Preparation of Noninfectious Arbovirus Antigens

Brand, Orville M.; Allen, William P.

doi:10.1128/aem.20.3.298-302.1970

Applied Microbiology

1970

DOI: 10.1128/aem.20.3.298-302.1970

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Preparation of Noninfectious Arbovirus Antigens

Orville M. Brand¹,

William P. Allen²

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Cited by 4 publications

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“…pellets are resuspended to 1/50 volume in borate-buffered saline (pH 9.0) containing 0.2% bovine serum albumin. Sodium azide to 0.1% may be added as a preservative, and the preparation may be rendered noninfectious by incubation with an equal volume of 0.2 M tris(hydroxymethyl)aminomethane-0.2 M f3-propiolactone (BPL; Koch-Light) at 4 C for 4 days (2).…”

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confidence: 99%

Rapid Preparation of Hemagglutinins of Togaviruses from Infected Cell Culture Fluids

Della-Porta

Westaway

1972

Applied Microbiology

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Antigens in infected cell culture fluids can be easily concentrated by polyethylene glycol precipitation to yield suitable hemagglutinating and complement-fixing antigens for several togaviruses. Although accumulation of hemagglutinin (HA) of Japanese B encephalitis (JBE) virus in infected cell culture fluids has been described (5, 8), little or no HA activity has been recovered for other togaviruses (9, 10). [The term togavirus was recommended by the Subcommittee of Vertebrate Viruses (1) to the International Committee on Nomenclature of Viruses for the arboviruses belonging to serogroups A and B.] We routinely prepare HA of slowgrowing group B and other togaviruses for HAinhibition (HI) tests by concentrating infected culture fluids by using polyethylene glycol (PEG) precipitation (7, 15). Confluent monolayers of PS cells or Vero cells are inoculated with 1/100 infected mouse brain stock for 1 hr, washed, and incubated at 37 C in Eagle's medium or medium 199 supplemented with 0.2% bovine serum albumin. Culture methods, cell lines, and virus strains employed were described previously (6, 13, 14). When cytopathic effect develops, harvested fluids are clarified at 10,000 x g for 10 min at 4 C and then made 8% with respect to PEG 6000 (Koch-Light Ltd., Colnbrook, Bucks, England) by adding 40% (w/v) stock solution at pH 7.8 in Hanks salt solution. After 1 hr at 4 C, virus is sedimented at 10,000 x g for 30 min, the fluid is decanted and drained, and This investigation was supported by a grant from the National Health and Medical Research Council of Australia.

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Rapid Preparation of Hemagglutinins of Togaviruses from Infected Cell Culture Fluids

Della-Porta

Westaway

1972

Applied Microbiology

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Inactivation of African horse-sickness virus by betapropiolactone and by pH

Parker

1975

Archives of Virology

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The inactivation of several types of African horse sickness virus (AHSV) by pH and by betapropiolactone (BPL) was studied. At 19 degrees - 22 degrees C the virus was stable between pH 6.0 and 10.4, whether suspended in mouse brain or in serumfree buffer. Below pH 5.6 and above pH 10.9, more than 99 per cent of infectivity was inactivated within 15 minutes. The addition of 50 per cent serum did not influence pH stability. Disinfection in the presence of citric acid and caustic soda is briefly discussed. Inactivation by BPL was complete within 30 minutes at 37 degrees C, yet incomplete after 15 hours at 4 degrees C. Types 3 and 9 virus grown in suckling mouse brain and types 1, 3 and 9 produced in pig kidney cells were equally susceptible to 0.1 per cent BPL, more than 99.9 per cent being inactivated. The effectiveness of BPL was reduced at least 10-fold by the addition of 50 per cent serum. No infective virus was detected following incubation of either tissue culture virus with 0.2 per cent BPL or of mouse brain virus with 0.3 per cent BPL. Virus suspensions exposed to 0.3 per cent BPL required buffering with Tris of at least 0.05 molar strength in order to maintain the pH within an acceptable range. Inactivated antigens prepared with 0.4 per cent or lower concentrations of BPL were immunogenic in guinea pigs.

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Development of an algorithm for production of inactivated arbovirus antigens in cell culture

Goodman

Russell

Velez

et al. 2014

Journal of Virological Methods

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Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

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Preparation of Noninfectious Arbovirus Antigens

Cited by 4 publications

References 1 publication

Rapid Preparation of Hemagglutinins of Togaviruses from Infected Cell Culture Fluids

Rapid Preparation of Hemagglutinins of Togaviruses from Infected Cell Culture Fluids

Inactivation of African horse-sickness virus by betapropiolactone and by pH

Development of an algorithm for production of inactivated arbovirus antigens in cell culture

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