Renfu Yin

Systematic selection of housekeeping genes for gene expression normalization in chicken embryo fibroblasts infected with Newcastle disease virus

Gene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize gene expression. Here we describe the first systematic selection and evaluation of suitable HKGs for gene expression analysis in chicken embryo fibroblasts (CEF) infected with NDV adapted to the guidelines from Gorzelniak and Ferguson. Our results indicate that ACTB, HPRT1 and HMBS were valuable and stable HKGs, while 18S RNA, GAPDH and SHDA are considerably regulated during the course of infection and thus precluded for normalization. Normalizing the infection dependent gene IFN-a and the infection independent gene B2M to inappropriate HKGs consequently misleads to significant errors in estimating their regulations. Our study emphasizes that even the most popular HKGs like 18S RNA and GAPDH can lead to divergent and inaccurate data interpretation of significant magnitude if not carefully analyzed for stability before.

Inhibition of Newcastle disease virus replication by RNA interference targeting the matrix protein gene in chicken embryo fibroblasts.

Newcastle disease (ND) is an infectious viral disease of birds caused by the Newcastle disease virus (NDV), also known as avian paramyxovirus type 1 (AMPV-1), which leads to severe economic losses in the poultry industry worldwide. In this study, the application of RNA interference (RNAi) for inhibiting the replication of NDV in cell culture by targeting the viral matrix protein gene (M) is described. Two M-specific shRNA-expressing plasmid constructs, named pS(M641) and pS(M827), were evaluated for antiviral activity against the NDV strain NA-1 by cytopathic effects (CPE), virus titration and real-time RT-PCR. After 36h of infection, both pS(M641) and pS(M827) reduced virus titers by 79.4- and 31.6-fold, respectively, and they down-regulated mRNA expression levels of the matrix protein gene M by 94.6% and 84.8%, respectively, in chicken embryo fibroblast (CEF) cells, while only pS(M641) significantly decreased CPE, compared to the control group. These results indicated that the M gene 641 and 827 sites represent potential antiviral therapy targets, and RNAi targeting of the M gene could not only represent an effective treatment in Newcastle disease but also aid as a method for studying the replication of NDV.

Selection and evaluation of stable housekeeping genes for gene expression normalization in carbon nanoparticle-induced acute pulmonary inflammation in mice

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use of unconfirmed housekeeping genes (HKGs) could lead to misinterpretation of the expression of genes of interest (GOI). In this study, the stability and suitability of 11 frequently used housekeeping genes, namely 18S rRNA, ACTB, B2M, CYPA, GADPH, GUSB, HMBS, HPRT1, RPL13A, SDHA and TBP in 36 lung tissues isolated from either wild-type (WT) mice or p50 knock out (p50-/-) mice or p105 knock-out (p105-/-) mice which were treated with either carbon nanoparticle (CNP) or H(2)O or non-treated, have been validated by geNorm, NormFinder and BestKeeper programs. The expression levels of ACTB, GUSB and RPL13A were the most constant in lung tissues across three genotypes and three kinds of treatments. A set of three most stable genes is found sufficient to be used as housekeeping genes for lung tissues in studies of similar design.

Development of a reverse genetics system based on RNA polymerase II for Newcastle disease virus genotype VII

Abstract Newcastle disease virus (NDV) has only a single serotype but diversified genotypes. Genotype VII strains are the prevalent currently circulating genotype worldwide, and in particular, these strains cause outbreaks in waterfowl. In this study, a reverse genetics system for highly virulent NDV isolated from goose flocks was developed independent of conventional T7 RNA polymerase. Infectious virus was successfully generated by an RNA polymerase II promoter to drive transcription of the full-length virus antigenome. A green fluorescent protein (GFP)-expressing virus was generated by inserting an additional transcription cassette coding for the enhanced GFP between the P and M genes of the genome. The expression of GFP was confirmed by western blotting and fluorescence microscopy. The replication kinetics and pathogenicity of the recombinant viruses are indistinguishable from the parental wild-type virus. This reverse genetics system will provide a powerful tool for the analysis of goose-origin NDV dissemination and pathogenesis, as well as preparation for genotype-matched NDV attenuated vaccines.

Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings

Abstract Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds.

Pulmonary DWCNT exposure causes sustained local and low-level systemic inflammatory changes in mice

Carbon nanotubes (CNTs) represent promising vectors to facilitate cellular drug delivery and to overcome biological barriers, but some types may also elicit persistent pulmonary inflammation based on their fibre characteristics. Here, we show the pulmonary response to aqueous suspensions of block copolymer dispersed, double-walled carbon nanotubes (DWCNT, length 1-10 μm) in mice by bronchoalveolar lavage (BAL) analysis, and BAL and blood cytokine and lung antioxidant profiling. The intratracheally instilled dose of 50 μg DWCNT caused significant pulmonary inflammation that was not resolved during a 7-day observation period. Light microscopy investigation of the uptake of DWCNT agglomerates revealed no particle ingestion for granulocytes, but only for macrophages. Accumulating macrophage, multinucleated macrophage and lymphocyte numbers in the alveolar region further indicated ineffective resolution with chronification of the inflammation. The local inflammatory impairment of the lung was accompanied by pulmonary antioxidant depletion and haematological signs of systemic inflammation. While the observed inflammation during its acute phase was dominated by neutrophils and neutrophil recruiting cytokines, the contribution of macrophages and lymphocytes with related cytokines became more significant after day 3 of exposure. This study confirms that acute pulmonary toxicity can occur on exposure of high doses of DWCNT agglomerates and offers further insight for improved nanotube design parameters to avoid potential long-term toxicity.

Genetic diversity of the genotype VII Newcastle disease virus: identification of a novel VIIj sub-genotype

Newcastle disease (ND) is a highly contagious disease of poultry caused by Newcastle disease virus (NDV). Multiple genotypes of NDV have been circulating worldwide and NDV is continuously evolving, resulting into more diversity. Of multiple viral genotypes, VII is particularly important given that it had been associated with most recent ND outbreaks worldwide. In this study, an epidemiological investigation performed in northeastern China during 2014–2015 showed that 11 genotype VII isolates amounted to 55 percent in a total number of NDV isolates. Therefore, to evaluate the genetic diversity worldwide and epidemiological distribution in China of genotype VII NDV, a phylogenetic analysis based on the 1255 complete F gene sequences showed that VII is the most predominant genotype worldwide. A further detailed characterization on genotype VII was conducted based on the 477 complete F gene sequences from 11 isolates and 466 reference viruses available in GenBank. The results demonstrated that VII can be further divided into 8 sub-genotypes (VIIb, VIId–VIIj), indicating its complex genetic diversity. It is worthy of note that the isolation rate of VIIj is increasing recently. It emphasizes the necessity to pay close attention to the epidemiological dynamic of genotype VII NDV and highlights the importance of vaccination program.

High Genetic Diversity of Newcastle Disease Virus in Wild and Domestic Birds in Northeastern China from 2013 to 2015 Reveals Potential Epidemic Trends

ABSTRACT Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is one of the most important viral diseases of birds globally, but little is currently known regarding enzootic trends of NDV in northeastern China, especially for class I viruses. Thus, we performed a surveillance study for NDV in northeastern China from 2013 to 2015. A total 755 samples from wild and domestic birds in wetlands and live bird markets (LBMs) were collected, and 10 isolates of NDV were identified. Genetic and phylogenetic analyses showed that five isolates from LBMs belong to class I subgenotype 1b, two (one from wild birds and one from LBMs) belong to the vaccine-like class II genotype II, and three (all from wild birds) belong to class II subgenotype Ib. Interestingly, the five class I isolates had epidemiological connections with viruses from southern, eastern, and southeastern China. Our findings, together with recent prevalence trends of class I and virulent class II NDV in China, suggest possible virus transmission between wild and domestic birds and the potential for an NDV epidemic in the future.

Expression of Raf kinase inhibitor protein is downregulated in response to Newcastle disease virus infection to promote viral replication

Newcastle disease virus (NDV) causes a severe and economically significant disease affecting almost the entire poultry industry worldwide. However, factors that affect NDV replication in host cells are poorly understood. Raf kinase inhibitory protein (RKIP) is a physiological inhibitor of c-RAF kinase and NF-κB signalling, known for their functions in the control of immune response as well as tumour invasion and metastasis. In the present study, we investigated the consequences of overexpression of host RKIP during viral infection. We demonstrate that NDV infection represses RKIP expression thereby promoting virus replication. Experimental upregulation of RKIP in turn acts as a potential antiviral defence mechanism in host cells that restricts NDV replication by repressing the activation of Raf/MEK/ERK and IκBα/NF-κB signalling pathways. Our results not only extend the concept of linking NDV-host interactions, but also reveal RKIP as a new class of protein-kinase-inhibitor protein that affects NDV replication with therapeutic potential.

CASCIRE surveillance network and work on avian influenza viruses

CASCIRE surveillance network and work on avian influenza viruses Yuhai Bi, Weifeng Shi, Jianjun Chen, Quanjiao Chen, Zhenghai Ma, Gary Wong, Wenxia Tian, Renfu Yin, Guanghua Fu, Yongchun Yang, William J. Liu, Chuansong Quan, Qianli Wang, Shenghu He, Xiangdong Li, Qianfeng Xia, Lixin Wang, Zhaohui Pan, Laixing Li, Hong Li, Wen Xu, Ying Luo, Hui Zeng, Lianpan Dai, Haixia Xiao, Kirill Sharshov, Alexander Shestopalov, Yi Shi, Jinghua Yan, Xuebing Li, Yingxia Liu, Fumin Lei, Wenjun Liu & George F. Gao