Estimating viral titres in solutions with low viral loads

Brownie, C.; Statt, J.; Bauman, Patricia; Buczynski, Greg; Skjolaas, K.A.; Lee, D.; Hotta, Jun‐ichi; Roth, Nathan

doi:10.1016/j.biologicals.2011.06.007

Cited by 19 publications

(19 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4B). As expected, this immune serum inhibited the homotypic sialidase most efficiently (NI 50 a All immune sera were produced in SPF chickens, except serum directed to the NA antigen of A/swine/Belzig/2/01 (H1N1), which was raised in SPF pigs. Animal groups (n ϭ 4/5) were immunized with the vaccines listed.…”

Section: Expression Of Functionally Active Neuraminidasementioning

confidence: 64%

“…Serial 2-fold diIutions of immune sera (50 l) were incubated in quadruplicate for 1 h at room temperature with 50 l of MEM containing 100 TCID 50 To analyze the inhibition of virus entry by immunofluorescence, MDCK cells were grown on 12-mm glass coverslips and incubated for 1 h at 4°C with 0.5 TCID 50 /cell of A/chicken/Yamaguchi/7/04 (H5N1). Subsequently, the cells were washed with PBS, incubated with MEM for 5 h at 37°C, fixed, and permeabilized as described above.…”

Section: Methodsmentioning

confidence: 99%

“…In the case of LPAIV, infected cells were detected by immunostaining with a monoclonal antibody directed to the NP antigen (49). Virus titers were calculated according to the Spearman-Kärber method and expressed as 50% tissue culture infectious doses (TCID 50 )/ml (50).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

Halbherr¹,

Ludersdorfer²,

Ricklin³

et al. 2015

J Virol

View full text Add to dashboard Cite

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCEThe neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

show abstract

Section: Expression Of Functionally Active Neuraminidasementioning

confidence: 64%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

Halbherr¹,

Ludersdorfer²,

Ricklin³

et al. 2015

J Virol

View full text Add to dashboard Cite

show abstract

“…The Vero E6 plates were scored microscopically for cytopathic changes (cpe). Viral titers were expressed as TCID 50 per ml as calculated with the Kärber method [31]. The highly attenuated vaccinia virus (VACV) strain MVA (clonal isolate F6, CEF passage 584) was used for this study.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Doel

Volz²,

Roose

et al. 2014

PLoS Negl Trop Dis

View full text Add to dashboard Cite

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.

show abstract

“…In cases where only a few positive cultures were found and the Spearman–Kaerber method was not applicable, the most probable number (MPN) method, a Poisson-based maximum likelihood method, was used [34, 35]. The MPN method estimated the virus concentration that could be transferred to TCID 50 [36, 37]. …”

Section: Methodsmentioning

confidence: 99%

Pathogen Safety of a New Intravenous Immune Globulin 10% Liquid

et al. 2017

View full text Add to dashboard Cite

BackgroundThe manufacturing process of a new intravenous immune globulin (IVIG) 10% liquid product incorporates two dedicated pathogen safety steps: solvent/detergent (S/D) treatment and nanofiltration (20 nm). Ion-exchange chromatography (IEC) during protein purification also contributes to pathogen safety. The ability of these three process steps to inactivate/remove viruses and prions was evaluated.ObjectivesThe objective of this study was to evaluate the virus and prion safety of the new IVIG 10% liquid.MethodsBovine viral diarrhea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), mouse encephalomyelitis virus (MEV), porcine parvovirus (PPV), and pseudorabies virus (PRV) were used as models for common human viruses. The hamster-adapted scrapie strain 263K (HAS 263K) was used for transmissible spongiform encephalopathies. Virus clearance capacity and robustness of virus reduction were determined for the three steps. Abnormal prion protein (PrPSc) removal and infectivity of the samples was determined.ResultsS/D treatment and nanofiltration inactivated/removed enveloped viruses to below detection limits. IEC supplements viral safety and nanofiltration was highly effective in removing non-enveloped viruses and HAS 263K. Overall virus reduction factors were: ≥9.4 log10 (HIV-1), ≥13.2 log10 (PRV), ≥8.2 log10 (BVDV), ≥11.7 log10 (MEV), ≥11.6 log10 (PPV), and ≥10.4 log10 (HAS 263K).ConclusionTwo dedicated and one supplementing steps in the manufacturing process of the new IVIG 10% liquid provide a high margin of pathogen safety.

show abstract

Estimating viral titres in solutions with low viral loads

Cited by 19 publications

References 8 publications

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Pathogen Safety of a New Intravenous Immune Globulin 10% Liquid

Contact Info

Product

Resources

About