43 results on '"Yuanmei Ma"'
Search Results
2. A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein
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Anzhong Li, Jingyou Yu, Mijia Lu, Yuanmei Ma, Zayed Attia, Chao Shan, Miaoge Xue, Xueya Liang, Kelsey Craig, Nirajkumar Makadiya, Jennifer J. He, Ryan Jennings, Pei-Yong Shi, Mark E. Peeples, Shan-Lu Liu, Prosper N. Boyaka, and Jianrong Li
- Subjects
Science - Abstract
Current Zika virus (ZIKV) subunit vaccine development largely focuses on prM and E proteins, and the role of NS1 for immune response and protection is unclear. Here, Li et al. develop an attenuated VSV-based vaccine expressing a ZIKV prM-E-NS1 polyprotein and characterize immune response and protection in mice.
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- 2018
- Full Text
- View/download PDF
3. Efficient Production of Human Norovirus-Specific IgY in Egg Yolks by Vaccination of Hens with a Recombinant Vesicular Stomatitis Virus Expressing VP1 Protein
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Yang Zhu, Yuanmei Ma, Mijia Lu, Yu Zhang, Anzhong Li, Xueya Liang, and Jianrong Li
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Human norovirus ,IgY ,vesicular stomatitis virus (VSV)-based vaccine ,Microbiology ,QR1-502 - Abstract
Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.
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- 2019
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4. Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United States
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Yuanmei Ma, Yu Zhang, Xueya Liang, Fangfei Lou, Michael Oglesbee, Steven Krakowka, and Jianrong Li
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Microbiology ,QR1-502 - Abstract
ABSTRACT A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine. IMPORTANCE Porcine coronaviruses (CoVs) are major viral infectious diseases of swine. Examples of porcine CoVs include porcine transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine respiratory coronavirus (PRCV). In February 2014, another porcine CoV, porcine deltacoronavirus (PdCV), emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease.
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- 2015
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5. Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model
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Yuanmei Ma, Stefan Niewiesk, Kristen French-Kim, Basavaraj Binjawadagi, Kelsey A Brakel, Jianrong Li, M.R. Watts, and Olivia Harder
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viruses ,Respiratory Syncytial Virus Infections ,Antibodies, Viral ,medicine.disease_cause ,Article ,Virus ,Immunity ,Respiratory Syncytial Virus Vaccines ,medicine ,Animals ,Sigmodontinae ,Cotton rat ,Ebola Vaccines ,Neutralizing antibody ,Glycoproteins ,General Veterinary ,General Immunology and Microbiology ,biology ,Ebola vaccine ,Immunogenicity ,Public Health, Environmental and Occupational Health ,Hemorrhagic Fever, Ebola ,biology.organism_classification ,Antibodies, Neutralizing ,Virology ,Infectious Diseases ,Respiratory syncytial virus (RSV) ,Vesicular stomatitis virus ,Respiratory Syncytial Virus, Human ,biology.protein ,Molecular Medicine ,Vesicular Stomatitis ,Viral Fusion Proteins - Abstract
Respiratory syncytial virus (RSV) is one of the most important causes of respiratory disease in infants, immunocompromised individuals, and the elderly. Natural infection does not result in long-term immunity, and there is no licensed vaccine. Vesicular stomatitis virus (VSV) is a commonly used vaccine vector platform against infectious diseases, and has been used as a vector for a licensed Ebola vaccine. In this study, we expressed the RSV fusion (F) protein, the RSV F protein stabilized in either a pre-fusion or a post-fusion configuration, the attachment glycoprotein (G), or the G and F proteins of RSV in combination in a VSV vector. Cotton rats were immunized with these recombinants intranasally or subcutaneously to test immunogenicity. RSV F stabilized in either a pre-fusion or a post-fusion configuration proved to be poorly immunogenic and protective when compared to unmodified F. RSV G provided partial protection and moderate levels of neutralizing antibody production, both of which improved with intranasal administration compared to subcutaneous inoculation. The most successful vaccine vector was VSV expressing both the G and F proteins after intranasal inoculation. Immunization with this recombinant induced neutralizing antibodies and provided protection from RSV challenge in the upper and lower respiratory tract for at least 80 days. Our results demonstrate that co-expression of F and G proteins in a VSV vector provides synergistic effects in inducing RSV-specific neutralizing antibodies and protection against RSV infection.
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- 2021
6. Codon-optimization of the respiratory syncytial virus (RSV) G protein expressed in a vesicular stomatitis virus (VSV) vector improves immune responses in a cotton rat model
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Kelsey A. Brakel, Yuanmei Ma, Rashmi Binjawadagi, Olivia Harder, Mauria Watts, Jianrong Li, Basavaraj Binjawadagi, and Stefan Niewiesk
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Immunity ,Respiratory Syncytial Virus Infections ,Vesiculovirus ,Antibodies, Viral ,Antibodies, Neutralizing ,Vesicular stomatitis Indiana virus ,GTP-Binding Proteins ,Virology ,Respiratory Syncytial Virus, Human ,Respiratory Syncytial Virus Vaccines ,Animals ,Sigmodontinae ,Codon ,Vesicular Stomatitis ,Viral Fusion Proteins - Abstract
Respiratory syncytial virus is an important cause of pneumonia in children, the elderly, and immunocompromised individuals. The attachment (G) protein of RSV generates neutralizing antibodies in natural RSV infection which correlate with protection against disease. The immune response to RSV is typically short-lived, which may be related to the heavy glycosylation of RSV-G. In order to improve its immunogenicity, we expressed G protein mutants in a vesicular stomatitis virus (VSV) vector system and tested their ability to protect cotton rats from RSV challenge. We found that the most protective construct was codon-optimized RSV-G, followed by wild-type G and membrane-bound G. Constructs which expressed the G protein with reduced glycosylation or the secreted G protein provided either partial or no protection. Our results demonstrate that modifications to the G protein are not advantageous in a VSV vector system, and that an intact, codon-optimized G is a superior vaccine candidate.
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- 2022
7. Mucosal Delivery of Recombinant Vesicular Stomatitis Virus Vectors Expressing Envelope Proteins of Respiratory Syncytial Virus Induces Protective Immunity in Cotton Rats
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Yuanmei Ma, Jianrong Li, Olivia Harder, Stefan Niewiesk, Mark E. Peeples, Rashmi Binjawadagi, Basavaraj Binjawadagi, and Kelsey A Brakel
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Immunology ,Genetic Vectors ,Respiratory System ,Respiratory Syncytial Virus Infections ,Biology ,Vaccines, Attenuated ,Microbiology ,Virus ,Viral vector ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Viral Envelope Proteins ,Virology ,Vaccines and Antiviral Agents ,Respiratory Syncytial Virus Vaccines ,Administration, Mucosal ,Animals ,030212 general & internal medicine ,Vector (molecular biology) ,Sigmodontinae ,Neutralizing antibody ,030304 developmental biology ,0303 health sciences ,Immunity, Cellular ,Attenuated vaccine ,Immunogenicity ,Vesiculovirus ,biology.organism_classification ,Recombinant Proteins ,Immunity, Humoral ,Disease Models, Animal ,Vesicular stomatitis virus ,Insect Science ,Respiratory Syncytial Virus, Human ,biology.protein ,Immunization ,Viral Fusion Proteins - Abstract
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 10(3) PFU. In contrast, doses greater than 10(6) PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection. IMPORTANCE Even after decades of intensive research efforts, a safe and efficacious RSV vaccine remains elusive. Expression of heterologous antigens from rVSV vectors has demonstrated several practical and safety advantages over other virus vector systems and live attenuated vaccines. In this study, we developed safe and efficacious vaccine candidates by expressing the two major immunogenic RSV surface proteins in rVSV vectors and delivering them mucosally in a prime-boost regimen. The main immune parameter responsible for protection was the antibody response. These vaccine candidates induced complete protection of both the upper and lower respiratory tracts.
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- 2021
8. Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses
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Youling Gao, Jinyan Gu, Hui Cai, Yunjian Lu, Jiyong Zhou, Mijia Lu, Anzhong Li, Yuanmei Ma, Howard H. Gu, and Jianrong Li
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RNA Caps ,S-Adenosylmethionine ,Guanine ,RNA methylation ,Swine ,viruses ,Immunology ,Gene Expression ,Viral Nonstructural Proteins ,medicine.disease_cause ,Virus Replication ,Methylation ,Microbiology ,Cell Line ,Interferon Lambda ,03 medical and health sciences ,Exon ,Interferon ,Virology ,Gene expression ,Chlorocebus aethiops ,medicine ,Animals ,Vero Cells ,030304 developmental biology ,Coronavirus ,0303 health sciences ,Binding Sites ,biology ,030306 microbiology ,Porcine epidemic diarrhea virus ,RNA ,biology.organism_classification ,Immunity, Innate ,Genome Replication and Regulation of Viral Gene Expression ,Viral replication ,Insect Science ,Exoribonucleases ,Interferon Type I ,Mutation ,RNA, Viral ,Interferons ,medicine.drug - Abstract
The 5′ cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2′-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses. IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.
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- 2020
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9. A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein
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Mark E. Peeples, Jianrong Li, Prosper N. Boyaka, Ryan N. Jennings, Jingyou Yu, Miaoge Xue, Yuanmei Ma, Zayed Attia, Pei Yong Shi, Mijia Lu, Anzhong Li, Xueya Liang, Nirajkumar Makadiya, Chao Shan, Kelsey Craig, Shan-Lu Liu, and Jennifer J. He
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0301 basic medicine ,Male ,viruses ,General Physics and Astronomy ,Viral Nonstructural Proteins ,Antibodies, Viral ,Zika virus ,Mice ,0302 clinical medicine ,Viral Envelope Proteins ,Vaccines, DNA ,030212 general & internal medicine ,lcsh:Science ,Antigens, Viral ,Mice, Inbred BALB C ,Vaccines, Synthetic ,Multidisciplinary ,biology ,Zika Virus Infection ,Vaccination ,virus diseases ,humanities ,3. Good health ,medicine.anatomical_structure ,Vesicular stomatitis virus ,Cytokines ,Female ,Antibody ,T cell ,Science ,Genetic Vectors ,Vaccines, Attenuated ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Immune system ,Th2 Cells ,Antigen ,medicine ,Animals ,Polyproteins ,Viral Vaccines ,General Chemistry ,Vesiculovirus ,Zika Virus ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Virology ,Antibodies, Neutralizing ,Disease Models, Animal ,030104 developmental biology ,Immunization ,biology.protein ,Th17 Cells ,lcsh:Q - Abstract
Current efforts to develop Zika virus (ZIKV) subunit vaccines have been focused on pre-membrane (prM) and envelope (E) proteins, but the role of NS1 in ZIKV-specific immune response and protection is poorly understood. Here, we develop an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate is attenuated in mice, where a single immunization induces ZIKV-specific antibody and T cell immune responses that provide protection against ZIKV challenge. Co-expression of prM, E, and NS1 induces significantly higher levels of Th2 and Th17 cytokine responses than prM-E. In addition, NS1 alone is capable of conferring partial protection against ZIKV infection in mice even though it does not induce neutralizing antibodies. These results demonstrate that attenuated rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection and highlights an important role for NS1 in ZIKV-specific cellular immune responses., Current Zika virus (ZIKV) subunit vaccine development largely focuses on prM and E proteins, and the role of NS1 for immune response and protection is unclear. Here, Li et al. develop an attenuated VSV-based vaccine expressing a ZIKV prM-E-NS1 polyprotein and characterize immune response and protection in mice.
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- 2018
10. Isolation and characterization of Chinese porcine epidemic diarrhea virus with novel mutations and deletions in the S gene
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Weihuan Fang, Jiang Sheng, Jing Sun, Shao Chunyan, He Haijian, Houhui Song, Yingshan Zhou, Yuanmei Ma, Li Qunjing, Shaobo Ba, Xiaodu Wang, Wu Yuan, and Lin Shi
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0301 basic medicine ,China ,Antigenicity ,Genotype ,Swine ,Spike gene ,Mutant ,Virulence ,Viral Plaque Assay ,Virus Replication ,Microbiology ,Article ,Genetic diversity ,Virus ,Epitopes ,03 medical and health sciences ,Chlorocebus aethiops ,Pathogenicity ,Animals ,Vero Cells ,Gene ,Phylogeny ,Swine Diseases ,Genetics ,General Veterinary ,biology ,Phylogenetic tree ,Molecular epidemiology ,Porcine epidemic diarrhea virus ,Genetic Variation ,General Medicine ,biology.organism_classification ,030104 developmental biology ,Amino Acid Substitution ,Mutation ,Spike Glycoprotein, Coronavirus ,Coronavirus Infections - Abstract
Highlights • We successfully isolated two novel PEDV strains, PEDV-LA1 and PEDV-LY4-98 in China. • The two novel PEDV isolates shared higher identities with United States strains than with South Korean strains. • Three unique amino acid substitutions were identified in the S1 N terminal domain of the PEDV-LY4-98 strain., Porcine epidemic diarrhea (PEDV) has raised growing concerns in the pig-breeding industry because it has caused significant economic losses. To better understand the molecular epidemiology and genetic diversity of PEDV field isolates, in this study, the complete spike (S) and ORF3 genes of 17 PEDV variants in Zhejiang, China during 2014 to 2017, were characterized and analyzed. Phylogenetic analysis based on the S gene and ORF3 gene of these 17 novel PEDV strains and PEDV reference strains indicated that all the PEDV strains fell into two groups designated G1 and G2. Notably, the strains identified in 2014–2015 were in G2, while the other five strains identified from 2016 to 2017 were in G1. Sequencing and phylogenetic analyses showed that recently prevalent Chinese PEDV field strains shared higher identities with United States strains than with South Korean strains. Compared with classical vaccine strains, a series of deletions and frequently occurring mutations were observed in the receptor binding domains of our PEDV strains. Besides, we successfully isolated and reported the genetic characterization two novel PEDV strains, PEDV-LA1 and PEDV-LY4-98, found on the Chinese mainland, which had significant variations in the S gene. Meanwhile, the virulence of the new mutants may be changed, the PEDV-LY4-98 strain, which has multiple mutations in the signal peptide-encoding fragment of the S gene showed delayed cytopathic effects and smaller plaque size compared with strain PEDV-LA1, which lacks these mutations. Three unique amino acid substitutions (L7, G8, and V9) were identified in the SP-encoding fragment of the S1 N-terminal domain of the PEDV-LY4-98 S protein compared with the S proteins of all the previous PEDV strains. The animal experiment revealed that these two novel strains were high pathogenic to neonatal pigs. Whether these amino acids substitutions and the N-glycosylation site substitutions influence the antigenicity and pathogenicity of PEDV remains to be investigated. Meanwhile, amino acid substitutions in the neutralizing epitopes may have conferred the capacity for immune evasion in these PEDV field strains. This study improves our understanding of ongoing PEDV outbreaks in China, and it will guide further efforts to develop effective measures to control this virus.
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- 2018
11. Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus
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Mark E. Peeples, Zhike Lu, Stefan Niewiesk, Yunsheng Xu, Mijia Lu, Olivia Harder, Chuan He, Jianrong Li, Jiyong Zhou, Phylip Chen, Anzhong Li, Miaoge Xue, Boxuan Simen Zhao, Yuanmei Ma, Xueya Liang, and Zijie Zhang
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Male ,0301 basic medicine ,Adenosine ,Methyltransferase ,Live attenuated vaccines ,Science ,viruses ,General Physics and Astronomy ,Respiratory Syncytial Virus Infections ,Biology ,Virus-host interactions ,Antibodies, Viral ,Vaccines, Attenuated ,Virus Replication ,Article ,General Biochemistry, Genetics and Molecular Biology ,Virus ,03 medical and health sciences ,0302 clinical medicine ,Chlorocebus aethiops ,Gene expression ,Respiratory Syncytial Virus Vaccines ,Animals ,Humans ,Sigmodontinae ,lcsh:Science ,Vero Cells ,Gene knockdown ,Multidisciplinary ,Attenuated vaccine ,Virulence ,General Chemistry ,Methylation ,Virology ,Up-Regulation ,3. Good health ,030104 developmental biology ,Viral replication ,Viral infection ,A549 Cells ,Respiratory Syncytial Virus, Human ,030220 oncology & carcinogenesis ,Vero cell ,RNA ,lcsh:Q ,Female ,HeLa Cells - Abstract
N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses., Here, Xue et al. identify N6-methyladenosine (m6A) modification sites in RNAs of respiratory syncytial virus (RSV) and show that these sites, particularly sites in the transcript encoding for the viral glycoprotein, affect virus replication in primary human cells and cotton rats.
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- 2019
12. Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases
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Lanying Du, Shibo Jiang, Yuan Zheng, Jian Shang, Fang Li, Yusen Zhou, Yang Yang, Jianrong Li, Chang Liu, and Yuanmei Ma
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0301 basic medicine ,Proteases ,Swine ,Cathepsin L ,Proteolysis ,medicine.disease_cause ,Microbiology ,Biochemistry ,Cathepsin B ,Cell Line ,03 medical and health sciences ,Viral entry ,medicine ,Animals ,Humans ,Protease inhibitor (pharmacology) ,Molecular Biology ,Coronavirus ,biology ,medicine.diagnostic_test ,Porcine epidemic diarrhea virus ,Cell Biology ,Virus Internalization ,Cysteine protease ,Virology ,030104 developmental biology ,Spike Glycoprotein, Coronavirus ,biology.protein ,Lysosomes - Abstract
Porcine epidemic diarrhea coronavirus (PEDV) is currently devastating the United States pork industry by causing an 80–100% fatality rate in infected piglets. Coronavirus spike proteins mediate virus entry into cells, a process that requires the spike proteins to be proteolytically activated. It has been a conundrum which proteases activate PEDV entry. Here we systematically investigated the roles of different proteases in PEDV entry using pseudovirus entry, biochemical, and live virus infection assays. We found that the PEDV spike is activated by lysosomal cysteine proteases but not proprotein convertases or cell surface serine proteases. Extracellular trypsin activates PEDV entry when lysosomal cysteine proteases are inhibited. We further pinpointed cathepsin L and cathepsin B as the lysosomal cysteine proteases that activate the PEDV spike. These results advance our understanding of the molecular mechanism for PEDV entry and identify potential antiviral targets for curbing the spread of PEDV.
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- 2016
13. Efficient Production of Human Norovirus-Specific IgY in Egg Yolks by Vaccination of Hens with a Recombinant Vesicular Stomatitis Virus Expressing VP1 Protein
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Anzhong Li, Yuanmei Ma, Mijia Lu, Jianrong Li, Yu Zhang, Xueya Liang, and Yang Zhu
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Gene Expression Regulation, Viral ,0301 basic medicine ,viruses ,030106 microbiology ,lcsh:QR1-502 ,Immunoglobulins ,Chicken Cells ,vesicular stomatitis virus (VSV)-based vaccine ,Biology ,Antibodies, Viral ,medicine.disease_cause ,Article ,Virus ,lcsh:Microbiology ,Microbiology ,03 medical and health sciences ,Antigen ,Virology ,IgY ,medicine ,Animals ,Humans ,Caliciviridae Infections ,Viral Structural Proteins ,Norovirus ,Vaccination ,Immunization, Passive ,Viral Vaccines ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Egg Yolk ,Gastroenteritis ,Kinetics ,030104 developmental biology ,Infectious Diseases ,Immunization ,Vesicular stomatitis virus ,Blood Group Antigens ,biology.protein ,Immunoglobulin Y ,Capsid Proteins ,Female ,Human norovirus ,Antibody ,Vesicular Stomatitis ,Chickens - Abstract
Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °, C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.
- Published
- 2019
14. Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation
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Roberto M. Uribe, Yuanmei Ma, Doug Culbertson, Jianrong Li, Nuttapong Phantkankum, David H. Kingsley, John H. Hughes, and Erin DiCaprio
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0301 basic medicine ,Swine ,030106 microbiology ,Virus Attachment ,Quantitative Reverse Transcriptase PCR ,Biology ,Real-Time Polymerase Chain Reaction ,medicine.disease_cause ,Fragaria ,Microbiology ,Virus ,Disease Outbreaks ,03 medical and health sciences ,medicine ,Animals ,Humans ,Tulane virus ,Caliciviridae Infections ,Virus quantification ,Infectivity ,Immunomagnetic Separation ,Gastric Mucins ,Ligand binding assay ,Norovirus ,RNA ,General Medicine ,Virus Internalization ,Virology ,Gastroenteritis ,030104 developmental biology ,Gamma Rays ,Virus Inactivation ,Food Science - Abstract
Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR.
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- 2016
15. Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms
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Jan Vinjé, Yuanmei Ma, Hannah Bolinger, Julia L. Sharp, Angela Fraser, Thomas Yeargin, Erin DiCaprio, Cortney M. Leone, Jianrong Li, Kitwadee Rupprom, Xiuping Jiang, Chaoyi Tang, Donald W. Schaffner, Elbashir Araud, and Muthu Dharmasena
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0301 basic medicine ,South carolina ,Male ,Infection risk ,Restaurants ,Food Handling ,South Carolina ,030106 microbiology ,Food Contamination ,010501 environmental sciences ,Environment ,medicine.disease_cause ,01 natural sciences ,Microbiology ,Article ,Disease Outbreaks ,03 medical and health sciences ,Retail food ,Environmental health ,Odd ratio ,Prevalence ,Medicine ,Humans ,Toilet Facilities ,Strategic ,0105 earth and related environmental sciences ,Caliciviridae Infections ,Ohio ,Toilet ,New Jersey ,business.industry ,Norovirus ,Gastroenteritis ,Disinfection ,Defence & Security Studies ,Bathrooms ,Door handle ,Fomites ,Female ,business ,Food Science ,Contaminated food - Abstract
Although transmission of human norovirus in food establishments is commonly attributed to consumption of contaminated food, transmission via contaminated environmental surfaces, such as those in bathrooms, may also play a role. Our aim was to determine the prevalence of human norovirus on bathroom surfaces in commercial food establishments in New Jersey, Ohio, and South Carolina under nonoutbreak conditions and to determine characteristics associated with the presence of human norovirus. Food establishments (751) were randomly selected from nine counties in each state. Four surfaces (underside of toilet seat, flush handle of toilet, inner door handle of stall or outer door, and sink faucet handle) were swabbed in male and female bathrooms using premoistened macrofoam swabs. A checklist was used to collect information about the characteristics, materials, and mechanisms of objects in bathrooms. In total, 61 (1.5%) of 4,163 swabs tested were presumptively positive for human norovirus, 9 of which were confirmed by sequencing. Some factors associated with the presence of human norovirus included being from South Carolina (odd ratio [OR], 2.4; 95% confidence interval [CI], 1.2 to 4.9; P < 0.05) or New Jersey (OR, 1.7; 95% CI, 0.9 to 3.3; 0.05 < P < 0.10), being a chain establishment (OR, 1.9; 95% CI, 1.1 to 3.3; P < 0.05), being a unisex bathroom (versus male: OR, 2.0; 95% CI, 0.9 to 4.1; 0.05 < P < 0.10; versus female: OR, 2.6; 95% CI, 1.2 to 5.7; P < 0.05), having a touchless outer door handle (OR, 3.3; 95% CI, 0.79 to 13.63; 0.05 < P < 0.10), and having an automatic flush toilet (OR, 2.5, 95% CI, 1.1 to 5.3; 0.05 < P < 0.10). Our findings confirm that the presence of human norovirus on bathroom surfaces in commercial food establishments under nonoutbreak conditions is a rare event. Therefore, routine environmental monitoring for human norovirus contamination during nonoutbreak periods is not an efficient method of monitoring norovirus infection risk.
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- 2018
16. Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo
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Yuanmei Ma, Yu Zhang, Jing Sun, Xueya Liang, Jianrong Li, and Hui Cai
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Paramyxoviridae ,Immunology ,chemistry.chemical_element ,Plasma protein binding ,Zinc ,Virus Replication ,Microbiology ,Cell Line ,Viral Proteins ,Pneumovirinae ,Human metapneumovirus ,Virology ,Animals ,Humans ,Paramyxovirinae ,Metapneumovirus ,Sigmodontinae ,Paramyxoviridae Infections ,biology ,biology.organism_classification ,Disease Models, Animal ,Viral replication ,chemistry ,Insect Science ,Pathogenesis and Immunity ,Female ,Mutant Proteins ,Protein Multimerization ,Carrier Proteins ,Protein Binding - Abstract
Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo . These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. IMPORTANCE The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Despite major efforts, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that the zinc binding activity of M2-1 is essential for virus replication and pathogenesis in vivo . Recombinant hMPVs that lacked zinc binding activity were not only defective in replication in the upper and lower respiratory tract but also triggered a strong protective immunity in cotton rats. Thus, inhibition of M2-1 zinc binding activity can lead to the development of novel, live attenuated vaccines, as well as antiviral drugs for pneumoviruses.
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- 2015
17. Receptor Usage and Cell Entry of Porcine Epidemic Diarrhea Coronavirus
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Xueya Liang, Fang Li, Shibo Jiang, Chang Liu, Jianrong Li, Guiqing Peng, Yuanmei Ma, Yang Yang, Lanying Du, Qianqian Qi, and Jian Tang
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Swine ,Immunology ,Virulence ,Virus Attachment ,Biology ,CD13 Antigens ,medicine.disease_cause ,Microbiology ,Virology ,Chiroptera ,medicine ,Animals ,Humans ,Receptor ,Coronavirus ,Cell entry ,Aminopeptidase N ,Porcine epidemic diarrhea virus ,Haplorhini ,Virus Internalization ,Pathogenicity ,biology.organism_classification ,N-Acetylneuraminic Acid ,Virus-Cell Interactions ,Epidemic diarrhea ,Insect Science ,Receptors, Virus ,Receptors, Coronavirus - Abstract
Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N -acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread.
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- 2015
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18. Mucosal Delivery of Recombinant Vesicular Stomatitis Virus Vectors Expressing Envelope Proteins of Respiratory Syncytial Virus Induces Protective Immunity in Cotton Rats.
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Binjawadagi, Basavaraj, Yuanmei Ma, Binjawadagi, Rashmi, Brakel, Kelsey, Harder, Olivia, Peeples, Mark, Jianrong Li, and Niewiesk, Stefan
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RESPIRATORY syncytial virus , *VESICULAR stomatitis , *HUMAN metapneumovirus infection , *ANTIBODY titer , *VIRUSES , *IMMUNITY , *PROTEINS - Abstract
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 10³ PFU. In contrast, doses greater than 106 PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection. [ABSTRACT FROM AUTHOR]
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- 2021
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19. Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain
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Xiaodong Zhang, Yu Zhang, Yehia M. Saif, Yongwei Wei, Lian Yu, Jianrong Li, Xingmeng Lu, Qingzhong Yu, Abdul Rauf, Yuanmei Ma, Jing Sun, and Konstantin Shilo
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Turkeys ,viruses ,Cross Protection ,Immunology ,Antibodies, Viral ,Vaccines, Attenuated ,Microbiology ,Virus ,Measles virus ,Human metapneumovirus ,Virology ,Vaccines and Antiviral Agents ,Animals ,Metapneumovirus ,Lung ,Poultry Diseases ,Attenuated vaccine ,Paramyxoviridae Infections ,biology ,Viral Vaccine ,Viral Vaccines ,Methyltransferases ,Viral Load ,biology.organism_classification ,Antibodies, Neutralizing ,Avian pneumovirus ,Trachea ,Viral replication ,Insect Science - Abstract
Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2′-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S -adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2′-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. IMPORTANCE Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2′- O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2′- O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.
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- 2014
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20. Heat Shock Protein 70 Enhances Mucosal Immunity against Human Norovirus When Coexpressed from a Vesicular Stomatitis Virus Vector
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Yuanmei Ma, Xueya Liang, Yongwei Wei, Stefan Niewiesk, Yue Duan, Michael Oglesbee, and Jianrong Li
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viruses ,T-Lymphocytes ,Genetic Vectors ,Immunology ,Biology ,Vaccines, Attenuated ,Microbiology ,Virus ,Mice ,Immunity ,Heat shock protein ,Virology ,Vaccines and Antiviral Agents ,Animals ,HSP70 Heat-Shock Proteins ,Immunity, Mucosal ,Mice, Inbred BALB C ,Vaccines, Synthetic ,Attenuated vaccine ,Viral Vaccine ,Norovirus ,virus diseases ,Viral Vaccines ,Vesiculovirus ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Immunoglobulin A ,Vaccination ,Gastrointestinal Tract ,Capsid ,Vesicular stomatitis virus ,Immunoglobulin G ,Insect Science ,Vagina ,Capsid Proteins ,Female - Abstract
Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 10 6 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 10 6 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo , thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.
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- 2014
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21. hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus
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Jianrong Li, Yuanmei Ma, Yaoling Shu, Mi Young Kim, Michael Oglesbee, and Yu Zhang
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Male ,Genetically modified mouse ,viruses ,Blotting, Western ,Immunology ,Apoptosis ,Mice, Transgenic ,Biology ,Real-Time Polymerase Chain Reaction ,Virus Replication ,Microbiology ,Vesicular stomatitis Indiana virus ,Measles virus ,Mice ,Vesicular Stomatitis ,Interferon ,Immunity ,Virology ,medicine ,Animals ,HSP70 Heat-Shock Proteins ,RNA, Messenger ,Cell Proliferation ,Neurons ,Immunity, Cellular ,Reverse Transcriptase Polymerase Chain Reaction ,Macrophages ,Brain ,biology.organism_classification ,Mice, Inbred C57BL ,Toll-Like Receptor 4 ,Viral replication ,Vesicular stomatitis virus ,Insect Science ,Interferon Type I ,Pathogenesis and Immunity ,Interferon type I ,medicine.drug - Abstract
The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro , hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.
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- 2013
22. Epidemiology, Prevention, and Control of the Number One Foodborne Illness
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Yuanmei Ma, Jianrong Li, Erin DiCaprio, and John H. Hughes
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Microbiology (medical) ,medicine.medical_specialty ,Epidemiology ,Psychological intervention ,medicine.disease_cause ,Antiviral Agents ,Article ,Vaccine development ,Foodborne Diseases ,Animal model ,Anti-Infective Agents ,Detection methods ,Medicine ,Humans ,Intensive care medicine ,Acute gastroenteritis ,Caliciviridae Infections ,Biodefense ,business.industry ,Norovirus ,Viral Vaccines ,Virology ,Prevention and control strategies ,Infectious Diseases ,Foodborne illness ,Human norovirus ,business - Abstract
Human norovirus (NoV) is the number one cause of foodborne illness. Despite tremendous research efforts, human NoV is still poorly understood and understudied. There is no effective measure to eliminate this virus from food and the environment. Future research efforts should focus on developing: (1) an efficient cell culture system and a robust animal model, (2) rapid and sensitive detection methods, (3) novel sanitizers and control interventions, and (4) vaccines and antiviral drugs. Furthermore, there is an urgent need to build multidisciplinary and multi-institutional teams to combat this important biodefense agent.
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- 2013
23. Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses
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Xianjun Dai, Yuanmei Ma, Jianrong Li, Haiqiang Chen, Erin DiCaprio, John H. Hughes, Xinhui Li, Fangfei Lou, David H. Kingsley, and Schaffner, DW
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0301 basic medicine ,Genotype ,Food Handling ,viruses ,030106 microbiology ,Sus scrofa ,Genome, Viral ,Biology ,Immunomagnetic separation ,medicine.disease_cause ,Real-Time Polymerase Chain Reaction ,Microbiology ,Applied Microbiology and Biotechnology ,Vaccine Related ,Pascalization ,03 medical and health sciences ,0404 agricultural biotechnology ,fluids and secretions ,Antigen ,Biodefense ,Genetics ,medicine ,Animals ,Humans ,Viral ,Genome ,Ecology ,Strain (chemistry) ,Immunomagnetic Separation ,Prevention ,Gastric Mucins ,Norovirus ,virus diseases ,04 agricultural and veterinary sciences ,Foodborne Illness ,040401 food science ,Virology ,Reverse transcription polymerase chain reaction ,Infectious Diseases ,Emerging Infectious Diseases ,High pressure ,Food Microbiology ,Capsid Proteins ,Digestive Diseases ,Food Science ,Biotechnology - Abstract
Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagated in vitro . In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1 > GII.6 > GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H 2 O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters. IMPORTANCE Human norovirus (HuNoV) is a leading cause of foodborne disease worldwide. Noroviruses are highly diverse, both antigenically and genetically. Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivable in vitro . Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix.
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- 2016
24. Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce
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Erin DiCaprio, Yuanmei Ma, Jianrong Li, John H. Hughes, and Anastasia Purgianto
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viruses ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Biology ,medicine.disease_cause ,Plant Roots ,Applied Microbiology and Biotechnology ,Virus ,Microbiology ,medicine ,Animals ,Humans ,Virus quantification ,Ecology ,Inoculation ,ved/biology ,Sequence Analysis, DNA ,Lettuce ,Viral Load ,Virus Internalization ,biology.organism_classification ,Virology ,Caliciviridae ,Plant Leaves ,Titer ,Shoot ,Food Microbiology ,Norovirus ,RNA, Viral ,Plant Shoots ,Food Science ,Biotechnology ,Murine norovirus - Abstract
Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 10 6 RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 10 6 to 2 × 10 6 PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (10 5 to 10 6 RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (10 1 to 10 3 PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (10 5 to 10 6 PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.
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- 2012
25. Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)
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Elbashir Araud, Fangfei Lou, Jianrong Li, David H. Kingsley, Erin DiCaprio, Yuanmei Ma, Yu Gao, John H. Hughes, and Schaffner, DW
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0301 basic medicine ,Oyster ,Hot Temperature ,viruses ,030106 microbiology ,ved/biology.organism_classification_rank.species ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Microbiology ,Virus ,Vaccine Related ,Foodborne Diseases ,03 medical and health sciences ,Tissue culture ,biology.animal ,Biodefense ,medicine ,2.2 Factors relating to the physical environment ,Animals ,Humans ,Cooking ,Aetiology ,Crassostrea ,Enterovirus ,Shellfish ,Infectivity ,Ecology ,biology ,ved/biology ,Prevention ,Norovirus ,biology.organism_classification ,Foodborne Illness ,Virology ,Infectious Diseases ,Emerging Infectious Diseases ,Good Health and Well Being ,Capsid ,Food Microbiology ,Virus Inactivation ,Digestive Diseases ,Infection ,Food Science ,Biotechnology ,Murine norovirus - Abstract
Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.
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- 2015
26. Two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus
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Yu Zhang, Steven Krakowka, Guiping Wang, Yuanmei Ma, Jianrong Li, Aiqing Jia, Michael Oglesbee, Xueya Liang, Houhui Song, and Andrew J. Niehaus
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0301 basic medicine ,Swine ,viruses ,Cross Reactions ,medicine.disease_cause ,Antibodies, Viral ,Microbiology ,Cross-reactivity ,Epitope ,Article ,Cell Line ,03 medical and health sciences ,Antigen ,medicine ,Animals ,Vero Cells ,Swine Diseases ,General Veterinary ,biology ,Porcine epidemic diarrhea virus ,Viral nucleocapsid ,General Medicine ,Nucleocapsid Proteins ,biology.organism_classification ,Virology ,Specific Pathogen-Free Organisms ,Porcine deltacoronavirus ,Coronavirus ,Intestines ,030104 developmental biology ,Vero cell ,biology.protein ,Antibody ,Coronavirus Infections - Abstract
Highlights • No cross-neutralization was detected between PEDV and PdCV. • A two-way cross-reactivity was detected between PEDV and PdCV. • Conserved epitope(s) in viral N proteins may contribute to antigenic cross-reactivity. • Prevention of PEDV and PdCV will require the development of separate virus-specific vaccine products., Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PdCV) cause indistinguishable clinical signs and pathological changes in swine. Here we investigated the antigenic relationship between PEDV and PdCV. We provide the first evidence that conserved epitope(s) on the respective viral nucleocapsid proteins cross-react with each other although virus neutralization cross-reactivity was not observed. As a practical matter, prevention of these two very similar diseases of swine will require the development of separate virus-specific vaccine products.
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- 2015
27. Inactivation of a Human Norovirus Surrogate, Human Norovirus Virus-Like Particles, and Vesicular Stomatitis Virus by Gamma Irradiation
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Yuanmei Ma, Kurtis Feng, Erin Divers, and Jianrong Li
- Subjects
Virosomes ,viruses ,ved/biology.organism_classification_rank.species ,Biology ,medicine.disease_cause ,Fragaria ,Applied Microbiology and Biotechnology ,Virus ,Microbiology ,Foodborne Diseases ,Virus-like particle ,Spinacia oleracea ,medicine ,Humans ,Mononegavirales ,Microbial Viability ,Ecology ,ved/biology ,Norovirus ,Vesiculovirus ,Lettuce ,Sterilization (microbiology) ,Rhabdoviridae ,biology.organism_classification ,Virology ,Disinfection ,Gamma Rays ,Vesicular stomatitis virus ,Food Microbiology ,Virus Inactivation ,Food Science ,Biotechnology ,Murine norovirus - Abstract
Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.
- Published
- 2011
28. Vesicular Stomatitis Virus as a Vector To Deliver Virus-Like Particles of Human Norovirus: a New Vaccine Candidate against an Important Noncultivable Virus
- Author
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Jianrong Li and Yuanmei Ma
- Subjects
viruses ,T-Lymphocytes ,Immunology ,Genetic Vectors ,Administration, Oral ,Enzyme-Linked Immunosorbent Assay ,Antibodies, Viral ,Vaccines, Attenuated ,Microbiology ,Virus ,Feces ,Mice ,Virus-like particle ,Virology ,Vaccines and Antiviral Agents ,Animals ,Mononegavirales ,Administration, Intranasal ,Caliciviridae Infections ,Cell Proliferation ,Mice, Inbred BALB C ,Vaccines, Synthetic ,Attenuated vaccine ,biology ,Norovirus ,virus diseases ,Viral Vaccines ,Vesiculovirus ,Rhabdoviridae ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Caliciviridae ,Immunoglobulin A ,Vaccines, Virosome ,Capsid ,Vesicular stomatitis virus ,Insect Science ,Vagina ,Capsid Proteins ,Female - Abstract
Human norovirus (HuNoV) is a major causative agent of food-borne gastroenteritis worldwide. Currently, there are no vaccines or effective therapeutic interventions for this virus. Development of an attenuated vaccine for HuNoV has been hampered by the inability to grow the virus in cell culture. Thus, a vector-based vaccine may be ideal. In this study, we constructed a recombinant vesicular stomatitis virus (rVSV-VP1) expressing VP1, the major capsid protein of HuNoV. Expression of the capsid protein by VSV resulted in the formation of HuNoV virus-like particles (VLPs) that are morphologically and antigenically similar to native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 through intranasal and oral routes stimulated a significantly stronger humoral and cellular immune response than baculovirus-expressed VLP vaccination. Moreover, we demonstrated that mice inoculated with rVSV-VP1 triggered a comparable level of fecal and vaginal IgA antibody. Taken together, the VSV recombinant system not only provides a new approach to generate HuNoV VLPs in vitro but also a new avenue for the development of vectored vaccines against norovirus and other noncultivable viruses.
- Published
- 2011
- Full Text
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29. A Gnotobiotic Pig Model for Determining Human Norovirus Inactivation by High-Pressure Processing
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Yuanmei Ma, David H. Kingsley, Erin DiCaprio, Steven Krakowka, Xinhui Li, Jianrong Li, John H. Hughes, Haiqiang Chen, Mu Ye, and Fangfei Lou
- Subjects
Oyster ,Food Handling ,Swine ,Food Contamination ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Microbiology ,Foodborne Diseases ,biology.animal ,medicine ,Pressure ,Animals ,Germ-Free Life ,Humans ,Viral shedding ,Feces ,Caliciviridae Infections ,Shellfish ,Infectivity ,Ecology ,biology ,Norovirus ,Virology ,Ostreidae ,Small intestine ,Disease Models, Animal ,medicine.anatomical_structure ,Viral replication ,Cell culture ,Food Microbiology ,Virus Inactivation ,Food Science ,Biotechnology - Abstract
Human norovirus (NoV) is responsible for over 90% of outbreaks of acute nonbacterial gastroenteritis worldwide and accounts for 60% of cases of foodborne illness in the United States. Currently, the infectivity of human NoVs is poorly understood due to the lack of a cell culture system. In this study, we determined the survival of a human NoV genogroup II, genotype 4 (GII.4) strain in seeded oyster homogenates after high-pressure processing (HPP) using a novel receptor binding assay and a gnotobiotic pig model. Pressure conditions of 350 MPa at 0°C for 2 min led to a 3.7-log 10 reduction in the number of viral RNA copies in oysters, as measured by the porcine gastric mucin-conjugated magnetic bead (PGM-MB) binding assay and real-time RT-PCR, whereas pressure conditions of 350 MPa at 35°C for 2 min achieved only a 1-log 10 reduction in the number of RNA copies. Newborn gnotobiotic piglets orally fed oyster homogenate treated at 350 MPa and 0°C for 2 min did not have viral RNA shedding in feces, histologic lesions, or viral replication in the small intestine. In contrast, gnotobiotic piglets fed oysters treated at 350 MPa and 35°C for 2 min had high levels of viral shedding in feces and exhibited significant histologic lesions and viral replication in the small intestine. Collectively, these data demonstrate that (i) human NoV survival estimated by an in vitro PGM-MB virus binding assay is consistent with the infectivity determined by an in vivo gnotobiotic piglet model and (ii) HPP is capable of inactivating a human NoV GII.4 strain at commercially acceptable pressure levels.
- Published
- 2015
30. Attachment and localization of human norovirus and animal caliciviruses in fresh produce
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Erin DiCaprio, Yuanmei Ma, John H. Hughes, Jianrong Li, Anastasia Purgianto, and Xiangjun Dai
- Subjects
Food Handling ,viruses ,ved/biology.organism_classification_rank.species ,Food Contamination ,Biology ,medicine.disease_cause ,Microbiology ,Plant Roots ,Virus ,Cell wall ,Mice ,Onions ,medicine ,Animals ,Humans ,Viral rna ,Pathogen ,Epidermis (botany) ,ved/biology ,Norovirus ,General Medicine ,Lettuce ,Plant Leaves ,Shoot ,Chlorine ,Caliciviridae ,Food Science ,Murine norovirus - Abstract
Fresh produce is a high risk food for human norovirus (NoV) contamination. To help control this pathogen in fresh produce, a better understanding of the interaction of human NoV and fresh produce needs to be established. In this study the attachment of human NoV and animal caliciviruses (murine norovirus, MNV-1; Tulane virus, TV) to fresh produce was evaluated, using both visualization and viral enumeration techniques. It was found that a human NoV GII.4 strain attached efficiently to the Romaine lettuce leaves and roots and green onion shoots, and that washing with PBS or 200 ppm of chlorine removed less than 0.4 log of viral RNA copies from the tissues. In contrast, TV and MNV-1 bound more efficiently to Romaine lettuce leaves than to the roots, and simple washing removed less than 1 log of viruses from the lettuce leaves and 1–4 log PFU of viruses from roots. Subsequently, the location of virus particles in fresh produce was visualized using a fluorescence-based Quantum Dots (Q-Dots) assay and confocal microscopy. It was found that human NoV virus-like particles (VLPs), TV, and MNV-1 associated with the surface of Romaine lettuce and were found aggregating in and around the stomata. In green onions, human NoV VLPs were found between the cells of the epidermis and cell walls of both the shoots and roots. However, TV and MNV-1 were found to be covering the surface of the epidermal cells in both the shoots and roots of green onions. Collectively, these results demonstrate that (i) washing with 200 ppm chlorine is ineffective in removing human NoV from fresh produce; and (ii) different viruses vary in their localization patterns to different varieties of fresh produce
- Published
- 2015
31. Identification of aromatic amino acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus is essential for both guanine-N-7 and ribose 2'-O methyltransferases
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Yuanmei Ma, Yongwei Wei, Xiaodong Zhang, Songhua Hu, and Jianrong Li
- Subjects
Models, Molecular ,RNA Caps ,Methyltransferase ,Molecular Sequence Data ,Biology ,Methylation ,Article ,Conserved sequence ,Cell Line ,Substrate Specificity ,chemistry.chemical_compound ,Amino Acids, Aromatic ,Viral Proteins ,Virology ,Catalytic Domain ,Aromatic amino acids ,Animals ,Amino Acid Sequence ,Binding site ,Polymerase ,Conserved Sequence ,Messenger RNA ,Sequence Homology, Amino Acid ,RNA methylation, replication and gene expression ,RNA ,DNA-Directed RNA Polymerases ,Methyltransferases ,Vesiculovirus ,mRNA cap methyltransferase ,Molecular biology ,Recombinant Proteins ,Biochemistry ,chemistry ,Amino Acid Substitution ,Vesicular stomatitis virus ,Mutation ,biology.protein ,Mutagenesis, Site-Directed ,RNA, Viral - Abstract
Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.
- Published
- 2010
32. Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases.
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Chang Liu, Yuanmei Ma, Yang Yang, Yuan Zheng, Jian Shang, Yusen Zhou, Shibo Jiang, Lanying Du, Jianrong Li, and Fang Li
- Subjects
- *
PORCINE epidemic diarrhea virus , *CORONAVIRUS diseases , *PROTEOLYTIC enzymes , *LYSOSOMAL storage diseases , *MICROBIOLOGICAL assay , *PORK industry , *INFECTIOUS disease transmission - Abstract
Porcine epidemic diarrhea coronavirus (PEDV) is currently devastating the United States pork industry by causing an 80-100% fatality rate in infected piglets. Coronavirus spike proteins mediate virus entry into cells, a process that requires the spike proteins to be proteolytically activated. It has been a conundrum which proteases activate PEDV entry. Here we systematically investigated the roles of different proteases in PEDV entry using pseudovirus entry, biochemical, and live virus infection assays. We found that the PEDV spike is activated by lysosomal cysteine proteases but not proprotein convertases or cell surface serine proteases. Extracellular trypsin activates PEDV entry when lysosomal cysteine proteases are inhibited. We further pinpointed cathepsin L and cathepsin B as the lysosomal cysteine proteases that activate the PEDV spike. These results advance our understanding of the molecular mechanism for PEDV entry and identify potential antiviral targets for curbing the spread of PEDV. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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33. mRNA Cap Methylation Influences Pathogenesis of Vesicular Stomatitis Virus In Vivo.
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Yuanmei Ma, Yongwei Wei, Xiaodong Zhang, Yu Zhang, Hui Cai, Yang Zhu, Shilo, Konstantin, Oglesbee, Michael, Krakowka, Steven, Whelan, Sean P. J., and Jianrong Li
- Subjects
- *
MRNA guanylyltransferase , *VESICULAR stomatitis , *IMMUNE system , *METHYLTRANSFERASES , *ADENOSYLMETHIONINE , *MICROBIAL virulence - Abstract
One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2′-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452- 456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSVK1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2′-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2′-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. IMPORTANCE Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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34. hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus.
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Mi Young Kim, Yuanmei Ma, Yu Zhang, Jianrong Li, Yaoling Shu, and Oglesbeea, Michael
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- *
VESICULAR stomatitis , *RNA virus infections , *HEAT shock proteins , *INTERFERONS , *MACROPHAGES , *GENE expression - Abstract
The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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35. Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce.
- Author
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DiCaprio, Erin, Yuanmei Ma, Purgianto, Anastasia, Hughes, John, and Jianrong Li
- Subjects
- *
NOROVIRUSES , *CALICIVIRUSES , *LETTUCE , *MICROBIOLOGY , *MICROBIAL ecology , *REVERSE transcriptase polymerase chain reaction , *RNA , *MICROBIOLOGICAL assay - Abstract
Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 x 106 RNA copies/ml ofahumanNoV geno-group II genotype 4 (GII.4) strain or 1 X 106 to 2 X 106 PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (105 to 106 RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (101 to 103 PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (105 to 106 PFU/g) at day 3 or 7 post-inoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
36. Vesicular Stomatitis Virus as a Vector To Deliver Virus-Like Particles of Human Norovirus: a New Vaccine Candidate against an Important Noncultivable Virus.
- Author
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Yuanmei Ma and Jianrong Li
- Subjects
- *
NOROVIRUSES , *PREVENTION of communicable diseases , *CELL culture , *LABORATORY mice , *MIRIDAE - Abstract
Human norovirus (HuNoV) is a major causative agent of food-borne gastroenteritis worldwide. Currently, there are no vaccines or effective therapeutic interventions for this virus. Development of an attenuated vaccine for HuNoV has been hampered by the inability to grow the virus in cell culture. Thus, a vector-based vaccine may be ideal. In this study, we constructed a recombinant vesicular stomatitis virus (rVSV-VP1) expressing VP1, the major capsid protein of HuNoV. Expression of the capsid protein by VSV resulted in the formation of HuNoV virus-like particles (VLPs) that are morphologically and antigenically similar to native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 through intranasal and oral routes stimulated a significantly stronger humoral and cellular immune response than baculovirus-expressed VLP vaccination. Moreover, we demonstrated that mice inoculated with rVSV-VP1 triggered a comparable level of fecal and vaginal IgA antibody. Taken together, the VSV recombinant system not only provides a new approach to generate HuNoV VLPs in vitro but also a new avenue for the development of vectored vaccines against norovirus and other noncultivable viruses. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
37. Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica).
- Author
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Araud, Elbashir, DiCaprio, Erin, Yuanmei Ma, Fangfei Lou, Yu Gao, Kingsley, David, Hughes, John H., and Jianrong Li
- Subjects
- *
AMERICAN oyster , *FOOD microbiology , *ENTEROVIRUSES , *BIOACCUMULATION , *ROTAVIRUSES - Abstract
Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin- magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for>6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
38. A Gnotobiotic Pig Model for Determining Human Norovirus Inactivation by High-Pressure Processing.
- Author
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Fangfei Lou, Mu Ye, Yuanmei Ma, Xinhui Li, DiCaprio, Erin, Haiqiang Chen, Krakowka, Steven, Hughes, John, Kingsley, David, and Jianrong Li
- Subjects
- *
GERMFREE animals , *LABORATORY swine , *NOROVIRUSES , *VIRUS inactivation , *GASTROENTERITIS - Abstract
Human norovirus (NoV) is responsible for over 90% of outbreaks of acute nonbacterial gastroenteritis worldwide and accounts for 60% of cases of foodborne illness in the United States. Currently, the infectivity of human NoVs is poorly understood due to the lack of a cell culture system. In this study, we determined the survival of a human NoV genogroup II, genotype 4 (GII.4) strain in seeded oyster homogenates after high-pressure processing (HPP) using a novel receptor binding assay and a gnotobiotic pig model. Pressure conditions of 350 MPa at 0°C for 2 min led to a 3.7-log10 reduction in the number of viral RNA copies in oysters, as measured by the porcine gastric mucin-conjugated magnetic bead (PGM-MB) binding assay and real-time RT-PCR, whereas pressure conditions of 350 MPa at 35°C for 2 min achieved only a 1-log10 reduction in the number of RNA copies. Newborn gnotobiotic piglets orally fed oyster homogenate treated at 350 MPa and 0°C for 2 min did not have viral RNA shedding in feces, histologic lesions, or viral replication in the small intestine. In contrast, gnotobiotic piglets fed oysters treated at 350 MPa and 35°C for 2 min had high levels of viral shedding in feces and exhibited significant histologic lesions and viral replication in the small intestine. Collectively, these data demonstrate that (i) human NoV survival estimated by an in vitro PGM-MB virus binding assay is consistent with the infectivity determined by an in vivo gnotobiotic piglet model and (ii) HPP is capable of inactivating a human NoV GII.4 strain at commercially acceptable pressure levels. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
39. Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo.
- Author
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Hui Cai, Yu Zhang, Yuanmei Ma, Jing Sun, Xueya Liang, and Jianrong Li
- Subjects
- *
PHYSIOLOGICAL effects of zinc , *HUMAN metapneumovirus infection , *VIRAL replication , *PARAMYXOVIRUSES , *HISTIDINE , *RECOMBINANT DNA - Abstract
Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
40. Receptor Usage and Cell Entry of Porcine Epidemic Diarrhea Coronavirus.
- Author
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Chang Liu, Jian Tang, Yuanmei Ma, Xueya Liang, Yang Yang, Guiqing Peng, Qianqian Qi, Shibo Jiang, Jianrong Li, Lanying Du, and Fang Li
- Subjects
- *
PORCINE epidemic diarrhea virus , *PORK industry , *PROTEIN receptors , *AMINOPEPTIDASES , *SIALIC acids , *DIARRHEA , *TRANSMISSIBLE gastroenteritis in swine , *SWINE - Abstract
Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
41. Inactivation of a Human Norovirus Surrogate, Human Norovirus Virus-Like Particles, and Vesicular Stomatitis Virus by Gamma Irradiation.
- Author
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Kurtis Feng, Divers, Erin, Yuanmei Ma, and Jianrong Li
- Subjects
- *
NOROVIRUSES , *VESICULAR stomatitis , *FOOD preservation , *FOODBORNE diseases , *FOOD contamination , *VIRAL proteins - Abstract
Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
42. Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses.
- Author
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Fangfei Lou, DiCaprio, Erin, Xinhui Li, Xianjun Dai, Yuanmei Ma, Hughes, John, Haiqiang Chen, Kingsley, David H., and Jianrong Li
- Subjects
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NOROVIRUSES , *REVERSE transcriptase polymerase chain reaction , *GENOTYPES , *BINDING site assay , *VIRAL receptors - Abstract
Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagated in vitro. In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1>GII.6>GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H2O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
43. Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain.
- Author
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Jing Sun, Yongwei Wei, Rauf, Abdul, Yu Zhang, Yuanmei Ma, Xiaodong Zhang, Shilo, Konstantin, Qingzhong Yu, Saif, Y. M., Xingmeng Lu, Lian Yu, and Jianrong Li
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METHYLTRANSFERASES , *BIRD diseases , *VIRAL vaccines , *VIRUS diseases in poultry , *ADENOSYLMETHIONINE , *INFECTIOUS disease transmission - Abstract
Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2′-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2′-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
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