Congratulations to Lexi Keene and Marylee Kapuscinski for their award-winning talks at this year's Rocky Mountain Virology Club Meeting.

Lexi earned the 2nd place Graduate Student Talk Award for her talk titled RNA viral metagenomics of 100-year-old Drosophila melanogaster museum specimens

Marylee earned the 1st place Graduate Student Talk Award for her talk titled Minigenome Melees: A novel high throughput method to evaluate reassortment potential between segmented RNA viruses

Marylee is pictured with the 2022 meeting's keynote speaker, Ana Fernandez-Sesma

Great job you two!

I've posted about the RMVC meeting before. It's one of my favorite meetings each year.

A paper published online on Jan 20, 2020 in the Journal of Medical Virology, titled Homologous recombination within the spike glycoprotein of the newly identified coronavirus 2019-nCoV may boost cross-species transmission from snake to human raises the intriguing possibility that a new coronavirus of substantial public health concern, 2019-nCoV, may have originated in snakes.

The authors make two primary claims. First, that 2019-nCoV is a recombinant virus. Second, that "...our evolutionary analysis suggest for the first time that snake is the most probable wildlife animal reservoir for the 2019-nCoV."

Recombination occurs when two viruses exchange genetic material. I'm not going to evaluate the claim that this virus may be recombinant.

I will however, consider the evidence that this virus may have originally been transmitted to humans from snakes. As I describe below, there is no direct evidence to support this claim, and the indirect evidence is flimsy.

Many pathogens can be transmitted from other animals to humans. Snakes were apparently one of the types of animals sold at the seafood market that has been linked to many of the initial human cases of 2019-nCoV. So it is plausible that this virus could have been from snakes.

There is no direct evidence that this type of coronavirus infects snakes. The authors do not identify a similar virus in snakes, and no similar coronaviruses have been identified previously in snakes.

(My lab has studied respiratory viruses of snakes that are belong to the same umbrella virus group as 2019-nCoV, but these are distant relatives).

The main line of evidence the authors present to support the claim that this virus may have originated in snakes is indirect, and based on codon usage patterns. Codons are DNA base triplets that encode a single amino acid in a protein. The same amino acid may be encoded by different codons, so there is flexibility in how a particular protein sequence could be encoded. Different organisms tend to use codons in a characteristic way. Viruses that matched their codon usage to that of their host could have an evolutionary advantage.

So if, as seems likely, 2019-nCoV was recently transmitted to humans from other animals, then it might be possible to discern the original host by comparing codon usage patterns of the virus to candidate hosts.

The authors compare the relative codon usage of 2019-nCoV with several candidate hosts, corresponding to some of the animals that were sold in the seafood market that has been linked to this outbreak and to the most closely related cornavirus to 2019-nCoV, bat-SL-CoVZC45, which was identified in a bat in 2017. The authors then calcualte the squared Euclidian distance between as a measure of how similar these codon usage patterns are between the various species. The results are shown in figure 3 of the paper:

The "heatmap" in panel A depicts the frequencies with which codons are used in different organisms. Each column of the table is one organism. Each row is one of 61 possible codons. A redder color indicates that a particular codon is used more often in that organism. A green color means that codon is used less frequently. The heirarchical branching at the top of the figure indicates the extent to which these patterns are similar in different species. You can see that the two similar viruses (the two left-most columns) have closely related codon usage patterns, as expected. The two snakes have relatively similar patterns, and the mammals have similar patterns.

The claim that snakes are the "most probable wildlife" origin of 2019-nCoV rest on the bar graph shown in panel B which shows the Euclidian distance between the codon usage frequency vectors of 2019-nCoV and the various possible hosts. The distance between 2019-nCoV and the 2 snakes is ~12-15, whereas the distance between that virus and the mammals is >25. At first glance, this seems convincing.

But consider the case of the other coronavirus analyzed, bat-SL-CoVZC45. This virus was isolated from a Rhinolophus sinicus bat in China in 2017. It's codon usage pattern is essentially identical to that of 2019-nCoV. Consequently, the distances between it's codon usage pattern and the candidate hosts will be essentially identical to those shown in panel B. By the logic presented here, the bat-SL-CoVZC45 virus, from a Rhinolophus sinicus bat, is more likely to have originated from a snake than from a Rhinolophus sinicus bat (which is one of the mammalian species analyzed).

It is possible to imagine a scenario where previously unknown snake coronaviruses independently transmitted to bats and to humans. But in my opinion, a simpler and more likely explanation is that this kind of codon usage analysis has limited predictive value to pinpoint a virus's exact host in a situation like this.

It remains possible that snakes are the source of 2019-nCoV, but the data presented here is not convincing evidence of that.

Snake Nidovirus Research in the Stenglein Lab

The Stenglein lab was among the first to discover nidoviruses in snakes and to link them to severe respiratory disease. Over the last 4 years, research on nidoviruses and their role in disease has continued in our laboratory. We have confirmed that nidoviruses cause respiratory disease in snakes and have made additional contributions to understanding the disease and how it is spread. Currently, we are focused on answering the following questions related to python nidoviruses:

1. Which snake species are susceptible to infection?
   
2. Which snake species are susceptible to disease (some types of snakes may be infected but might not get sick)
3. What fraction of infected snakes get sick, and to what extent?
4. Why do some types of snakes get infected but not others?
   
5. How is infection transmitted from snake to snake?
   
6. Can some snakes recover from infection?
   
7. How can infection best be diagnosed?
8. What management strategies would be most effective at reducing or eliminating disease from a collection?

We are pursuing a multi-pronged approach to answer these questions that relies on sampling of naturally-infected snakes in private collections, and laboratory experiments. We are particularly interested in sampling collections of mixed snakes species with a history of nidovirus-caused respiratory disease. We never disclose the identity of collections.

Our laboratory does not currently study anti-viral therapies or vaccine development for this disease. There are several reasons for this: 1) similar respiratory viruses in humans and animals also lack effective therapeutics due to the nature of these viruses, 2) there are no effective commercially available vaccines for any diseases in snakes, and 3) these studies are usually very costly and often unsuccessful. We feel it is more practical and effective to investigate population characteristics of the disease in the hopes of generating appropriate management strategies for reduction and prevention of infection and spread.

 

Frequently Asked Questions

What are nidoviruses?

Nidoviruses are a group of related viruses known to cause a severe and sometimes fatal respiratory disease in snakes. These viruses are cousins of SARS and MERS coronaviruses that affect humans, which can also cause severe respiratory disease.

What snakes are susceptible to nidovirus infection and disease?

This is one of the primary questions our laboratory is trying to answer. So far, nidoviruses have been detected predominately in pythons and some boas, but it is our goal to determine all the snake species that are susceptible and how severely the disease affects them. There is no evidence that this virus can infect or cause disease in humans.

How many strains of virus are there and how do they differ in virulence?

Viruses that have complete genome sequencing data (a method for determining different strains of virus) have been isolated from ball pythons, an Indian rock python, and green tree pythons. The nidoviruses from ball and Indian rock pythons are relatively similar, but are quite different from those isolated from green tree pythons. Although these viruses have not been taxonomically categorized, it is likely there are multiple strains and these may affect snake species differently. Our research will attempt to address this question through the sampling of high numbers and a wide range of snakes of species.

What are the clinical signs of nidovirus infection?

The clinical signs can vary from mild and non-specific to severe respiratory signs or sudden death. Typically, snakes will begin to have increased amounts of clear mucus in the mouth and nose (A) and the gums may become reddened. This can progress to wheezing, breathing with the mouth open (B), more rapid breathing, or coughing. Other signs include a poor or non-existent appetite, weight loss, decreased activity level, dehydration, and spending more time on the bottom of the cage (if a perching snake). Secondary bacterial infections may also play a role in disease, so mouth rot could be evidence of an underlying nidovirus infection (although there are many other causes for this as well).

How long are snakes usually infected?

This is currently unknown. Some snakes remain nidovirus positive for over a year. Other snakes succumb to disease and die within 6-12 months of being infected. There is also evidence in our laboratory that some snakes can clear the infection following 6-12 months of infection, but the data to support this remains anecdotal and this is not clearly established. Overall, this disease is chronic and usually results in long-term infection in snakes.

How is this disease transmitted?

Our laboratory has found virus in the oral/nasal cavity, lungs, and feces. Therefore, transmission is thought to occur by direct or indirect exposure to respiratory secretions (similar to the common cold) or to feces from infected snakes.

How can nidovirus infection be treated?

Currently, there is no specific treatment for nidovirus infection in snakes, nor is a vaccine available. This is similar to other viral diseases of snakes, and in fact in most species, including humans. Only rare viral diseases have been treated with anti-viral therapy and with varying success. Also, there are no effective commercially available vaccines for any snake diseases, despite attempts at vaccine production. Typical treatment includes supportive care and antibiotics to prevent secondary bacterial infections.

How long does the virus stay infectious in the environment?

This has not been tested for snake nidoviruses. However, SARS coronavirus, a closely related virus, is relatively stable outside of the host for up to 4 days. For SARS, heating and UV irradiation can eliminate infectious virus in the environment, as can common disinfectants (e.g. accelerated hydrogen peroxide, alcohol-based disinfectants with greater than 79% ethanol, and solutions with at least 0.050% of triclosan, 0.12% of Chloroxylenol (PCMX), 0.21% of sodium hypochlorite bleach, 0.23% of pine oil, or 0.10% of a quaternary compound with 79% of ethanol). It is unknown if snake nidoviruses are similarly affected by these disinfectant and antiseptic methods, but compounds listed above are good places to start when sterilizing cages and areas that have been exposed to nidovirus positive snakes or their excreta. These viruses may also be carried as fomites on hands, clothes, and instruments, therefore, it is important to use appropriate protective gear and washing of items before using between infected and uninfected snakes. Ideally, nidovirus positive snakes would be isolated/quarantined in a separate area and have designated instruments and protective gear only used with these snakes.

Where can I find recent literature about snake nidovirus?

Stenglein 2014 - Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius. (Web) (PDF)

Uccellini 2014 - Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius). (Web)

Bodewes 2014 - Novel divergent nidovirus in a python with pneumonia. (Web)

Marschang 2017 - Detection of nidoviruses in live pythons and boas. (Web)

Dervas 2017 - Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis). (Web)

Hoon-Hanks 2018 - Respiratory disease in ball pythons (Python regius) experimentally infected with ball python nidovirus. (Web) (PDF)

How can I learn more about the Stenglein lab?

If you would like to know more about the Stenglein lab and the research we do, or you believe you have a collection that might be suitable for our study, please don’t hesitate to contact us:

Mark Stenglein - The head of the laboratory that conducts this research

Laura Hoon-Hanks - The veterinary researcher in charge of the nidovirus project

What can I do to support this research?

We appreciate your support - please see below for donation instructions:

To financially support this research:

If you are interested in financially supporting our research, your support would be utilized in multiple ways:

1. The purchase of snake sampling supplies (e.g. swabs, tubes, virus media) and the shipping costs, which we provide free-of-charge to the private collections we are studying.
2. The processing and testing of samples for nidovirus.
3. The analysis of nidovirus positive samples by multiple modalities to further characterize the virus and the disease it causes.

• No funds will be used to pay the researchers. This funding will go directly to supporting affected collections enrolled in the study and financing the experiments necessary to study this virus.*

Donation Instructions

1. Go to https://advancing.colostate.edu/CVMBS/MIP/GIVE
2. Enter the amount you would like to donate in the "Microbiology, Immunology & Pathology Department Research" field.
3. Click "Next"
4. Enter in your contact information and click "Next".
5. Select "Add Notes About My Gift" and enter "Stenglein lab nidovirus research" in the text field.
6. Proceed to payment information.

Thank you! We appreciate your support!

Link to workshop material

I'm delighted to be participating in the inaugural Genomics of Disease in Wildlife Workshop, which is being held starting today at Colorado State University. We just had our kick off reception, and I'm very impressed by the other instructors and the workshop participants. It seems like a great group.

I'm going to be leading several of the sections, on genomics, sequencing, accessing online resources, de novo assembly, and metagenomics.

I'm putting all of the material (lectures, exercises) I've developed for this workshop up on GitHub, here.

Shaun has been selected to participate in the GAUSSI program. Congratulations, Shaun!

The GAUSSI program at CSU is an NSF-funded program that aims to teach biologists more computing and computer scientists more biology. It offers 1-year fellowships to PhD students and a series of modular 1 and 2 credit courses that allow trainees to pursue a tailored curriculum.

I co-teach a class in the GAUSSI program (with Carol Wilusz and Dan Sloan), that teaches students how to design and perform experiments using next generation sequencing.

Our paper on viruses in wild Anopheles gambiae mosquitoes was recently published in Virology and is one of several articles higlighted in its issue. We had the opportunity to write a blog post on Virology Highlights Blog , (motto: 'Chosen by editors / Written by researchers'). It was fun to write about the paper in a less formal than usual context and to explain some of the back story and challenges we faced..

There are several additional reasons that I am happy about the publication of this paper. First, it's my first 'last author' paper, which, I guess, means I'm in charge!?! Second, it is a product of our lab's growing connections with the other labs in the AIDL. I am truly excited about the opportunity to be part of this fantastic group and it was great to work with the Ebel, Foy, and Brackney labs here. Finally, this publication spawned some exciting follow-up projects that we are pursuing. Keep tuned for developments on that front.

An anopheline mosquito (image credit: CDC)

This weekend marked the 16th annual meeting of the Rocky Mountain Virology Club. This meeting draws virologists from Colorado and surrounding states and keynote speakers from further away. There is also usually a good contignent of prion researchers from the Prior Research Center at CSU. This year's keynote speaker was Jon Towner from the CDC, who gave a fantastic talk on his research on filovirus reservoirs.

The RMVC is one of my favorite meetings ever. First of all, I am always blown away by the great science. There are lots of opportunities for students and postdocs to give talks and the atmosphere is engaged but friendly. The meeting is also a reasonable size (~60 people or so?). You get a chance to talk with most everyone, including the keynote speakers, which can be tricky at bigger meetings. The meeting schedule is also a lot more relaxed than a typical meeting. A lot of meetings have content scheduled for 12-14 hours a day, which I find to be too much for my brain to absorb. At talk-crammed meetings I usually end up spacing out midday. Here, there is plenty of time for socializing and a break in the middle of Saturday afternoon. This provides a great opportunity to go explore CSU's mountain campus, aka Pingree Park, where the meeting is held. Pingree Park is absolutely beautiful and about a 1.5 hour drive from Fort collins.

Pingree Park (image credit: CSU)

The aspens are usually in peak color (image credit: RMVC)

Some folks bring their families and another unexpected feature of the meeting is really great and free child care (thanks, Joel Rovnak and the organizers!). Each year, Lori, the woman who runs the day care, has the kids do a science-themed art project.

Here's an 'arenavirus' model that my daughter Dahlia made at the 2014 meeting. (It's in my office now and is a useful prop when I'm talking to people about viral glycoproteins :)):

All of the kids' virus models were entered in a competition. The virologists had to guess what kind of viruses these were.

I encourage everyone in my lab to go to this meeting and hope to see you up there too!

Today is Joey's last day in the lab. He's starting as a bioinformatician at Somalogic in Boulder, CO.

This is the first time a lab member has left the lab, and it's bittersweet. I know that one of my jobs as a PI is to train postdocs and help them to get jobs, so in a sense this is exactly as it should be. But we are sad to see Joey go, and wish him well!