Post by Nadica (She/Her) on Jun 15, 2024 6:23:26 GMT
Inactivation of Avian Influenza A(H5N1) Virus in Raw Milk at 63°C and 72°C - Published June 14, 2024
TO THE EDITOR:
In March 2024, highly pathogenic avian influenza (HPAI) virus of A(H5N1) clade 2.3.4.4b was detected in mammalian livestock, mostly dairy cattle, in the United States and has now been reported in dairy herds in nine U.S. states.1 High titers of infectious virus and genome copies of HPAI A(H5N1) virus have been found in milk from these infected dairy cows.2 Molecular testing has also revealed the presence of HPAI A(H5N1) genetic material in approximately 20% of the samples obtained from retail pasteurized milk products, but researchers have been unable to culture virus from these samples.3 In this study, we measured the stability of HPAI A(H5N1) virus in raw milk at 63°C and 72°C, the temperatures most commonly used in commercial pasteurization.4
We diluted HPAI A(H5N1) virus A/mountain lion/MT/1/2024 (clade 2.3.4.4b; Global Initiative on Sharing All Influenza Data [GISAID] accession number, EPI_ISL_19083124) in raw (unpasteurized) cow’s milk to 106 50% tissue-culture infectious doses (TCID50) per milliliter of medium. We heat-treated the milk in a temperature-controlled heat block at 63°C and 72°C. We quantified infectious virus by means of end-point titration in Madin–Darby canine kidney cells, using 1:10 serial dilutions. HPAI A(H5N1) genome copies were quantified by quantitative reverse-transcriptase–polymerase-chain-reaction (qRT-PCR) assay.
We inferred individual titers and virus half-lives in a Bayesian framework, as described previously5 (see the Supplementary Appendix, available with the full text of this letter at NEJM.org), modeling the virus titers found in a hemagglutination assay as Poisson-distributed. At 63°C, HPAI A(H5N1) virus was inactivated from initial titers of 106 TCID50 per milliliter to undetectable levels within 2 minutes (Figure 1A). We estimated the half-life of infectious virus to be 4.5 seconds (95% credible interval, 3.5 to 5.8) at 63°C (Figure 1B and Table S1 in the Supplementary Appendix). At 72°C, we observed a decrease in virus titers from approximately 105 to approximately 101 TCID50 per milliliter within 5 seconds and then very low titers (<10 TCID50 per milliliter, at the boundary of detectability) until the 20-second time point had been reached; no viable virus was found at later time points (Figure 1A). These data are not consistent with simple exponential decay, and therefore we do not report a half-life. At both temperatures, HPAI A(H5N1) virus genome copy numbers measured by qRT-PCR decreased by less than 1 log over a period of 30 minutes at 63°C or a period of 15 seconds at 72°C (Fig. S1).
FIGURE 1
Inactivation of Highly Pathogenic Avian Influenza A(H5N1) Virus in Raw Milk at 63°C and 72°C.
We emphasize that these measurements reflect experimental conditions, and direct measurements of virus in milk from infected cows that has been heated with commercial pasteurization equipment are needed. Given our findings, however, heat treatment at 63°C would yield a decrease in infectious viral titer by a factor of 1010 within 2.5 minutes (95% credible interval, 1.9 to 3.2), so standard bulk pasteurization of 30 minutes at 63°C has a large safety buffer. At 72°C, we measured a decrease in infectious virus by a factor of 104 within 5 seconds; however, we also detected infectious virus just above the assay limit of detection for as long as 20 seconds at 72°C. This finding indicates the potential for a relatively small but detectable quantity of HPAI A(H5N1) virus to remain infectious in milk after 15 seconds at 72°C if the initial titer is sufficiently high, at least under these specific experimental conditions.
Our findings highlight the need for research on HPAI A(H5N1) virus in dairy production. Replication of these findings and extension to other dairy products is needed, including the study of milk from infected dairy cows with commercial pasteurization equipment, because treatment conditions can alter the effectiveness of heat inactivation.5 One limitation of our study is the use of raw-milk samples spiked with HPAI A(H5N1) virus, since raw milk from infected cows may have a different composition or may contain cell-associated virus that could have an effect on inactivation kinetics. Lastly, although gastrointestinal infections with HPAI A(H5N1) virus have been reported in several mammalian species,2 the dose dependence of the probability of human infection through ingestion of HPAI A(H5N1) virus in milk is unknown.