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Volume 30, Number 11—November 2024
Research Letter

Influenza A(H5N1) Virus Resilience in Milk after Thermal Inactivation

C. Joaquin Caceres, L. Claire Gay, Flavio Cargnin Faccin, Dikshya Regmi, Roberto Palomares, and Daniel R. PerezComments to Author 
Author affiliation: University of Georgia College of Veterinary Medicine, Athens, Georgia, USA

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Figure 1

Heat treatment of influenza virus in milk. A–C) We diluted influenza A viruses in Opti-Mem control media (Fisher Scientific, https://www.fishersci.com) or commercial off-the-shelf pasteurized whole milk and heat-treated samples of different volumes at the times and temperatures shown; we calculated time from the moment the sample was placed in the heat block. A sandwich design in a heat block ensured uniform temperature exposure. After treatment, we chilled samples on ice for 5 minutes, adjusted them to a final volume of 200 μL, and titrated by TCID50 in MDCK cells (10). Results are shown for reverse genetics wild-type strain A/Puerto Rico/8/1934 (H1N1) (A); Vietnam/1203/04, a reverse genetics virus carrying the H5 hemagglutinin and N1 neuraminidase segments from A/Vietnam/1203/2004 (H5N1) in the background of PR/8/34, with the H5 segment modified with a monobasic cleavage site (Δ) (B); and a field isolate of the wild-type highly pathogenic strain A/turkey/Indiana/3707-003/2022 (H5N1) (C). D, E) A/Puerto Rico/8/1934 (H1N1) strain was spiked on pasteurized (D) and unpasteurized (E) milk samples at the times and temperatures shown. Circles indicate individual measurements; error bars indicate 95% CIs. Light gray shaded area indicates log10 TCID50 value of 1. NS, not significant; TCID50, 50% tissue culture infectious dose.

Figure 1. Heat treatment of influenza virus in milk. A–C) We diluted influenza A viruses in Opti-Mem control media (Fisher Scientific, https://www.fishersci.com) or commercial off-the-shelf pasteurized whole milk and heat-treated samples of different volumes at the times and temperatures shown; we calculated time from the moment the sample was placed in the heat block. A sandwich design in a heat block ensured uniform temperature exposure. After treatment, we chilled samples on ice for 5 minutes, adjusted them to a final volume of 200 μL, and titrated by TCID50 in MDCK cells (10). Results are shown for reverse genetics wild-type strain A/Puerto Rico/8/1934 (H1N1) (A); Vietnam/1203/04, a reverse genetics virus carrying the H5 hemagglutinin and N1 neuraminidase segments from A/Vietnam/1203/2004 (H5N1) in the background of PR/8/34, with the H5 segment modified with a monobasic cleavage site (Δ) (B); and a field isolate of the wild-type highly pathogenic strain A/turkey/Indiana/3707-003/2022 (H5N1) (C). D, E) A/Puerto Rico/8/1934 (H1N1) strain was spiked on pasteurized (D) and unpasteurized (E) milk samples at the times and temperatures shown. Circles indicate individual measurements; error bars indicate 95% CIs. Light gray shaded area indicates log10 TCID50 value of 1. NS, not significant; TCID50, 50% tissue culture infectious dose.

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