利用合成 DNA 控制 H5N1 禽流感研究

In recent months, the US FDA, CDC, USDA, and WHO have been closely tracking a US epidemic of the highly pathogenic avian Influenza (HPAI) A/H5N1 subtype in cows. HPAI H5N1 has long been viewed as a virus with pandemic potential but has not yet made a sustained jump to humans¹.

Virology and public health researchers must closely study and monitor this outbreak to help slow the spread between cattle and stay ahead of evolutionary developments that increase human transmission risks.

Here, we briefly discuss H5N1 and the current status of the bovine epidemic while also describing how synthetic nucleic acid controls support viral research progress.

H5N1 and The Unfolding Epidemic

Since the HPAI H5N1 subtype was first identified in 1996, it has been the focus of many research studies and surveillance activities². Many Influenza strains, including H5N1, originate in wild waterfowl and other avian hosts, which act as the primary natural reservoir of the virus. These viruses predominantly take root and replicate in a bird's intestinal tract following fecal-oral inoculation. Though other avian flu viruses usually cause little to no disease in birds, HPAI is characterized by higher morbidity and mortality levels and more pronounced respiratory infections in these species.

"At present, there are no documented cases of human-to-human transmission"

The virus also occasionally jumps into humans. At present, there are no documented cases of human-to-human transmission, but between January 2003 and 5月 2024, global records indicate there have been roughly 900 cases of HPAI H5N1 infection in humans with a total case fatality rate of over 50%³.

Accordingly, researchers and public health officials pay close attention when HPAI H5N1 infections occur in livestock and companion animals.

H5N1 Outbreak in the US

Since early 2022, H5N1 has been detected in ~10,000 wild birds in the US and has affected over 100 million poultry across 48 states, leading to massive culls and financial burden^4-6.

In late March 2024, the USDA first reported a multi-state outbreak of HPAI H5N1 in dairy cows⁷. As of July 31st, agricultural and public health researchers have detected H5N1 in dairy cattle and milk samples from over 170 herds across 13 states⁸. Adding to concerns, four humans have contracted the virus following exposure to dairy cows, representing the first documented instances of H5N1 leaping to humans from a mammal host. This is an important development because it indicates the virus may be evolving traits that make it easier to infect humans. While it is rare for avian viruses to jump directly to humans, sustained human Influenza outbreaks typically occur following transmission from birds to other mammals and then to humans⁹.

H5N1 Symptoms & Impact

Infected dairy cows show significant decreases in milk production, which coincides with altered milk color and viscosity¹⁰. Some cows also show mild respiratory symptoms and nasal discharge. Though HPAI H5N1 is associated with high morbidity and mortality in birds, this has not yet been observed in dairy cattle. Most cows can recover with treatment, and cull rates have stayed at 2% or less, mitigating some economic harm for dairy farms¹⁰.

"The estimated economic impact to Dairy farms is about $100 to $200 per cow over 2 to 3 weeks"

Still, affected farms face financial losses associated with reduced milk production (~10-20%), veterinary treatments, cattle movement restrictions, and, potentially, decreased demand associated with consumer concerns^11,12. The American Association of Bovine Practitioners estimated the economic impact to be about $100 to $200 per cow over 2 to 3 weeks¹³. Given that there are ~9.4 million dairy cows in the US, this presents a sizable economic risk¹⁴. It’s worth noting that the financial effect on poultry farms has also been pronounced. Halfway through 2024, nearly as many birds have been culled to mitigate H5N1 as in all of 2023⁶.

In humans, infected dairy workers have exhibited relatively minor symptoms, including conjunctivitis and those associated with acute respiratory infections^15,16. Yet, as more animal cases occur, the risks of additional human infections rise, and with it, human outbreak potential.

Viral Transmission Research

Several questions remain about the current outbreak. For example, researchers have yet to determine definitively how this new H5N1 strain transmits, but multiple lines of evidence suggest milk may play a key role on dairy farms. Experts suspect that milking machines may have contributed to the spread¹⁷. Testing has revealed relatively high viral loads in milk from infected cattle. Infected udders and milk may contaminate milking machines, which are often not sterilized between cows. As it stands, udder-to-udder transmission appears to be the primary route¹⁸.

However, the bovine H5N1 strain has spilled back into wild birds and poultry while also infecting cats and other mammals, suggesting that some transmission may occur independently of milking machines¹⁹.

Evidence suggests respiratory droplets may not be a significant source of spread

A recent study showed that H5N1 isolated from cow milk could infect mice and ferrets (a model organism for studying human Influenza infection) following both oral and intranasal inoculation²⁰. Milk H5N1 could also bind Influenza receptor glycans found abundantly in human upper respiratory tracks, a common prerequisite to human infection. This initial study also reported inefficient respiratory droplet transmission in ferrets, in agreement with a CDC ferret study that showed effective transmission through direct contact and weaker transmission through respiratory droplets²¹. Another recent study preprint also showed calves could become infected through aerosol containing the virus²². However, low viral shedding from the respiratory tract may indicate it's not a significant source of spread.

Together, this data suggests that H5N1 does not currently spread efficiently through respiratory aerosolized droplets (such as those that linger in the air following a sneeze or cough). However, further evolution of the virus could accumulate additional mutants required for improved respiratory transmission.

A significant amount of research is needed to better understand this virus, not only how it spreads but how it affects various hosts and how it is likely to evolve over time, especially given its historically high mortality rate³. To answer the remaining questions about the H5N1 strain, researchers need reliable experimental tools to study and track this novel outbreak. Essential to this effort are molecular controls that help validate and qualify essential assay methods, like nucleic acid amplification tests (NAATs). To support this research, Twist offers fully synthetic H5N1 controls for quantitative reverse transcription PCR (RT-qPCR), digital PCR, other NAAT formats, and next-generation sequencing (NGS).

Why Synthetic Controls?

Viral NAATs and NGS require positive controls to confirm that an assay can effectively detect specific viruses in samples. They also serve as a point of comparison and calibration, enabling genetic material quantification to determine values such as viral load.

Historically, researchers sourced positive controls from real-world and cultured samples known to contain the virus. However, these samples often contain live viruses, increasing handling risks. Furthermore, RNA viruses, like Influenza, evolve quickly. The accumulation of mutations in live viruses can limit nucleic acid amplification, assay signal, and standardization potential, complicating result interpretation.

Synthetic nucleic acid controls are a safer and more reliable tool for virology researchers since they lack live viruses, cannot evolve, and are in set quantities. Using our proprietary nucleic acid synthesis technology, Twist Bioscience offers accurate synthetic H5N1 HA and NA genes for use as positive controls since these genes are critical for subtyping Influenza through NAATs.

Synthetic H5N1 controls cover 99.9% of bases in the HA and NA genes. Using well-established manufacturing processes, Twist can generate H5N1 controls with low error rates (>99% accuracy) at any scale and rapidly deliver them in various formats to our research customers. Twist can also easily customize viral controls, allowing researchers to quickly update their assays to track changing features like new viral variants.

Since fully synthetic controls have no risk of accidental live virus contamination or release, Twist’s synthetic nucleic acid controls can be used in a BSL-1 lab, reducing the inherent infrastructural and process barriers that come with stricter BSL requirements.

As a leading voice for global biosecurity, Twist understands the value of widespread, low-cost monitoring for HPAI, the power of positive controls in ensuring the accuracy of these tests, and the importance of limiting security challenges posed by transfer and use of live virus for use as positive controls. By providing a synthetic positive control containing only the HA and NA genome segments, researchers can ensure the accuracy of their NAATs while safeguarding against biosecurity risk.

Conclusion

The H5N1 virus circulating in cows poses a clear agricultural and pandemic risk. Rapid, large-scale research is needed to advance our understanding of this virus and to further work toward human outbreak preparedness. Given the experimental scale and speed these studies require, researchers need molecular controls that simplify their work without compromising safety and efficacy. Twist’s synthetic nucleic acid controls provide that solution for H5N1 research, empowering assay development and deployment.

If you need controls for your virology research, check out our synthetic viral controls.

TWIST SYNTHETIC NUCLEIC ACID H5N1 CONTROLS ARE FOR RESEARCH USE ONLY (RUO), NOT FOR DIAGNOSTIC PROCEDURES.

参考文献

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