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Posts Tagged ‘microbiology’

How many bacteria/viruses are actually removed by the body’s natural defenses while we are sick?

March 10, 2015 Leave a comment

The answer to this question is completely dependent on the context of infection. For instance, when you brush your teeth, you introduce bacteria from your mouth into your blood stream and become temporarily bacteremic. Fortunately, the immune system clears these bacteria quickly. In this daily event, thousands of bacteria enter the bloodstream. After brushing, bacteria in the blood were below 104 cfu/ml, as estimated by PCR, and exact numbers are nearly impossible to quantify (Lockhart et al. 2008).

In contrast,  Ebolavirus replicates to ~107 – 108 virions/ml in the blood of non-human primates (Bente et al. 2009). The average person has ~5 liters of blood. Very conservatively, an Ebola survivor will have cleared ~5 x 1010 virions from just the blood. That’s 50 billion virions.

Keep in mind that this is also just a snapshot of a moment during infection. Viruses are constantly propagating, and a single virally infected cell can produce thousands of viral particles in a single day. It’s possible that over the course of a 2 week infection, 1011 – 1012 virions may be produced and cleared (it’s probably much higher). While this number is staggering, it somehow pales in comparison to the magnitude of the U.S. debt, which currently stands at ~$18 trillion, or $1.8 x 1013.

Geisbert et al. 2004 Nat. Med.

Geisbert et al. 2004 Nat. Med.

I also estimated the rough number of virions from the liver and spleen, two organs with significant ebola titer, and the total is nowhere near the magnitude of virions in the blood.* Additionally, ebola virus is being shed through mucosa and other bodily fluids. People generate ~ 4 liters of mucus daily in the gastrointestinal tract. I could not find an accurate estimate of Ebola viral titers in the mucus, but it’s unlikely to be near the levels of that in the blood. Granted, mucus could still contribute significantly to viral titer. There’s also the possibility that other organs harbor high titers of ebola virus, especially the gut.

*Approximately 104 – 105 virions/gram were detected in the liver and spleen using a mouse model for Ebola (Bray et al. 1996). Given that the average mass of a human liver is 1.5kg and a spleen is 140g (Molina et al. 2012), and using conservative estimates, about 1.6 x 107 virions from the spleen and liver alone, over 1000 times less than that from the blood.

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What happens under your skin when a mosquito bites

August 7, 2013 Leave a comment

Although mosquito tongues are analogous to needles, they are actually mobile and flexible structures. After puncturing the skin, the tongue searches for a blood vessel to feast on. Here’s what happens when a mosquito bites you:

Also fascinating is:

“When the mosquitoes were infected with the Plasmodium parasites that cause malaria, they spent more time probing around for blood vessels. It’s not clear why—the parasites could be controlling the insect’s nervous system or changing the activity of genes in its mouthparts. Either way, the infected mosquitoes give up much less readily in their search for blood, which presumably increases the odds that the parasites will enter a new host.”

It’s also possible that the mosquito’s tongue mobility or sucking ability is hindered when colonized by Plasmodium, causing the mosquito to spend a longer time searching for blood vessels, and increasing the probability of a Plasmodium to leave the mosquito and enter a new host. Maybe Yellow Fever and Dengue elicit a similar response?

Full article from natgeo here:

Here’s what happens inside you when a mosquito bites

Coughing is commonly not from the common cold

August 5, 2013 Leave a comment

A leading cause of respiratory disease in children is Respiratory Syncytial Virus (RSV), producing over 3 million cases of lower respiratory illness and about 100,000 deaths annually (Nair et al. 2010). RSV is classified as a virus in the family Paramyxoviridae, which are all non-segmented negative sense RNA viruses. Other viruses that utilize this method of replication include Mumps virus, Human Metapneumovirus (hMPV), and  Henipavirus, the inspiration of the movie Contagion. Like most viruses that cause respiratory symptoms, including viruses that cause common cold, numbers of RSV infections increase in winter months when people spend more time indoors (Florida is weird).

MMWR RSV 2007

Nearly all children will encounter RSV, but only 2-3% will require hospitalization. However, the real trouble is re-infection. Typically after your immune system develops antibodies against an antigen, it can recall the “memory” of the infection to produce specific antibodies and lymphocytes to prevent re-infection by the same agent. However, this “memory” can fade, and is of particular concern with RSV.

Challenge experiments have shown that 73% of adults became reinfected a second time within 26 months, and 43% became reinfected a third time with apparent symptoms in the majority of the cases (Hall et al. 1991). In short, the virus is capable of reinfecting healthy individuals, and immunity is relatively short-lived. This also makes it particularly difficult to produce a vaccine that does not need to be administered annually.

An appropriate adaptive immune response requires the activity of dendritic cells (DCs). In short, DCs are antigen presenter cells, and direct the T-cell and B-cell response at a particular target. DCs move from a site of infection to the lymph nodes in order to activate the proliferation of the proper lymphocytes to defend against a particular pathogen (a short video is provided below). DC migration is directed primarily by chemokines, or signalling proteins that the DC recognizes through the receptor CCR7. Without CCR7, the DC’s ability to detect chemokines is severely diminished, and without migration to the lymph node, downstream activation of the adaptive immune response is absent.

In the 2011 paper in PLOS Pathogens by Nouen et al., the authors demonstrate that RSV infection leads to a decrease in the expression of CCR7 in DCs during infection. RSV alters dendritic cell migration, and reduces DC migration to the lymph nodes.I This finding suggests that RSV is capable of regulating the immune response by reducing the activation of lymphocytes. Measles virus, another paramyxovirus, has also been shown inhibit DC migration through the modulation of CCR7 expression.

I would again like to highlight the incredible adaptability of pathogens to persist in an environment. Viruses have evolved to persist in even the harshest of all environments, including the human immune system, which is tasked with the very specific job of fighting these viruses.

Despite being known as a clinically significant human pathogen since the 1950s, a vaccine for RSV is still unavailable.  However, a better understanding of how RSV modulates immunity gives us a clearer picture of why RSV vaccine development is so difficult. In contrast to EbolavirusRSV is very well adapted to humans, and rarely kills its host, but the disease burden of RSV is certainly significant.

Fortunately, basic research into mechanisms of pathogenesis is leading us in the right direction. But what you may believe to be a “common cold” (which may actually be caused by any number of viruses including rhinovirus, adenovirus, or coronavirus) can be RSV. Persistent infection should have you worried, but at least it’s not Ebola.

The frightening Filoviridae

July 22, 2013 1 comment

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Ebola hemorrhagic fever is caused by Ebola virus, a filamentous virus that is part of the family Filoviridae. Ebolavirus is of particular concern to global health as a result of killing as many as 90% of its victims. The Hot Zone tells in great detail the discovery and fear driven by Filoviruses, I highly suggest it.* This high mortality can be attributed to its ability to avoid detection by the immune system. Normally the human adaptive and innate immune system are capable of recognizing specific markers, or antigens, from pathogens. These antigens trigger an immune response that can clear the infection. The adaptability and complexity of the immune system is astounding, and can be better understood with the above wikipedia links.** While the immune system plays a large role in infectious disease, the star of the game is Ebolavirus.

Viruses are characterized by their parasitism, and the inability to metabolize and replicate in the absence of a host. Because of this, viruses hijack host cell machinery in order to reproduce. But first, the virus must gain entry into a cell. Viruses have found various ways to enter their host cells. For example, HIV entry is characterized by fusion of the viral envelope to the membrane of the cell, without any endocytosis. In contrast, most viruses utilize a process of endocytosis, where fusion of the virus to the cell membrane triggers a response by the cell to engulf the virus. How Viruses Hijack Endocytic Machinery.

After entering a cell, Ebolavirus uses host proteins, along with a few of its own, to make copies of itself. These new viral copies leave the cell in search of another host to infect. However, after leaving the cell, the viral particles are exposed to the dangers of the human immune system. The mechanism by which Ebolavirus evades the immune system has been dubbed antigenic subversion. The paper by Mohan et al. at Emory University is here, and open access: Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus.

Viruses are subject to natural selection and evolution, similar to all organisms. Many pathogens undergo antigenic variationwhere the antigens of the microbe change such that the immune system no longer recognizes it as a pathogen, allowing it to reside in its host undetected. Alternatively, antigenic subversion is a mechanism by which Ebolavirus actively “hides” from the immune system by telling the infected cell to secrete an antigen (secreted GlycoProtein, sGP) that effectively undermines the ability of the immune system to recognize the capsid of the virus. The excreted antigens sequester the antibodies directed toward finding and clearing viral particles coated in GP1,2, the capsid glycoprotein responsible for attachment of the virus to the host cell. Without an appropriate antibody response, clearance of the virus is virtually impossible and free viral particles roam the body destroying cell after cell. This is where it gets heavy.

The translated products that contribute to antigenic subversion are a result of transcriptional editing. The viral polymerase carried by Ebolavirus is prone to slippage, and 20% of the time, the viral RNA polymerase creates a mRNA transcript that allows for the translation of open reading frames as a single protein, the fusion GP1,2 protein. 80% of the time, the polymerase creates a transcript that carries a stop codon before the second open reading frame, creating the secreted sGP protein. (PNAS Sanchez 1996)

Furthermore, antibodies against sGP harbor low reactivity with GP1,2. However, antibodies against GP1,2 react strongly with sGP. Because sGP is simply a truncated version of GP1,2, this suggests that the conformations of sGP create epitopes that differ from those on GP1,2. These conformational changes alter the immune response, leading to the production of antibodies that can strongly opsonize sGP, but weakly bind GP1,2.

Many pathogens employ a decoy mechanism, where they create an antigen that absorbs the effects of the antagonizing antibodies. However, this differs in that the “decoy” antigen is not only sequestering antibodies, but also altering the immune response to create ineffective and poorly protective antibodies against sGP when the immune system would be better off producing anti-GP1,2 antibodies.

A 3 minute discussion about the entry of Ebolavirus:

*A crappily formatted, but readable, PDF version of the book is here.

**I tried to keep it concise, but introduction to viruses is no brief task. I should do a pathogen 101, in addition to an immune system 101. I would like to delve into specific molecular mechanisms, but it’s easier to think about than it is to write about. Next time.

An introduction to emerging infectious diseases

July 17, 2013 Leave a comment

Viral_infections_and_involved_speciesWhile most of my research is directed toward antibiotic resistance in bacteria, the vast majority of the news in the popular media suggests that the most prevalent diseases are caused by cancer and viral agents. While concerns of bacterial drug resistance are on the rise, the fear of an influenza pandemic or an ebola outbreak creates excellent headlines.

Additionally, bacteria are relatively slow killers and infections are generally treatable (although some are not). A virus may have an incubation period of about two or three weeks with mild flu-like symptoms. During this time you may believe you have acquired the seasonal flu, and that it will pass. You hug your kids, and walk around the office touching doorhandles, shaking hands, and using the company copier. By the time you’re hemmorhaging and you realize it’s not just the flu, it’s too late, and you may have infected others.

The first thing you learn in microbiology is that bacteria are ubiquitous, but these single celled organisms are not alone in their pervasiveness. There’s a saying that for each bacterial species, there exists at least one virus that is capable of infection. Viruses are extremely prevalent. In a single milliliter of sea water, there are roughly 10 million viral particles, and about 15 times as many viruses as bacteria (a fun read). Despite the prevalence of viruses, most are incapable of infecting humans. Many bacterial infections are closely associated with immunocompromised individuals, and immunosuppression is often caused by a virus, such as HIV (opportunistic infections and AIDS).

Viral research has lead to incredible advances in medicine. Because general hygiene has decreased the incidences of bacterial caused diseases such as plague and tularemia, scientific efforts have been directed toward understanding viruses. Vaccines for smallpox and polio have saved an immeasurable number of lives. Viruses may even be key to a future cure for cancer or other genetic diseases, as viruses have been engineered as tools to deliver gene therapies (an easy to read microbe wiki link on viral based gene therapies). Hopefully I can shed light on some interesting aspects of emerging infectious diseases.

*I began writing this as a brief introduction to highlight some of the research in the field of microbiology, virology and immunology. I had too much to say, and hopefully my next post will cover this paper about how ebola evades the immune response.

Briefly on Curiosity

May 22, 2013 Leave a comment

It’s pretty obvious that there isn’t enough time in the day.

052213 Insert

Conferences tend to leave you with more questions than answers, and that’s science. With every answer, you have ten more questions. But there just isn’t enough time to investigate everything you want to know. Weird and inexplicable things happen all the time in science. Sometimes curiosity can get the best of you, and drive you on a tangent to delve deeper and deeper into a story with no apparent end. And that’s the best part.

Sadly, science funding is now in a place where curiosity is rarely funded, and translational biomedical science has the upper hand. However, curiosity is what got us here. Exhibit A: Leeuwenhoek wanted to look at little things, and with his crude hand-crafted microscopes, he did just that.

Books and their facts rarely give you an idea of what is still left to discover. Just think that each sentence in your book is the summary of a scientist’s contributions throughout his entire life. Most scientists rarely make breakthrough discoveries, and  barely contribute a sentence to your book. However, their curiosity at least gives them a chance, and each mechanism that is seemingly understood just raises more questions about how or why.

But curiosity must have direction. For example, I wanted to clone a gene in a specific orientation, and without going into detail, the gel above shows 9 plasmids with incorrect orientation, and 1 correct. It is interesting that I have a 10% success given that the gene I want to clone can be inserted in only two directions, giving me a 50/50 chance of the correct orientation. This discrepancy in orientation could be due to a variety of effects, but I simply don’t have time to investigate this phenomenon, especially when all I really want is my clone.

The questions in science are the best part, and we take them for granted. Sure, you study an important protein, that when defective causes Alzheimer’s. But keep in mind the big picture. Your protein is encoded by DNA, regulated transcriptionally and translationally, and has interacting partners within the cell. All these processes, so little time. Focus, and good luck.

 

Why a PhD?

December 4, 2012 4 comments

phd081508sMy students frequently ask me “do you have to do research over the break?” No. I WANT to do research over the break.

There’s a fundamental problem with students in the sciences. They don’t care about anything but grades and “getting a good job.” They don’t get exposed to research and simply have no idea what it means. It’s upsetting. Each of them seems to think they will simply go to medical school and become a doctor. Health care is sensationalized by the media, while the real life savers are in the background doing research. Doing real science.

There was a discussion on the ASM page of LinkedIn that I stumbled upon. The thread was about “the future of the Ph.D.” That’s when I saw this mind blower: “While it’s true there are some jobs that require the degree, there are a lot more that will consider you overqualified if you get it. If you want a secure career look elsewhere.”

WHAT ARE YOU TALKING ABOUT? This is incredibly aggravating. You get a Ph.D because you absolutely love science. Not for fame, or money (there is none in science), not for some “secure career.” You do it because you can’t live without the thrill of the discovery. You have to love the chase, to be enthralled with the unknown. You have to know that with each thing you find out, there are a million more intricacies behind it.

It’s hard to believe that most people are heading into their adult lives seeking degrees for some sort of pay increase or stability. Look at what science has given us. We take it all for granted.

Food for thought: The US military receives roughly $680 billion per year. The NIH only receives $30 billion annually. How is this sustainable?

“The reason you do it, is because you can’t do without it.”