists. In addition, no rebound in viral replication was observed after treatment withdrawal, indicating a long-lasting inhibitory effect. These results suggest that LXR agonists decrease the transcriptional activity of cccDNA. In contrast, no significant anti-HBV effect was observed in HepG2-derived cell lines. Interestingly, LXR agonist treatment strongly reduced cholesterol 7α-hydroxylase 1 (CYP7A1) mRNA levels. Knockdown of CYP7A1 gene expression with small interfering RNA inhibited HBV activity in PHHs, suggesting CYP7A1 as a potential factor contributing to the antiviral effects of LXR agonists. CONCLUSIONS: We found that activation of the LXR pathway with synthetic LXR agonists could elicit potent anti-HBV activity in PHHs, possibly through sustained suppression of cccDNA transcription. Our work highlights the therapeutic potential of targeting the LXR pathway for the treatment of chronic HBV infection.

Resveratrol affects Zika virus replication in vitro.

Zika virus (ZIKV) infection is a serious public health concern. ZIKV infection has been associated with increased occurrences of microcephaly among newborns and incidences of Guillain-Barré syndrome among adults. No specific therapeutics or vaccines are currently available to treat and protect against ZIKV infection. Here, a plant-secreted phytoalexin, resveratrol (RES), was investigated for its ability to inhibit ZIKV replication in vitro. Several RES treatment regimens were used. The ZIKV titers of mock- and RES-treated infected cell cultures were determined using the focus-forming assay and the Zika mRNA copy number as determined using qRT-PCR. Our results suggested that RES treatment reduced ZIKV titers in a dose-dependent manner. A reduction of >90% of virus titer and ZIKV mRNA copy number was achieved when infected cells were treated with 80 µM of RES post-infection. Pre-incubation of the virus with 80 µM RES showed >30% reduction in ZIKV titers and ZIKV mRNA copy number, implying potential direct virucidal effects of RES against the virus. The RES treatment reduced >70% virus titer in the anti-adsorption assay, suggesting the possibility that RES also interferes with ZIKV binding. However, there was no significant decrease in ZIKV titer when a short-period of RES treatment was applied to cells before ZIKV infection (pre-infection) and after the virus bound to the cells (virus internalization inhibition), implying that RES acts through its continuous presence in the cell cultures after virus infection. Overall, our results suggested that RES exhibited direct virucidal activity against ZIKV and possessed anti-ZIKV replication properties, highlighting the need for further exploration of RES as a potential antiviral molecule against ZIKV infection.

Development of a reverse transcription-loop- mediated isothermal amplification (LAMP) assay for the rapid detection of onion yellow dwarf virus.

Onion yellow dwarf virus (OYDV) is one of the most important viral pathogens of onion. In particular, on 'Rossa di Tropea' onion, granted with Protected Geographical Indication (PGI) trademarks, this pathogen represents the most limiting biotic stress in terms of spread, severity of symptoms and damage, and its detection is necessary to preserve high quality standards and avoid yield losses. A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed for detection of OYDV. The specificity, sensitivity, repeatability and reproducibility of the assay were validated according to EPPO standard PM7/98 (2). Diagnostic specificity, diagnostic sensitivity and diagnostic accuracy were determined in both leaf and bulb tissues. To enhance the feasibility of a LAMP-based method for field diagnosis, several nucleic acid extraction methods were compared to simplify sample preparation. The results showed the reliability of the method for OYDV detection, with a limit of detection (LOD) comparable to real time reverse transcription polymerase chain reaction (RT-qPCR). The ease of sample preparation, and the more than acceptable LOD, indicated that the RT-LAMP assay could be used in plant pathology laboratories with limited facilities and resources, as well as directly in the field. This work was carried out in the frame of "SI.ORTO" project.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Efficacy of Combination Treatment with Sodium Metasilicate and Sodium Hypochlorite for Inactivation of Norovirus on Fresh Vegetables.

In recent years, fresh vegetables have frequently been associated with the foodborne transmission of enteric viruses, such as human norovirus (NoV). Therefore, several studies have focused on developing methods to inactivate foodborne viruses for preventing outbreaks of foodborne illnesses. Sodium hypochlorite (NaOCl) is commonly used as a disinfectant, but results in undesirable effects on the appearance and taste of foods and can generate toxic byproducts when it exceeds the allowable concentration. Here, we evaluated the efficacy of a range of NaOCl concentrations (50-1000 ppm) for reducing the amounts of human NoV (NoV GII.4) on lettuce (Lactuca sativa), celery (Apium graveolens L.), and white cabbage (Brassica oleracea ssp. capitata). In addition, the combination treatment of NaOCl and sodium metasilicate (SMS, 0.1-0.5%) pentahydrate was evaluated for its ability to decrease the populations of NoV GII.4 in the three food samples. An immunomagnetic separation procedure combined with reverse transcription quantitative polymerase chain reaction was used for virus detection. For lettuce, celery, and cabbage, the NoV GII.4 recovery rates were 57.3% ± 6.5%, 52.5% ± 1.7%, and 60.3% ± 3.9%, respectively, using a glycine/NaCl elution buffer (0.25 M glycine/0.14 M NaCl, pH 9.5). The reductions of NoV GII.4 were 3.17, 3.06, and 3.27 log10 genomic copies/µL for lettuce, celery, and cabbage, respectively, at 1000 ppm NaOCl, while a reduction of ∼3 log10 genomic copies/µL was obtained when the samples were treated with a combination of 100 ppm NaOCl and 0.4% SMS pentahydrate. Taken together, these results demonstrated that combined treatment with NaOCl and SMS pentahydrate was an efficient strategy to reduce the concentration of NaOCl for control of NoV GII.4 contamination in fresh vegetables.

Development of a fast and efficient method for hepatitis A virus concentration from green onion.

Hepatitis A virus (HAV) can cause serious liver disease and even death. HAV outbreaks are associated with the consumption of raw or minimally processed produce, making it a major public health concern. Infections have occurred despite the fact that effective HAV vaccine has been available. Development of a rapid and sensitive HAV detection method is necessary for an investigation of an HAV outbreak. Detection of HAV is complicated by the lack of a reliable culture method. In addition, due to the low infectious dose of HAV, these methods must be very sensitive. Current methods rely on efficient sample preparation and concentration steps followed by sensitive molecular detection techniques. Using green onions which was involved in most recent HAV outbreaks as a representative produce, a method of capturing virus particles was developed using carboxyl-derivatized magnetic beads in this study. Carboxyl beads, like antibody-coated beads or cationic beads, detect HAV at a level as low as 100 pfu/25g of green onions. RNA from virus concentrated in this manner can be released by heat-shock (98°C 5min) for molecular detection without sacrificing sensitivity. Bypassing the RNA extraction procedure saves time and removes multiple manipulation steps, which makes large scale HAV screening possible. In addition, the inclusion of beef extract and pectinase rather than NP40 in the elution buffer improved the HAV liberation from the food matrix over current methods by nearly 10 fold. The method proposed in this study provides a promising tool to improve food risk assessment and protect public health.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes.

AIM: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on polyinosinic-polycytidylic acid (poly I:C)-triggered intracellular innate immunity against hepatitis C virus (HCV) in hepatocytes. METHODS: A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain (JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular mRNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon (IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells. RESULTS: Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFN-stimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION: Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Sensitivity enhancement of graphene/zinc oxide nanocomposite-based electrochemical impedance genosensor for single stranded RNA detection.

An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10-11M to 1.0×10-6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10-12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization.

Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection.

Standard molecular methods for plant virus diagnosis require the purification of RNA or DNA extracts from a large number of samples, with sufficient concentration and quality for their use in PCR, RT-PCR, or qPCR analysis. Most methods are laborious and use either hazardous and/or costly chemicals. A previously published protocol for RNA isolation from several plant species yields high amounts of good quality RNA-DNA mixture in a simple, safe and inexpensive manner. In the present work, this method was tested to obtain RNA-DNA extracts from leaves of tomato, potato and three species of citrus, and was compared with two commercial kits. The results demonstrated that this protocol offers at least comparable nucleic acid quality, quantity and purity to those provided by commercial phenol-based or spin column systems and that are suitable to be used in PCR, RT-PCR and qPCR for virus and viroid detection. Because of its easy implementation and the use of safe and inexpensive reagents, it can be easily implemented to work in plant virus and viroid detection in different plant species.

An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies.

A rabies vaccine adjuvanted with saponins from leaves of the soap tree (Quillaja brasiliensis) induces specific immune responses and protects against lethal challenge.

Quillaja brasiliensis (Quillajaceae) is a saponin producing species native from southern Brazil and Uruguay. Its saponins are remarkably similar to those of Q. saponaria, which provides most of the saponins used as immunoadjuvants in vaccines. The immunostimulating capacities of aqueous extract (AE) and purified saponin fraction (QB-90) obtained from leaves of Q. brasiliensis were favorably comparable to those of a commercial saponin-based adjuvant preparation (Quil-A) in experimental vaccines against bovine herpesvirus type 1 and 5, poliovirus and bovine viral diarrhea virus in mice model. Herein, the immunogenicity and protection efficacy of rabies vaccines adjuvanted with Q. brasiliensis AE and its saponin fractions were compared with vaccines adjuvanted with either commercial Quil-A or Alum. Mice were vaccinated with one or two doses (on days 0 and 14) of one of the different vaccines and serum levels of total IgG, IgG1 and IgG2a were quantified over time. A challenge experiment with a lethal dose of rabies virus was carried out with the formulations. Viral RNA detection in the brain of mice was performed by qPCR, and RNA copy-numbers were quantified using a standard curve of in vitro transcribed RNA. All Q. brasiliensis saponin-adjuvanted vaccines significantly enhanced levels of specific IgG isotypes when compared with the no adjuvant group (P ≤ 0.05). Overall, one or two doses of saponin-based vaccine were efficient to protect against the lethal rabies exposure. Both AE and saponin fractions from Q. brasiliensis leaves proved potent immunological adjuvants in vaccines against a lethal challenge with a major livestock pathogen, hence confirming their value as competitive or complementary sustainable alternatives to saponins of Q. saponaria.

A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.

Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Maori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.