scholarly journals A biosafety level 2 surrogate for studying SARS-CoV-2 survival in food processing environmental biofilms

2021 ◽  
Author(s):  
Austin Blake Featherstone ◽  
Sapna Chitlapilly Dass
Keyword(s):  
Hepatitis Virus ◽  
Fold Increase ◽  
Meat Processing ◽  
Mixed Species ◽  
Murine Hepatitis Virus ◽  
Viral Particles ◽  
Processing Facility ◽  
Processing Plants ◽  
Level 2 ◽  
Biosafety Level

Meat processing plants have been at the center of the SARS-CoV-2 pandemic. There are several factors that contribute to the persistence of SARS-CoV-2 in meat processing plants and one of the factors is the formation of a multi-species biofilm with virus. Biofilm can act as a reservoir in protecting, harboring, and dispersing SARS-CoV-2 from biofilm to the meat processing facility environment. We used Murine Hepatitis Virus (MHV) as a surrogate for SARS-CoV-2 virus and meat processing facility drain samples to develop mixed-species biofilms on commonly found materials in processing facilities (Stainless-Steel (SS), PVC and tiles). The results showed that MHV was able to integrate into the environmental biofilm and survived for a period of 5 days at 7C. There was no significate difference between the viral-environmental biofilm biovolumes developed on different materials SS, PVC, and tiles. There was a 2-fold increase in the virus-environmental biofilm biovolume when compared to environmental biofilm by itself. These results indicate a complex virus-environmental biofilm interaction which is providing enhanced protection for the survival of viral particles with the environmental biofilm community.

scholarly journals Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells

2022 ◽  
Vol 5 (1) ◽  
pp. 5
Author(s):  
Tautvydas Shuipys ◽  
Naim Montazeri
Keyword(s):  
Cell Lines ◽  
Hepatitis Virus ◽  
Plaque Assay ◽  
Culture Collection ◽  
Murine Hepatitis Virus ◽  
Virus Particles ◽  
Pathogenic Viruses ◽  
Overlay Assay ◽  
Level 2 ◽  
Biosafety Level

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Cleaning Conveyor Belts in the Chicken-Cutting Area of a Poultry Processing Plant with 45°C Water

2014 ◽  
Vol 77 (3) ◽  
pp. 496-498 ◽  
Author(s):  
V. M. SOARES ◽  
J. G. PEREIRA ◽  
C. M. ZANETTE ◽  
L. A. NERO ◽  
J. P. A. N. PINTO ◽  
...  
Keyword(s):  
Conveyor Belt ◽  
Hot Water ◽  
Poultry Meat ◽  
Processing Plant ◽  
Meat Processing ◽  
Work Shift ◽  
Processing Facility ◽  
Processing Plants ◽  
Conveyor Belts ◽  
Poultry Processing

Conveyor belts are widely used in food handling areas, especially in poultry processing plants. Because they are in direct contact with food and it is a requirement of the Brazilian health authority, conveyor belts are required to be continuously cleaned with hot water under pressure. The use of water in this procedure has been questioned based on the hypothesis that water may further disseminate microorganisms but not effectively reduce the organic material on the surface. Moreover, reducing the use of water in processing may contribute to a reduction in costs and emission of effluents. However, no consistent evidence in support of removing water during conveyor belt cleaning has been reported. Therefore, the objective of the present study was to compare the bacterial counts on conveyor belts that were or were not continuously cleaned with hot water under pressure. Superficial samples from conveyor belts (cleaned or not cleaned) were collected at three different times during operation (T1, after the preoperational cleaning [5 a.m.]; T2, after the first work shift [4 p.m.]; and T3, after the second work shift [1:30 a.m.]) in a poultry meat processing facility, and the samples were subjected to mesophilic and enterobacterial counts. For Enterobacteriaceae, no significant differences were observed between the conveyor belts, independent of the time of sampling or the cleaning process. No significant differences were observed between the counts of mesophilic bacteria at the distinct times of sampling on the conveyor belt that had not been subjected to continuous cleaning with water at 45°C. When comparing similar periods of sampling, no significant differences were observed between the mesophilic counts obtained from the conveyor belts that were or were not subjected to continuous cleaning with water at 45°C. Continuous cleaning with water did not significantly reduce microorganism counts, suggesting the possibility of discarding this procedure in chicken processing.

scholarly journals Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

2020 ◽  
Author(s):  
Katharine H.D. Crawford ◽  
Rachel Eguia ◽  
Adam S. Dingens ◽  
Andrea N. Loes ◽  
Keara D. Malone ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Single Cycle ◽  
Viral Particles ◽  
Main Target ◽  
Human Sera ◽  
Level 3 ◽  
Valuable Complement ◽  
Level 2 ◽  
Biosafety Level

AbstractSARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

scholarly journals Recombinant Human Thymosin Beta-4 Protects against Mouse Coronavirus Infection

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Rui Yu ◽  
Yunyun Mao ◽  
Kai Li ◽  
Yanfang Zhai ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Ideal Model ◽  
Highly Pathogenic ◽  
Thymosin Beta 4 ◽  
N Terminus ◽  
Coronavirus Infection ◽  
Level 2 ◽  
Biosafety Level

Coronaviruses (CoVs) are enveloped and harbor an unusually large (30–32 kb) positive-strand linear RNA genome. Highly pathogenic coronaviruses cause severe acute respiratory syndrome (SARS) (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome (MERS) (MERS-CoV) in humans. The coronavirus mouse hepatitis virus (MHV) infects mice and serves as an ideal model of viral pathogenesis, mainly because experiments can be conducted using animal-biosafety level-2 (A-BSL2) containment. Human thymosin beta-4 (Tβ4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tβ4 regulates actin polymerization and functions as an anti-inflammatory molecule and an antioxidant as well as a promoter of wound healing and angiogenesis. These activities led us to test whether Tβ4 serves to treat coronavirus infections of humans. To test this possibility, here, we established a BALB/c mouse model of coronavirus infection using mouse CoV MHV-A59 to evaluate the potential protective effect of recombinant human Tβ4 (rhTβ4). Such a system can be employed under A-BSL2 containment instead of A-BSL3 that is required to study coronaviruses infectious for humans. We found that rhTβ4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host’s immune response, alleviating pathological damage, and promoting repair of the liver. These results will serve as the basis for further application of rhTβ4 to the treatment of human CoV diseases such as COVID-19.

scholarly journals Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 513 ◽  
Author(s):  
Katharine H. D. Crawford ◽  
Rachel Eguia ◽  
Adam S. Dingens ◽  
Andrea N. Loes ◽  
Keara D. Malone ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Single Cycle ◽  
Viral Particles ◽  
Human Sera ◽  
Level 3 ◽  
Vesicular Stomatitis ◽  
Valuable Complement ◽  
Level 2 ◽  
Biosafety Level

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

scholarly journals Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

2016 ◽  
Vol 90 (16) ◽  
pp. 7415-7428 ◽  
Author(s):  
Nicole R. Sexton ◽  
Everett Clinton Smith ◽  
Hervé Blanc ◽  
Marco Vignuzzi ◽  
Olve B. Peersen ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Fold Increase ◽  
Rna Polymerases ◽  
Coxsackie Virus ◽  
Murine Hepatitis Virus ◽  
Replication Fidelity ◽  
Replicase Proteins ◽  
Positive Sense

ABSTRACTPositive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex.IMPORTANCERNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

Genetic Predisposition To The Expression Of Lymphoid Procoagulant Activity (PCA) In Response To Viral Stimulation

1981 ◽  
Author(s):  
G A Levy ◽  
J L Leibowitz ◽  
T S Edgington
Keyword(s):  
Mononuclear Cells ◽  
Hepatitis Virus ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Murine Hepatitis Virus ◽  
Peripheral Blood Mononuclear ◽  
C3h Mice ◽  
Acute Necrotizing

A predisposing foctor in the induction of viral induced disease may be the genetic make-up of the host. Leukocytes from a variety of species uniformly generate increased amounts of procoagulant molecules in response to a variety of stimuli including antigen:antibody complexes, lipopolysaccharides (LPS), complement fragments C5a and lectins. Although the amount of increased PCA varies with the concentration and duration of contact with the stimulus, all individuals respond to equivalently these stimuli. The coronavirus family is a group of RNA viruses and includes the murine hepatitis virus (MHV) which causes strain dependent forms of disease in mice. BaIb/C and DBA/2 mice develop acute necrotizing hepatitis, C3H mice develop a mild chronic disease and the A strain develops no evidence of hepatitis. Peripheral blood mononuclear cells from BaIb/C and DBA/2 mice respond both in vitro and in vivo with a marked increase in procoagulant activity (PCA), with 106 PFU of MHV-3 invoking an 80-fold increase in PCA and 10 PFU a 10 fold increase in PCA. PBM from C3H strain mice respond to MHV with a 6.8-fold increase of PCA at 106 PFU of virus. In constrast, PBM from A strain mice do not respond to 10-106 PFU or virus. The PCA was a product of monocytes as demonstrated by direct cytologic assay, however induction of PCA required the presence of lymphocytes. Two types of PCA were found, an early increase of a novel cellular prothrombinase and later increase in thromboplastin activity. Thus, the expression of MHV induced PCA appears to be genetically restricted and also correlates with susceptibility to viral induction of disease. The existence of comparable genetic restrictions in man might relate the variable incidence of thrombotic diseases. Supported by NIH grants HL- 16411 and CA-28166.

Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

1987 ◽  
Vol 45 ◽  
pp. 908-909
Author(s):  
Alain R. Trudel ◽  
M. Trudel
Keyword(s):  
Electron Microscopy ◽  
Fold Increase ◽  
Observation Time ◽  
Clinical Samples ◽  
Maximum Speed ◽  
Viral Particles ◽  
Structural Details ◽  
Latex Spheres ◽  
Increased Sensitivity ◽  
Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

Methodical guidelines on the control of fasciolosis in cattle in the Kaluga region

2018 ◽  
Vol 12 (2) ◽  
pp. 108-116
Author(s):  
A.M. Ustinov ◽  
R.T. Safiullin ◽  
R.R. Safiullin
Keyword(s):  
Infected Animal ◽  
Fasciola Hepatica ◽  
Adult Stage ◽  
Federal District ◽  
Dosage Forms ◽  
Pond Snail ◽  
Meat Processing ◽  
Processing Plants ◽  
Causative Agents ◽  
Kaluga Region

Information is provided on the causative agents of fasciolosis and the developmental biology of Fasciola hepatica. Fasciolosis of cattle is widespread in the conditions of the Kaluga Region and the Central Federal District on farms of different forms of ownership. The greatest number of F. hepatica eggs in 1 g of feces in cattle is established in JSC "Iskra" - 175,4 specimens, the lowest - in the samples from cows from private farms - 87,6 sp. At the opening slaughter of cattle at meat processing plants region and slaughterhouses invasion of their fasciolae was 34.8%. The average number fascial one infected animal was 32.1 specimens, including adult - and larval specimens of 25.2 and 6.9 copies At veterinary-sanitary expertise and helminthological autopsy of the liver, gallbladder and bile ducts installed year-round invasion fasciolae mainly in the adult stage. Young animals of the current year are infected with fasciola in the pasture massly in August-September, and the extent of invasion in November was 4.3%, for winter-stabling months an average of 12.6%, in March - 17.9% and in April after de-worming - 5.9%.The data obtained in the study of the distribution of the small pond has shown that in the Kaluga region these mollusks are widespread. In this case, the invasion of the small pond snail by larval forms of fasciolae in different years has its own peculiarities, which are influenced by climatic factors.The different dosage forms of clozantel - Rolenol and Santel and Genamectin with mixed fascioliasis and hypodermatosis of bovine in the recommended dose against subcutaneous gadfly larvae - provided 100% efficacy. The tested dosage forms of clozantel are similar in effectiveness against subcutaneous gadfly larvae (EE-100%) and fasciol (EE-95 and 90%, IE-97.4 and 96.3%).The therapeutic and economic effectiveness of complex domestic preparations of Helmicide and Fascocida in spontaneous fascioliasis of cattle is studied.

Identification of antibiotics in meat. Flow cytometry capabilities

Vsyo o myase ◽  
2020 ◽  
pp. 56-60
Author(s):  
Zayko E.V. ◽  
◽  
Kuznetsova O.A. ◽  
Bataeva D.S. ◽  
Grudistova M.A. ◽  
...  
Keyword(s):  
Raw Materials ◽  
Animal Husbandry ◽  
Flow Cytometer ◽  
Poultry Meat ◽  
Meat Processing ◽  
Beta Lactams ◽  
Antimicrobial Substances ◽  
Chemotherapy Drugs ◽  
Processing Plants ◽  
Meat Samples

The problem of the uncontrolled use of antibiotics currently remains unresolved. Step-by-step monitoring of meat using modern methods will reduce the risk of using contaminated meat raw materials for food production. Qualitative monitoring will identify samples containing residual antimicrobial substances. The use of methods for identifying groups of antibiotics will help narrow the search for antibiotics by expensive chromatographic methods. A study was carried out of beef, pork and poultry meat, which is used in meat processing plants in the production of raw smoked sausages, using two methods. At the first stage, using a qualitative microbiological method, the raw meat was evaluated for the presence of antimicrobial substances, then their group was determined using a NovoCyte flow cytometer. According to the results of a study on a flow cytometer, it was found that out of 10 groups of antibiotics that can be determined by the tested method, the group of lincosamides was not found in all meat samples. The most common groups of chemotherapy drugs in pork were sulfonamides – 29.6 %, tetracycline group – 18.5 % and beta-lactams – 14.8 %, and in beef aminoglycosides – 36.7 %, phenicols – 30 % and beta-lactams – 13.3 %. In poultry meat samples, the most common were sulfonamides – 23.2 %, fenicols – 23.2 %, and beta-lactams – 16 %. Five groups of antibiotics were found in all studied types of meat: fenicols, β-lactams, macrolides, polypeptide antibiotics, and quinolones. This indicates their widespread use in animal husbandry and poultry farming.

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