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Posts Tagged ‘virology’

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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How Hantavirus Hurts

December 19, 2014 Leave a comment
Click me to get to the reference page

Annual Hantavirus cases. Blue bars are # of cases. Black line is case fatality rate. CDC

There have been 6 confirmed cases of hantavirus pulmonary syndrome, and 3 deaths caused by Hantavirus in New Mexico this year. Hantavirus pulmonary syndrome is characterized as severe flu-like symptoms during the early stages of disease, which further progress into shortness of breath and accumulation of fluid in the lungs. Between 11 and 48 cases of hantavirus pulmonary syndrome occur annually in the US. Despite the low frequency of Hantavirus infections in the US, the case fatality rate of hantavirus is roughly 40%. Hantavirus is endemic in most countries, and circulates in wild rodents, which is then transmitted to humans through contact with rodent feces. Transmission usually occurs after breathing in aerosolized feces. Interestingly, there has only been one reported case of person-to-person transmission of Hantavirus in South America. Evidence suggests that person-to-person transmission does not occur with Hantavirus in the US.

There are several species of Hantavirus, of which the Andes virus is the most well characterized and prevalent in South America. In the US, an isolate of Hantavirus from the Four Corners region was cleverly named Sin Nombre virus, which in spanish translates to “No Name virus.” In 1993, an outbreak of Sin Nombre Hantavirus in New Mexico resulted in 24 confirmed infections, and 12 deaths. Hantaviruses belong to a family of viruses called the Bunyaviridae that are segmented single-stranded negative-sense RNA enveloped viruses.

Click me for a link to the paper

Hantavirus replication. Vaheri et al. 2013

The genome of Hantaviruses consists of three segments of single-stranded RNA appropriately named the S, M and L segments for their respective lengths. The genome encodes at least four proteins, the nucleocapsid protein, two envelope glycoproteins, and the RNA-dependent RNA polymerase that is required for replication of the RNA genome. Hantaviruses are able to enter cells via binding to host cell B2-integrin proteins that are normally responsible for cell-to-cell adhesion. It is curious that while Hantaviruses are able to replicate in lung epithelial cells similar to influenza, Hantavirus infection alone does not kill these epithelial cells. This brings up many questions regarding Hantavirus pathogenesis, which is somewhat addressed in a recent paper by Braun et al.

A subset of immune cells called Natural Killer (NK) cells are important for recognition and removal of virus-infected cells through the production of enzymes that kill infected host cells. NK cells are part of the innate immune response that are activated during early infection, and have the capacity to recognize when cells are infected through binding specific cell surface proteins. More specifically, NK cells can distinguish between normal healthy conditions and aberrant expression of these cell surface proteins that signal to inhibit or activate NK cell functions. Additionally, NK cells can be activated by interferon or other stimulating cytokines such as IL-12, 15, and 18.

Infection with Hantavirus leads to increased numbers of NK cells, which makes sense in the context of infection. Braun et al. observed that this population of NK cells is also activated, expressing increased levels of CD69, an activation marker, in peripheral circulating NK cells. In addition, granzyme and perforin, two effector molecules of NK cells that are released to kill cells were also upregulated. This indicates that NK cells proliferate and are activated in the context of Hantavirus infection.

Importantly, NK cells are unable to respond to Hantavirus alone, and Hantavirus is also unable to infect NK cells. In contrast, NK cells are able to recognize Hantavirus-infected epithelial cells that respond to Hantavirus infection by producing IL-15. However, the NK cells that are activated by Hantavirus-infected cells are unable to kill Hantavirus-infected cells and are more efficient at killing uninfected cells.It has also been reported that the Hantavirus NP protein can inhibit the activity of granzyme, thus blocking death of Hantavirus-infected cells. This strange effect may partially explain Hantavirus pathology.

Lymphocytes

Diversity of lymphocytes and their development. Janeway’s Immunobiology

Normally, activated NK cells are inhibited by healthy cells due to regular levels of cell surface proteins that tell the NK cell to deactivate. However, NK cell activation by Hantavirus infection leads to nonspecific killing of host cells. This cell death may contribute to vascular permeablization that occurs during Hantavirus infection. While this model may explain some Hantavirus pathogenesis, it is highly simplified as NK cells are a small subset of the immune system. It is also improbable that NK cells are the sole mediator of Hantavirus pathogenesis.

There are currently no approved antivirals or vaccines that provide protection against Hantavirus infection in the US. While the translational aspects of virology are important, it is often the basic science that leads to breakthroughs. This paper describing the interactions between NK cells and Hantavirus doesn’t directly provide a vaccine. However, like all basic research, it betters our understanding of Hantavirus pathogenesis which is necessary to make advances in potential Hantavirus therapies. I would also argue that we learn more through basic research than through applied research although the effects of basic research may take longer to materialize.

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The unfounded fears of Influenza research

August 10, 2013 Leave a comment

Tweety has the fluAs if the H5N1 transmission papers weren’t talked about enough, now the likelihood of similar research with H7N9 has lead to more unfounded fears. The first report of a possible human to human transmission of H7N9 came out a few days ago, and rather than instill fear that scientists will “create a deadly virus just to prove they can,” this possibility of an epidemic should highlight the importance of studying such a pathogen.

This has been harped on by every scientist I know, but it apparently still needs to be said. Even if we tried to engineer a more virulent and transmissible virus, we would never be able to out-craft nature. Natural selection will take place, and an animal-to-human or human-to-human transmissible virus will eventually pass into humans, and it is our job to get ahead of it and find out where it is going. There isn’t a single pathogen we could engineer that nature wouldn’t be able to do better.

Maybe more importantly, nature does not stop playing the game even if we decide to quit. It is Jumanji, and we are the kids playing the game. Even if we want to leave all the horrors of the game behind, we can’t. We are but pawns in nature’s game of chess.

The value of understanding mechanisms behind a potential pandemic virus is immeasurable. In the highly possible scenario, where a few thousand people come down with serious illness caused by influenza H7N9, the worst possible thing we could say is “well you’re screwed because we stopped looking for a cure.” Even in the most extreme circumstance, where a lab strain is somehow released that is highly transmissible and virulent, the best way to combat the threat is to do more research. 

Biosecurity is something every lab takes very seriously. As researchers, we take precautions to prevent both samples from getting contaminated, as well as ourselves from getting infected. Scientists are humans, too. We would rather not get seriously ill, and thus we have measures to prevent exposure to pathogens.

The fear mongering will never end because layman-shocking sensationalized headlines sell. But like all trolls, they get off on your attention. So give them none, and leave the scientists and the ferrets to save your lives. After all, “it is a curious feature of our existence that we come from a planet that is very good at promoting life but even better at extinguishing it.” – Bill Bryson in A Short History of Nearly Everything.

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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.