We are studying West Nile virus because it is an excellent example of an “emerging virus.” These are viruses that were once found only in a very small area and had minimal impacts, but for several reasons have expanded and are now major health problems for humans, livestock and wildlife. This is not only the case for West Nile virus: other mosquito-borne viruses such as yellow fever virus, chikungunya virus, and Zika virus, that once impacted smaller groups of people, have recently caused massive outbreaks in Central and South America. By studying West Nile virus in the birds and mosquitoes that are responsible for its spread, we can better understand how new viruses emerge, how the virus changes over time, and how these changes can alter transmission potential and disease in wildlife and humans.

West Nile virus causes serious disease in humans, wild birds, horses and other animals. In humans, cases of West Nile virus are usually mild. However, a small percentage of people infected with West Nile virus develop neurologic disease, where brain inflammation can lead to seizures, paralysis and sometimes death. Since 1999, over 24,000 Americans have developed West Nile virus-associated neurologic disease and there have been more than 2,300 West Nile virus-associated human fatalities. More recently, scientific research has shown that survivors of West Nile virus neuroinvasive disease may have long-term problems including memory loss, tremors, and seizures.

Data generated from our research will likely have worldwide scientific impacts, but also local ones. That is because West Nile virus is a major problem right here in Colorado, where we live and work. West Nile virus transmission, and human cases of disease, occur every year in our community. This is why our efforts are also focused on minimizing the burden of West Nile virus to humans and other animals, including birds. We do this by conducting West Nile virus surveillance testing for local communities.

In addition to the threat of serious human disease, West Nile virus also causes serious disease in other animals. Disease and death as a result of West Nile virus has also been reported in birds, horses, sheep, llamas, alpacas, dogs, cats, alligators, squirrels and other animals.

West Nile virus has major and devastating impacts on wild bird populations. During some of the worst West Nile virus years, up to 72% of crows in some areas are estimated to have been killed by the virus. West Nile virus also affects other species of birds, including many birds of prey, like owls and hawks. Raptors infected with West Nile virus can experience blindness if they survive and the virus has significantly reduced the survival of nearly half of the bird species examined in North America, particularly native sparrows, vireos and finches.

Our work is focused on understanding the basic biology that causes new viruses to spread and become health problems. We hope that someday what we are learning can be used to help limit the effects of West Nile and other viruses like it.

Because our work specifically examines how West Nile virus circulates between mosquitoes and wild birds, we have been able to identify key details about how specific bird species are crucial to the maintenance of the virus in nature. Our research has revealed that when West Nile virus infects a variety of bird species, the virus changes in specific ways. Those changes, in turn, affect the severity of disease and its impacts on both humans and animals. Because of this, our work contributes significantly to the overall scientific understanding of West Nile and other viruses and aids in efforts to mediate their effects on humans and animals.

Yes, there are several. First, our research has allowed us to contribute to studies of new bird feeds — for example, seeds coated with ivermectin — that may directly help protect wild birds (and humans) from West Nile virus. Our work with West Nile virus in birds also has assisted in improving responses to other emerging viruses, such as Zika virus and Powassan virus. In summary, the work that we do with wild birds cannot be viewed in isolation, but is part of a larger effort to combat the problem of emerging infectious diseases impacting both public health and animal welfare.

Cell culture and computer modelling are both excellent tools for understanding the dynamics of living systems. However, these approaches have considerable limitations. Most importantly, these cultures only contain one type of cell, while whole organisms have many cell types. For this reason, studies of viruses that use only cell cultures can be misleading and do not mirror what the virus does in humans and animals. With respect to computer modelling, even the most powerful computers are unable to model the complexities of living systems. Without conducting controlled experiments involving the animals that are naturally infected, we don’t know how to best use future computer models.

Because animal research is necessary for the specific scientific questions our lab is trying to answer, we abide by the “3 Rs” of animal use in science: Replacement, Reduction and Refinement. When possible, we replace studies in birds by using cell culture experiments. We reduce our animal use by using the fewest possible that will provide meaningful data and repeatable (valid) results. We refine our treatment of animals by providing them with toys and puzzles for enrichment, and housing them together in groups, as they are social animals. It is a special privilege to work with wild animals — one that we take very seriously. Wild birds are one of the most valuable resources in our lab and each one is used for work that we consider to be crucial to our goals.

Our work with animals is subject to a tremendous amount of oversight. We cannot, on a whim, decide to conduct an animal experiment. Federal agencies require universities to implement specific protections for all animals used in research. Written protocols involving animals must include a scientific justification for working with a given species, a detailed list of procedures and drugs used to minimize pain and discomfort, and a description of the methods used to search for alternatives to animal research. Colorado State University maintains a supervisory committee that implements these protections. This committee is known as the Institutional Animal Care and Use Committee, or IACUC.

The IACUC includes a university bioethicist, at least one outside member of our local community, and an attending veterinarian who is responsible for the clinical care of all animals used for research at Colorado State. The IACUC reviews all studies involving animals, provides guidelines on pain management and humane treatment, and inspects all animal facilities for compliance twice a year. All of our work with animals has been reviewed and approved by the CSU IACUC and our funders. In addition, several veterinarians and laboratory animal technicians check on our birds at least once a day to make sure that they are healthy and well cared for.

Our work with birds is also reviewed and regulated by the US National Institutes of Health and state and federal wildlife agencies. Additionally, the animal facilities where we work undergo routine inspections by the Centers for Disease Control, United States Department of Agriculture and the Association for Assessment and Accreditation of Laboratory Animal Care.

Our laboratory captures wild birds in northern Colorado using nets. Birds are collected at locations where landowners and facilities have given us permission to remove them, particularly when they are considered to be a nuisance. Captured birds are kept in large, open rooms that allow for free flight and for groups to live together (see pictured).

Each room has branches for birds to perch on, and toys and snacks are provided for enrichment. Birds are given unlimited access to food and water. They are caged only during studies where strict bio-containment is required. Veterinarians or other trained personnel monitor the birds daily.

Our IACUC protocols cover four procedures for use with captive birds: blood collection, subcutaneous virus inoculation, virus inoculation via mosquito, and euthanasia.

• All blood samples from sparrows, robins and crows are collected using a small needle. This is, by far, the least traumatic and fastest way to collect blood from birds. Collecting blood from birds is required to both assess previous exposure to West Nile virus and monitor the level of virus in blood.
• We infect birds with West Nile virus by injecting virus just under the skin to mimic mosquito infection. The injection site is cleansed and the virus is delivered through the use of a small needle.
• We sometimes allow infected mosquitoes to feed on birds because the virus is transmitted in nature by mosquito bites. For this procedure, birds are placed over a carton with a mesh cover that contains mosquitoes for a period of 30 minutes, then the birds are returned to their enclosure. Birds, of course, receive a lot of mosquito bites in nature. In fact, several species of mosquitoes feed primarily on  birds, and are referred to as “ornithophilic”, or “bird-loving” mosquitoes. These types of mosquito are responsible for maintaining and transmitting Eastern equine encephalitis virus, St. Louis encephalitis virus, and other viruses in addition to West Nile virus.
• Because crows become ill due to West Nile virus infection, we minimize suffering by humanely euthanizing infected crows before they develop any signs of disease. In the unlikely event that a bird in our care were to become obviously sick, the experiment would be ended and the bird would be immediately euthanized. Birds are euthanized under the care of veterinarians according to procedures approved by the Panel on Euthanasia of the American Veterinary Medical Association.

All of our work with animals is supervised by people who have no vested interest in the success of our studies and are not affiliated with Dr. Ebel or his students or other trainees. As stated above, our work must first be approved by CSU’s IACUC before it takes place. Animal care and supervision is performed by the veterinarians and personnel of the CSU Lab Animal Resources group. We also meet with veterinarians from the university’s Lab Animal Resources department before we are allowed to work with birds. Our animal facilities undergo routine inspections for approval by the Centers for Disease Control, United States Department of Agriculture and the Association for Assessment and Accreditation of Laboratory Animal Care, which all have stringent rules on how animals are used in experimental settings.

West Nile virus has major impacts on crows, killing a very large percentage of some populations. Our first studies, conducted in the early 2000s, focused on naturally infected crows (and other crow relatives, such as blue jays) that were found dead and submitted to the New York State West Nile virus surveillance system. We learned a tremendous amount from these studies. Field-collected samples, however, have many limitations. We don’t know how long the bird was infected or how long the bird had been dead. To really understand the evolution of West Nile virus and how that affects its transmission and severity of disease, we need to know exactly what strain of virus the bird was infected by and how long it was infected. We can only do that in a controlled laboratory environment.

Wild crows and robins  are not endangered, threatened, or listed as a species of concern. Furthermore, the number of animals that we use in our studies is extremely small compared to the size of the whole population. Our studies have no significant impact on bird populations in Colorado.

It depends. In many cases, their captivity is only about three months. However, sometimes they need to remain in captivity for a longer period (up to around a year). For that reason, we are very dedicated to regularly providing them with fresh food, room to fly and perch, and lots of social and interactive toys.

Infecting birds with West Nile virus requires strict Animal Biosafety Level 3 (ABSL3) containment. Removing live animals from ABSL3 is not permitted by law. House sparrows, another species we work with in our research, are an invasive species that displaces native birds. That is another reason we do not release them after capture.