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Pei Z.,Hubei University | Jiang X.,University of California at Berkeley | Yang Z.,Taizhou Institute of Virology | Yang Z.,Jiangsu Affynigen Biotechnologies Inc. | And 11 more authors.
PLoS ONE | Year: 2015

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella- based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses. Copyright © 2015 Pei et al.


Yang Z.,Nanjing University | Yang Z.,Taizhou Institute of Virology | Yang Z.,Jiangsu Affynigen Biotechnologies Inc. | Mao G.,Nanjing University | And 18 more authors.
Virologica Sinica | Year: 2013

A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection. © 2013 Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg.


Yang Z.,Nanjing University | Yang Z.,Taizhou Institute of Virology | Yang Z.,Jiangsu Affynigen Biotechnologies Inc | Vu G.-P.,University of California at Berkeley | And 8 more authors.
Viruses | Year: 2014

RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application. © 2014 by the authors; licensee MDPI, Basel, Switzerland.


Gu H.,Nanjing University | Gu H.,Taizhou Affynigen Biotechnologies Inc. | Gu H.,Taizhou Institute of Virology | Qi X.,Nanjing University | And 16 more authors.
Journal of Applied Microbiology | Year: 2010

Aim: The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). Methods and Results: The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. Conclusions: Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. Significance and Impact of the Study: These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings. © 2009 The Authors.


Yang Z.,Nanjing University | Yang Z.,Taizhou Institute of Virology | Yang Z.,Jiangsu Affynigen Biotechnologies Inc | Reeves M.,University of California at Berkeley | And 9 more authors.
Viruses | Year: 2015

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant highly effective in inhibiting HCMV gene expression and blocking viral replication, and is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. gene-targeting agent for anti-HCMV therapy. © 2015 by the authors; licensee MDPI, Basel, Switzerland.


PubMed | Taizhou Institute of Virology, Hubei University, Jinan University and University of California at Berkeley
Type: | Journal: Scientific reports | Year: 2016

Infection of enterovirus 71 (EV71) and associated hand, foot, and mouth disease (HFMD) are recognized as emerging public health issues worldwide. Hundreds of thousands of children are annually infected with EV71 and develop HFMD in China alone. Studies of EV71 infection are critical to the treatment and prevention of the associated HFMD outbreaks. In this report, we studied an outbreak of 105 HFMD cases in Shawo Township of China between September to October 2012. More than 90% of cases were children younger than 9 years old, with over 50% of cases aged 3-6 years old. Laboratory studies detected a high prevalence of EV71 and suggested EV71 as the most common enterovirus causing HFMD in Shawo. Sequencing analysis showed that the EV71 strains from Shawo belong to the C4 subgenotype, and are phylogenetically more related to those from the distant city of Nanchang than those from the nearby city of Wuhan with distinct variations. More girls were found to be associated with EV71 in Shawo whereas more boys were associated with EV71 in Wuhan and Nanchang. Our studies further the understanding of the molecular epidemiological features of HFMD and infection by enteroviruses in China.


PubMed | Taizhou Institute of Virology, Hubei University, Jinan University and University of California at Berkeley
Type: | Journal: Scientific reports | Year: 2016

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.


PubMed | Taizhou Institute of Virology, Wuhan University, The Peoples Hospital of Taizhou, Nanjing University and University of California at Berkeley
Type: Journal Article | Journal: Viruses | Year: 2015

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.


PubMed | Taizhou Institute of Virology, Peoples Hospital of Taizhou, Nanjing University and University of California at Berkeley
Type: Journal Article | Journal: Viruses | Year: 2014

RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.

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