SJCEIRR researchers responded quickly to the influenza A(H5) outbreak in cattle. Read about their initial study below or in PDF format.

Purpose: To determine if infectious influenza viruses can be recovered from milk samples obtained from supermarkets using tissue culture and egg propagation methods.

Methods:
Samples:
Milk Samples: Four influenza A PCR (US CDC influenza A virus matrix (M) assay as described in¹) positive milk samples purchased from retail supermarkets were used. The sample with lowest cycle threshold (Ct) value, sample 1, was extracted and run in quadruplicate. Ct values; sample 1 (Ct 29), sample 9 (Ct 31), sample 10 (Ct 30), sample 13 (Ct 31). All samples were also positive with US CDC’s H5 B assay² and consequently confirmed as A(H5).

Cell Culture:
1. Madin-Darby Canine Kidney cells (MDCKs, ATCC, P35) were plated at 10⁶ cells/well of 6 well plates in growth medium (MEM, 5% fetal bovine sera, 1x penicillin/streptomycin/ amphotericin B, 1mM L-glutamine) overnight, resulting in confluent monolayers.

2. Cells (n=1 well per virus dilution) were washed 3x with sterile phosphate buffered saline (PBS), inoculated for 1 hr at 37°C with:

• 1mL milk sample prepared neat (undiluted) 
• 1mL milk sample diluted 1:4 in infection medium (MEM, 1% bovine serum albumin, 1x penicillin/streptomycin/amphotericin B, 1mM L-glutamine)
• or 1mL infection medium only (mock inoculated)

3. Inoculums were removed, monolayers were washed 3x with PBS, and replaced with 2mL infection medium. 
*No trypsin was added to the culture medium.

4. Cells were incubated for 72 hr, 37°C. Every 24 hr, supernatants were collected and assessed for hemagglutination activity (HA assay) as an indicator of virus particle presence, using chicken red blood cells (cRBCs).

Eggs:

1. 10-day old embryonated chicken eggs (n=3 per virus dilution) were inoculated with:

• 200 µL milk sample diluted 1:1 in antibiotic.
• 100 µL milk sample diluted 1:10 in antibiotic/PBS.
• 100 µL egg antibiotics/PBS (mock-inoculated)

2. Eggs were candled daily for 72 hr to assess cell viability. 

3. At 72 hpi, allantoic fluid was harvested from each egg and assessed for hemagglutination activity as an indicator of virus particle presence, using cRBCs.

Results:
Cell culture: No CPE was observed from cells inoculated with any milk sample at any timepoint. Similarly, no HA activity was detected at any time point. A positive control for HA activity was used and yielded >=128 HAU/50 µL.

Eggs: No egg death was observed with any milk sample at any timepoint. All allantoic fluid from 72 hpi incubated eggs was negative for HA activity (0 HA activity). A positive control for HA activity was used and yielded >=128 HAU/50 µL.

Second passage: We next collected cell culture supernatant and allantoic fluid from 72hpi samples and used these (neat without dilution) to inoculate additional cells and eggs as described above. No evidence of virus growth was detected at 72 hpi in these samples.

Conclusion:
No evidence for viable infectious influenza A virus was found in any of the four samples tested.

The information included here has been shared with U.S. government agencies responsible for monitoring and responding to the current H5N1 influenza outbreak in U.S. dairy cattle.

¹ Table 1 - Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2 - Volume 27, Number 7—July 2021 - Emerging Infectious Diseases journal - CDC
² protocols_influenza_virus_detection_feb_2021.pdf (who.int)