... were performed under biosafety level 2 conditions at the Missouri State University Vivarium. Unless specified, infection of cells by virus was performed at a multiplicity of infection (MOI) of 1 plaque-forming unit (pfu) per cell in a 6-well plate without trypsin supplementation. Influenza viruses are in the family Orthomyxoviridae and carry 8 negative-sense single-stranded RNA in their genome 1,2. 9. Influenza A Exposure Control Plan At week 6, under Animal Biosafety Level … Viral load – influenza virus titres in lung homogenates, nasal washes or BAL. Hence, this study shows that modifications of the PR8 background can optimize influenza vaccine seed viruses. matory cytokine and chemokine levels were relatively elevated in the bronchoalveolar lavage (BAL) supernatant from mice given the HP virus (Fig. All the work with the influenza viruses was performed in a Class II biosafety cabinet (BSC) in a biosafety level-2 (BSL-2) laboratory. ... 2009 H1N1 viruses were conducted under biosafety level 3 enhanced (BSL3+) containment in accordance with guidelines of the World Health Organization [15]. Influenza A(H5N1) viruses isolat-ed in Asia and A/Puerto Rico/8/34 (PR8) (H1N1) were Influenza viruses cause over 500,000 deaths worldwide 1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism 2.Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the … The chicken in vivo challenge experiment was conducted in biosafety level 3 containment facilities at the Friedrich-Loeffler-Institut (FLI), Greifswald-Insel Riems, Germany. 3 and table S1).The initial improvement in the backbone resulted from the use of genes from a PR8 variant (designated PR8x) that had been adapted over five passages to growth in MDCK … The virus was prepared as described previously (Kondoh et al. This book is organized to provide systematic coverage of all related issues and will serve equally well as both a textbook and a handbook for general reference. S13, B and C, and S14). By contrast, a low-dose infection with CA09 H1N1 influenza virus induced substantially higher levels of these mediators (fig. Charles River offers human influenza type A and B viruses, as well as Sendai virus (Cantell strain), supplied as allantoic fluid and sucrose gradient-purified. This Implementation Plan clarifies the roles and responsibilities of governmental and non-governmental entities, including Federal, State, local, and tribal authorities and regional, national, and international stakeholders, and provides ... Found inside – Page 94Specifically listed are : Influenza rinis A / PR8 / 34 Newcastle virus ... agreed that they are suitable for science experiments at a junior level . This two-volume work covers the molecular and cell biology, genetics and evolution of influenza viruses, the pathogenesis of infection, resultant host innate and adaptive immune response, prevention of infection through vaccination and ... TRA2A inhibits avian influenza virus YS replication and promotes human influenza virus PR8 replication. Found insideThis is a valuable resource for those seeking to embrace and implement biorisk management systems in their facilities and operations, including the biological research, clinical diagnostic, and production/manufacturing communities. All virus infections were carried out in a Biosafety Level 2 facility, except the influenza A/Vietnam/1203/04 (H5N1) virus experiment, which was conducted in a Biosafety Level 3 facility in CDC, Atlanta, GA, USA. When, in late 2011, it became public knowledge that two research groups had submitted for publication manuscripts that reported on their work on mammalian transmissibility of a lethal H5N1 avian influenza strain, the information caused an ... Influenza A viruses were inoculated into 10-day-old 92 embryonated chicken eggs at 37℃ for 3 days, whereas influenza B virus was amplified at 35℃ An influenza A virus (H7N9) anti-neuraminidase monoclonal ... and the remaining genes from PR8. An influenza virus strain, A/Puerto Rico/8/34 (H1N1; PR8) was used in this study. To investigate the role of IGF1 in IAV-mediated acute inflammatory lung injury, the expression of IGF1 and inflammatory cytokines was tested after IAV A/Puerto … At week 6, under Animal Biosafety Level 3 enhanced conditions, mice were anesthetized with avertin and inocu-lated intranasally with challenge viruses. PR8-huN1, PR8-avN1, and A/Vietnam/1203/04 were administered at doses of 10 and 100 50% MLD 50 (mouse lethal doses). METHODSVirus preparation. All work involving infectious H5N1 influenza was performed in government-approved biosafety level 3–enhanced containment facilities with experimental protocols in compliance with applicable federal statutes and institutional guidelines. Hence, there is an urgent need to develop universal vaccines against influenza virus. Animal Influenza, Second Edition is a comprehensive text on animal influenza. As reported elsewhere6,15, the Q226L/ G228S mutations led to increased binding to Siaa2,6Gal. International Reagent Resources (IRR) was established by the Centers of Disease Control (CDC). To date, almost all human cases have been linked to close contact with infected poultry. Keywords: Sheng Jiang san, Anti-influenza activity, Neuraminidase inhibition, Immune regulation Background Influenza is a contagious respiratory illness causing sea-sonal epidemics and occasional pandemics. A virulent strain of influenza A virus, A/PR/8/34 (H1N1) (PR8), was kindly provided by Dr. Yuelong-Shu (Chinese Center for Disease Control and Prevention). The infectious materials were handled in a biosafety level 2 facility under approved protocols in accordance with guidelines of Hokkaido University. S14), giving rise to overt lung inflammation that worsened over the course of viral infection, despite robust Il10 expression (figs. It is hoped that this book will stimulate our thinking, help us in designing new experiments, and possibly show avenues leading to the control of the diseases associated with influenza viruses. Influenza virus isolation, propagation, and characterization protocols are critical for completing reproducible basic research studies and for generating vaccine seed stocks. Mice inoculated with PR8 had viral titers peaking at 72 hours post inoculation (p.i.) Found inside – Page 399The use of LPAIV strains has fewer biosecurity and biosafety concerns in relation ... the six internal genes from an influenza A vaccine strain such as PR8, ... Specifications for personal protection are provided for both BSL2 enhanced and BSL3 24 enhanced biosafety levels, and guidance is provided on biosafety management and Unless specified, infection of cells by virus was performed at a multiplicity of infection (MOI) of 1 plaque-forming unit (pfu) per cell in a 6-well plate without trypsin supplementation. Some virus stocks were propagated in the allantoic cavity of embryonated hen eggs at 34°C for 48-72 h (A/PR/8) or 37°C for 24 h (SP-83). while mice given the PR8-PB1-F2(1918) virus reached high titers (equivalent to the peak of PR8) slightly earlier at 48 hours … Found inside – Page 306Cell culture-based influenza vaccine manufacturing demonstrated a little anticipated benefit during the 2009 H1N1 ... biosafety levels had been lowered. The death toll of influenza epidemics is between 250,000 to 500,000. The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. Fig.6. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Variants I202T/R220S, W153R/T160I, N169I/H184L/I217M and H130Q/ K157E resembled VN1203/PR8 in their binding to glycans, despite All experiments with live H5N1 virus were done in a biosafety level-3 containment laboratory. Found inside – Page 92... of HA∆ and NA plasmids with PR8 internal genes plasmids in Vero cells; ... The biosafety level requirement for each step is shown on the left column ... Individual Human Reference Antiserum to Influenza A/Indonesia/05/2005 (H5N1), High Titer, NR-33669.” Biosafety Level: 1 Appropriate safety procedures should always be used with this material. 3c) with PR8-H5N8 (PB2-627E). ... as a seasonal human influenza strain. However, the CVVs generated by using internal genes from PR8 are suitable for egg-based production platform. Influenza, commonly called "the flu", is an infectious disease caused by influenza viruses. Influenza A Virus (PR8 Strain) Document Title – if multipage document, update footer in page 2 also. Backbones for synthetic virus rescue. 2012). This book is an indispensable tool for anyone involved in the research, development, or manufacture of new or existing vaccines. It describes a wide array of analytical and quality control technologies for the diverse vaccine modalities. 293T, A549, sh-NMBR, sh-Luciferase and BMDM cells were seeded in 6-well plates at 37 °C in 5% CO 2. For the preparation of pandemic influenza CVVs, reverse genetic technology is a common method to generate high-growth reassortant viruses using six internal genes derived from A/Puerto Rico/8/34 (PR8) for egg-based manufacturing systems. However, they occasionally provide poor protection owing to antigenic mismatch. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, a, Binding of VN1203 mutants to sialylglycopolymers in solid-phase binding assays.A human virus (K173/PR8), an avian virus (VN1203/PR8) and mutant VN1203/PR8 viruses were compared for their ability to bind to sialylglycopolymers containing either α2,3–linked (blue) or α2,6-linked (red) sialic acids. Ten 6-week-old chickens (VALO SPF; Lohmann Tierzucht GmbH, Cuxhaven) were inoculated oculo-oronasally with 10 6 50% tissue culture infectious dose 50 (TCID 50) of either Gi-wt or Gi-NS-PR8 … Current influenza … x . All experiments with H5N1 subtypes were conducted under biosafety level 3, enhanced containment. We found that influenza virus infections reduced blood flow speed and decreased neutrophil motility. Shipra Sharma was supported by a fellowship from the Indian Medical Research Council, New Delhi, India. Page . All experiments with H5N1 subtypes were conducted under biosafety level 3, enhanced containment. A/Viet Nam/1203/2004 (H5N1) Virus Unique Identifier. Thrombocytopenia (low platelet count) is a commonly observed and sometimes life-threatening symptom during sepsis and severe influenza. Found inside – Page 52... is in Class 4 ) Corona viruses Influenza viruses ( all types except A / PR8 ... 17D vaccine strain BIOSAFETY LEVEL 3 AGENTS 3B 3B1 3B2 383 3BA Bacterial ... Swine flu is a present emerging infectious disease. The purpose of this book is to summarise and present the topics specifically relating to a new problem in our world, swine flu, in the forms that are unique in the present day. lethal dose; NA, neuraminidase; PR8, influenza virus A/Puerto Rico/8/34 (H1N1); PR8-avN1, reassortant PR8 ... lenge. Fouchier study reveals changes enabling airborne spread of H5N1. Strain Name. Found inside – Page 79... of BALB/cJ mice infected with influenza A/Puerto Rico/8/34 (H1N1) (PR8). ... performed in a Biosafety level 2 facility that is accredited by AALAAS. For assistance in determination of the appropriate containment level, please contact OEHS at 373-2769. Abstract. Choi, YK. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. IAV, A/California/07/2009 (H1N1) pdm09, FR‐201, was obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention. Live virus experiments using BJ501, CA07 and their mock-infected control, AF(P3), were performed in Biosafety Level 3 (P3) laboratory and live virus experiments using seasonal H1N1 and PR8 and their mock-infected control, AF(P2), were performed in Biosafety level 2 (P2) laboratory, under governmental and institutional guidelines. Influenza lung titers (Figure 1A,C), for both PR8 and PR8-PB1-F2(1918), initially increase exponentially reaching maximum titers of /ml lung homogenate and /ml lung homogenate, respectively . 14-17 For instance, it was reported in 14% of the hospitalized cases globally during the 2009 influenza pandemic. ... All studies involving IAV infection were carried out in biosafety level 2 (BSL2 and ABSL2) conditions and approved facility. Culture-Derived Influenza A/PR/8/34 (H1N1) Virus Stock and the Production Impact ... BSL-1 Biosafety level 1 BSL-2 Biosafety level 2 BTEC Biomanufacturing Training and Education Center ... PR8 Influenza A/Puerto Rico/8/1934 (H1N1) SRID Single-radial-immunodiffusion assay TCID 50 Tissue culture infectious dose - 50% Anti-viral therapy includes vaccines and a few anti-viral drugs. Found inside – Page 32Experiments with VN1203 / 04 and HK156 / 97 viruses were conducted in a biosafety level ( BSL ) 3+ containment facility . The immunogenicity and efficacy of a F.I. Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. BSL2 Exposure Control Plan: . We identified α‐actinin‐4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). YYMMDD. However vaccines are not always available in time, as demonstrated by the emergence of the new 2009 H1N1-type pandemic strain of swine origin (S-OIV) in April 2009, and the acquisition of resistance to neuraminidase inhibitors such … Found inside – Page 334These events have raised concerns about the potential of avian influenza viruses ... an egg-based vaccine in a biosafety level-2 production facility without ... A/Vietnam/1203/2004 or A/Puerto Rico/8/34 (H1N1; PR8) strain by using reverse genetics (15). All viruses of human or primate origin must be handled at Biosafety Level 2 or higher. Ripk2-/-, Trif-/-, Mavs-/-, Casp1/11 ... Mouse-adapted influenza A/PR/8/34 H1N1 virus (hereafter referred as PR8) stocks were prop-agated by allantoic inoculation of hen’s eggs with seed virus. Found inside – Page 39All influenza viruses were propagated on MDCK-II cells in infection medium [DMEM ... Experiments involving HPAIV were conducted in a biosafety level (BSL) 3 ... Vaccination with H7N2-PR8 virus alone protected mice from infection in the lungs, but CAN/04 was isolated from nasal turbinates (2.5 log 10 EID 50 /ml; Table Table5) 5) and spleen (1.2 log 10 EID 50 /ml) in one out of three mice. Color-flu viruses stably express high levels of a fluorescent protein fused with the NS1 viral protein, and show replicative ability and virulence similar to those of wild-type influenza … We further increased rescue efficiency by using improved influenza backbones (sets of genome segments encoding influenza virus proteins other than HA and NA) (Fig. In general, low-risk oncogenic viruses may be handled at Biosafety Level 1 and moderate risk oncogenic viruses may be handled at Biosafety Level 2. 3 and table S1).The initial improvement in the backbone resulted from the use of genes from a PR8 variant (designated PR8x) that had been adapted over five passages to growth in MDCK … Experiments using low-pathogenic North American viruses of avian origin (TK/VA/02 and H7N2-PR8) were conducted according to approved biosafety level 2 protocols. 3.3.1 Biosafety levels All laboratory facilities commensurate with each laboratory’s function and the recommended containment level for the agents or materials being handled, based on risk assessment as per the WHO biosafety guidelines. Generation of H7N9 CVVs by reverse genetics For safety, all experiments with H7N9 CVVs were con-ducted in biosafety level 3 (BSL-3) containment approved by the Taiwan CDC. K173/PR8 preferentially bound to Siaa2,6Gal. To understand the effect of trypsin on cell viability after infection by influenza virus, we performed a cell proliferation assay. As adults, the mice were place in reverse 12 hr light–dark cycles for 2 weeks before being infected with the influenza A virus (PR8). Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice. 5 | Rev. The remaining viruses were handled under BSL2 containment. Influenza A/Puerto Rico/8/34 (PR8; H1N1) and A/Vietnam/1203/04 (VN1203; H5N1) stock viruses were prepared in 10-day-old embryonated chicken eggs or MDCK cells, respectively. Larry Bowen. The workshop summary, The Threat of Pandemic Influenza: Are We Ready? addresses these urgent concerns. Color-flu viruses stably express high levels of a fluorescent protein fused with the NS1 viral protein, and show replicative ability and virulence similar to those of wild-type influenza viruses in mice (15–17). Body weights were monitored regularly and deaths noted daily. This system has been instrumental in the development of novel influenza vaccines and in the understanding of viral pathogenicity and the functions of viral proteins. This third edition aims to provide new and updated methods on animal influenza viruses as well as more advanced protocols that will guide the reader in designing research. In addition, research on influ enza virus has led to important advances in eukaryotic molecular and cellular biology and in immunology. A major focus of this book is the molecular biology of influenza virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CL(pro). coinfection with influenza A virus and. influenza infection each year in the United States (17) 23 Figure 2: Influenza viral structure shown with spherical morphology (24) 24 ... native PR8 in allantoic fluid 68 Figure 17: Reducing Western blot of recombinant A/Puerto Rico/8/1934 HA, NYMC X-162 and ... BSL: Biosafety Level MOI: Multiplicity of Infection . ACDP agrees that though most influenza viruses from human sources may be safely handled at containment level 2 (CL2) there are circumstances and activities when a higher containment level is required to protect human and animal health. This reporter-based 50 assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with 51 high-throughput compatible sample processing and analysis. increased the M2e-specific antibody level while also boosting stalk antibody levels. A subcommittee was formed to look at two aspects of the I’s influenza policy: re - examining the medical/scientific rationale for our requirement to offer the influenza vaccine to animal care personnel, and determining the appropriate biosafety level for work with mouse-adapted influenza strains (ex. The biosafety control measures that are proposed, defined as BSL2 22 enhanced (pandemic influenza vaccine), take this and also potential risks to animal health, into 23 account. Cells and viruses 293T cells were grown in Dulbecco's modified eagle medium (D-MEM) supplemented with 10% fetal Here, we screened A/Puerto Rico/8/34 (PR8… 941 1. The modified HA viral RNA segment was combined with the viral PB2, PB1, PA, NP, M, and NS viral RNA segments of a high-yield version of A/Puerto Rico/8/34 (H1N1) virus and the NA viral RNA segments of Gyr, NC09, or PR8, resulting in viruses that were exempt from select agent regulations and could be used under biosafety level 2 (BSL2) containment. The frequent reassortment of influenza virus may cause high x . 2. ... 2009 H1N1 viruses were conducted under biosafety level 3 enhanced (BSL3+) containment in accordance with guidelines of the World Health Organization [15]. Conventional influenza vaccines need to be designed and manufactured yearly. Found inside – Page 288It is not known whether a reassortant based on PR8 will be able to replicate in mice, ... Specifications for “BSL2 enhanced (pandemic influenza vaccine)” ... Some virus stocks were propagated in the allantoic cavity of embryonated hen eggs at 34°C for 48-72 h (A/PR/8) or 37°C for 24 h (SP-83). Figure 2 | Characterization of the receptor-binding properties of isolated viruses. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Found insideThe influenza virus poses a threat to human health and is responsible for global epidemics every year. Moreover, influenza is increasingly resistant to anti-viral drugs 3. Influenza A (H5N1) viruses isolated in Asia and A/Puerto Rico/8/34 (PR8) (H1N1) were propagated in embryonated chicken eggs or in Madin-Darby canine kidney (MDCK) cells. All work involving infectious H5N1 influenza was per-formed in government-approved biosafety level 3–enhanced containment facilities with experimental pro-tocols in compliance with applicable federal statutes and institutional guidelines. The acute inflammatory lung injury is an important cause of death due to influenza A virus (IAV) infection. Moreover, the expression levels of PB2, ... All experiments with YS/H5N1 virus were performed in an animal biosafety level 3 laboratory of Huazhong Agricultural University. All experiments with live influenza viruses were performed in an approved biosafety level-2 laboratory. As expected, the cHA Ca2 M2 strategy showed an enhanced M2e antibody titer and protected mice from a challenge virus with heterosubtypic HA and NA more e ectively than the repeated immunizations with PR8 Ca2 M2 virus (expressing the M2e epitope) alone. By contrast, a low-dose infection with CA09 H1N1 influenza virus induced substantially higher levels of these mediators (fig. This volume focuses on various methods used by researchers to study mucosal-associated invariant T (MAIT) cells in the aspects described below. The CDC-IRR resources are available to public health facilities, research institutions, and life science companies. In contrast, VN1203/ PR8 bound to only Siaa2,3Gal. However, the 2009 pandemic H1N1 virus infection is predominantly seen in children and adults (<35 years of age), but rarely in people older than 65 years of age. 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