H5N1 Influenza Virus in Raw Milk and Cheese: Stability & Risk
Okay, hereS a breakdown of the methods described in the provided text, focusing on the key experimental procedures. I’ll organize it into sections for clarity.
1. Luciferase assay (In Vitro)
* Purpose: To measure virus infectivity in cells.
* Procedure:
* A small volume (µl) of cell lysate (cells that may have been infected with the virus) was added to wells of a luminometer plate.
* Luciferase assay reagent (Nano-Glo from Promega) was added to each well (50 µl). Luciferase is an enzyme that produces light when it reacts with its substrate,and its activity is linked to viral infection.
* Luminescence (light output) was measured using a luminometer plate reader (BioTek Synergy LX).
* data Analysis: Luminescence was normalized to a mock-infected control (cells not exposed to the virus) and expressed as fold changes. This means the light output from infected cells was compared to the light output from uninfected cells to determine how much the virus increased luciferase activity.
2. Ferret Study (In Vivo – Oral Infectivity of HPAI H5N1)
* Animals: 20 ferrets (10 female, 10 male), 8-9 weeks old, influenza-negative.
* Housing: Individually housed in HEPA-filtered cages at an animal biosafety level 3 facility.
* Experimental Groups & Exposure Routes:
* Voluntary Feeding (n=4 per group):
* HPAI H5N1-spiked raw milk (5 ml)
* Company-made raw-milk cheese (naturally contaminated, 5g solid)
* Company-made raw-milk cheese suspension (10% suspension, 5 ml)
* Store-bought cheese (negative control, 5g)
* Direct Inoculation (n=2 per group):
* P/O (Oro-pharyngeal): 1 ml HPAI-spiked milk, administered while sedated and holding the animal upright.
* I/N (Intra-nasal): 1 ml HPAI-spiked milk, administered drop-by-drop into each nostril while sedated and holding the animal upright.
* Virus Titer: the virus in the spiked milk was adjusted to 5 x 104 EID50 (Egg Infectious Dose 50 - a measure of virus concentration) to match the titer in the naturally contaminated cheese.
* Feeding Schedule: Voluntary feeding groups received repeated doses on days 0, 3, 4, and 5 post-exposure (p.e.). Direct inoculation groups received a single dose.
* Monitoring:
* Clinical Parameters: Body temperature (transponder), body weight, and clinical signs where monitored daily.
* sample Collection:
* Nasal Swabs (NS), Oropharyngeal Swabs (OPS), Rectal Swabs (RS): Collected on days 0, 1, 3, 5, 7, 10, and 14 p.e. Stored in viral transport medium (VTM) at -80°C.
* Blood: Collected from the cranial vena cava on days 0, 7, and 14 p.e. Serum was separated,aliquoted,and stored at -20°C.
* Euthanasia: Animals were euthanized when they reached a humane endpoint or at the end of the study (day 14 p.e.).
* Ethics: The study was approved by the Cornell University Institutional Animal Care and Use committee (IACUC approval number 2024-0094).
3. Virus Neutralization Assay
* Purpose: To measure the levels of neutralizing antibodies against HPAI H5N1 in the ferret serum.
* Method: A virus neutralization assay was performed using a recombinant virus (rTX2/24-miniGFP2) that expresses a fluorescent protein (miniGFP2). (Details of the specific assay procedure are referenced to a previous publication).
Let me know if you’d like me to elaborate on any specific aspect of these methods!