Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection

Bovine viral diarrhea virus (BVDV) infects cattle and may lead to persistent infection (PI). The PI animals harbor BVDV throughout their life and become immune tolerant against BVDV. Thus, diagnosis of this virus in herd is highly important. Recombinant E2 protein expression (using pET-32a in Escherichia coli) was confirmed by SDS-PAGE and Western blotting; then purified by Ni⁺ affinity chromatography. Chickens were immunized with BVDV-E2 protein, and IgY antibodies were extracted from egg yolk by PEG-6000. The peak titer of anti-BVDV-E2-IgY was 1:128,000 after the fifth immunization. IgY-based enzyme-linked immuno sorbent assay (ELISA) and immunochromatographic assay (ICA) were further developed. Coincidence of ELISA and ICA test with RT-PCR was 95.45 and 90.91%, respectively. The anti-BVDV-E2 IgY could be used in routine screening of BVDV infection. Besides, it can also be applicable while licensing and/or using live vaccines; screening of imported products containing bovine serum and strong surveillance of BVDV outbreaks.     

Cytokines and persistent viral infections

Intracellular pathogens such as the human immunodeficiency virus, hepatitis C and B or Epstein–Barr virus often cause chronic viral infections in humans. Persistence of these viruses in the host is associated with a dramatic loss of T-cell immune response due to functional T-cell exhaustion. Developing efficient immunotherapeutic approaches to prevent viral persistence and/or to restore a highly functional T-cell mediated immunity remains a major challenge. During the last two decades, numerous studies aimed to identify relevant host-derived factors that could be modulated to achieve this goal. In this review, we focus on recent advances in our understanding of the role of cytokines in preventing or facilitating viral persistence. We concentrate on the impact of multiple relevant cytokines in T-cell dependent immune response to chronic viral infection and the potential for using cytokines as therapeutic agents in mice and humans.     

Cold chain and virus‐free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes

To prevent vaccine‐associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype‐2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus‐free and cold chain‐free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low‐cost, cold chain/poliovirus‐free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non‐toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site‐specific integration of CTB‐VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9–15‐fold in chloroplasts. GM1‐ganglioside receptor‐binding ELISA confirmed pentamer assembly of CTB‐VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1‐VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB‐VP1co, formulated with plant‐derived oral adjuvants, enhanced VP1‐specific IgG1, VP1‐IgA titres and neutralization (80%–100% seropositivity of Sabin‐1, 2, 3). In contrast, IPV single dose resulted in <50% VP1‐IgG1 and negligible VP1‐IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB‐VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low‐cost solution to eradicate polio.     

CRISPR/Cas系统在抗病毒研究中的应用

近年来,CRISPR/Cas系统因其效率高、靶向性强、易操作等优势,已被广泛应用于多种病毒研究中。本文首先简单介绍了CRISPR/Cas系统的分类,并比较了Cas9和Cas12a与Cas13a的特点;其次重点介绍了CRISPR/Cas9通过靶向破坏病毒基因组,或编辑宿主关于病毒生命周期的关键因子的策略在抗病毒方面的各种应用,CRISPR/Cas13a采用靶向破坏病毒基因组方法在抗病毒中的应用,以及CRISPR/Cas12a和CRISPR/Cas13a在病毒基因检测中的应用。最后讨论了CRISPR/Cas在病毒研究中面临的挑战,并讨论了CRISPR/Cas12a作为抗病毒工具的潜在应用前景。由于CRISPR/Cas系统自身的优势,预计该系统将会给病毒相关的疾病诊断和控制带来革命性的变化。     

Continuous Solvent/Detergent Virus Inactivation Using a Packed‐Bed Reactor

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two‐fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in‐line mixer and a packed‐bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in‐line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed‐bed reactor for continuous VI unites fully continuous processing with very low‐pressure drop and scalability.     

Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

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Virus-PLoc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells

Viruses can reproduce their progenies only within a host cell, and their actions depend both on its destructive tendencies toward a specific host cell and on environmental conditions. Therefore, knowledge of the subcellular localization of viral proteins in a host cell or virus-infected cell is very useful for in-depth studying of their functions and mechanisms as well as designing antiviral drugs. An analysis on the Swiss-Prot database (version 50.0, released on May 30, 2006) indicates that only 23.5% of viral protein entries are annotated for their subcellular locations in this regard. As for the gene ontology database, the corresponding percentage is 23.8%. Such a gap calls for the development of high throughput tools for timely annotating the localization of viral proteins within host and virus-infected cells. In this article, a predictor called "Virus-PLoc" has been developed that is featured by fusing many basic classifiers with each engineered according to the K-nearest neighbor rule. The overall jackknife success rate obtained by Virus-PLoc in identifying the subcellular compartments of viral proteins was 80% for a benchmark dataset in which none of proteins has more than 25% sequence identity to any other in a same location site. Virus-PLoc will be freely available as a web-server at http://202.120.37.186/bioinf/virus for the public usage. Furthermore, Virus-PLoc has been used to provide large-scale predictions of all viral protein entries in Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The results thus obtained have been deposited in a downloadable file prepared with Microsoft Excel and named "Tab_Virus-PLoc.xls." This file is available at the same website and will be updated twice a year to include the new entries of viral proteins and reflect the continuous development of Virus-PLoc.     

Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles

We describe the use of quantitative PCR (QPCR) to titer recombinant baculoviruses. Custom primers and probe were designed to gp64 and used to calculate a standard curve of QPCR derived titers from dilutions of a previously titrated baculovirus stock. Each dilution was titrated by both plaque assay and QPCR, producing a consistent and reproducible inverse relationship between C(T) and plaque forming units per milliliter. No significant difference was observed between titers produced by QPCR and plaque assay for 12 recombinant viruses, confirming the validity of this technique as a rapid and accurate method of baculovirus titration.