Tuesday, August 21, 2012

Xerox the available gene if the other important fellow is not available

Hello people,

   Welcome back readers for yet another episode of interesting microbiology. As always am back here to discuss some cool microbiology, some science and blah bla bla. Lately i have been listening to some interesting talks in ibioseminars and learning some interesting facts. The content is really appealing and i advice you to check it out.

Fig 1: Big bang Theory (Wikipedia)
    Science is ever changing. Older hypothesis or theories are challenged with better technologies and newer explanations are given grounds. Theories that were once considered path breaking are remodeled to break more paths. This applies to any branch of science. If can sum it up "Theories are best only until a newer one appears". Who doesn't know about the big bang? The origin of universe!!! And if i say the theory is now challenged! Well, thats what is in forefront news now. James Quach, the lead author of a new paper is the challenger. (Read more about it here)

    Another work that caught my brief attention is a work on hantavirus. Hantavirus is a negative sense RNA virus of the Bunyaviridae family. The virus is known to be pathogenic in humans and the infection is basically a zoonosis, mean to say acquired from animals. Rodents are the important source. The infection mainly leads to Hantavirus pulmonary syndrome (HPS) or Hemorrhagic fever with Renal syndrome (HFRS) (Source). Type I interferon (IFN) is known to be inhibitory against the Hantaviral replication. Obviously, the virus has evolved strategies to bypass this. How this is achieved is largely unknown. The paper published in Advances in Virology by Valery Matthys and Erich R. Mackow (Link), address this issue. The viral RNA is sensed by the MAVS system (See my previous post on MAVS). The pathway finally results in activation of a protein STING (Stimulator of interferon genes). For a detailed literature on STING go here. Gn proteins cytoplasmic tail (Gn-T) is proposed to be intefering with STING-TBK1-TRAF3. The fig 2 shown below shows the action.

Fig 2: Potential model of hantavirus Gn-T disruption of STING-TBK1-IRF3 complex formation
 
    The next story has to do with "Rift valley fever virus". Just as the above this also is a negative sense RNA virus of the Bunyaviridae. RVF causes zoonotic infection typically transmitted by mosquito bite (Aedes or culex). The paper titled "Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells" shows the potential of curcumin, a compound found in turmeric to inhibit RVF replication. The paper identifies that IKK complex in NF-kB pathway is important for viral replication and by down-regulating it they can impair the viral replication. The fig 3 illustrates the role of IKK complex formed from 3 subunits One thing that this implies to me is somehow the virus needs NFkB activation for its survival in contrast to the hantavirus that i talked about above.

Fig 3: NF-kB pathway (Source)
Photo 1: Aarthi Narayanan


     The down regulation of IKK complex was achieved using circumin. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2 mediated phosphorylation of the viral protein. The effect was shown against the MP-12 strain (lab strain) and the virulent ZH501 strain. “Curcumin is, by its very nature, broad spectrum,” Narayanan says. “However, in the published article, we provide evidence that curcumin may interfere with how the virus manipulates the human cell to stop the cell from responding to the infection.”. Co investigator Kylene Kehn-Hall adds “We are very excited about this work, as curcumin not only dramatically inhibits RVFV replication in cell culture but also demonstrates efficacy against RVFV in a mouse model.” (Source).

     One paper that needs to be discussed in a slight detail is by Tolu Oni etal. But first some background information for this paper.

   Mycobacterium tuberculosis is a common respiratory pathogen in many parts of the world. TB as nicknamed for tuberculosis, can often be very challenging to diagnose. Most of the diagnostic lab rely on a simple AFB staining technique (Not even fluorescence method), with low sensitivity to diagnose TB. The tests is sometimes accompanied by a simple mantoux test. Newer diagnostic methods are slowly making its way to diagnostics. For a concise list of newer diagnostic method in detection of TB check out my ppt below.



       Latent tuberculosis Infection (LTBI), is a difficult to diagnose situation. Often a lab was left with an option of mantoux test which cannot distinguish between a latent and past TB infection. The method was then replaced with interferon-gamma release assays (IGRA). Interferon Gamma Release Assays are theoretically a class of assays for viral and infectious diseases that measure the CMI (Cell mediated immunity) in infected individuals through the levels of interferon gamma released. In the event of infection, T cells from the individual will be senstized (via MHC proteins) to the antigens presented by cells of the infecting organism. T cells will thus be able to bind to foreign infecting cells, releasing interferon-gamma (Reference). Please note that mantoux test and IGRA, both detect CMI.

Fig 4: Mantoux test (Source)
      Now the question is what is the difference in terms of detection if both assays detect the same profile. The answer lies in the fact that, for TST (Tuberculin skin test) or mantoux test the complex mixture of different antigens used are not specific for M. tuberculosis. Hence a local immunologic activity at the site of the antigen deposition does not differentiate between an existing immune response elicited by bacille Calmette-Guérin (BCG) vaccination, past infection or cross reacting mycobacterium. IGRA is a newer generation assay, and is designed to be more specific. The assay uses specific M. tuberculosis antigens like early secretory antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and the TB7.7 antigens. This avoids false positives with previous vaccinations. And by the way, IFN-γ is produced by different cells of the immune system: CD4 T-cells, CD8 Tcells and NK cells. For more information on IGRA refer here. Quantiferon-Gold in-tube assay (QFT-IT) or T-spot are the commercially available IGRA.      Now let me give you a situation. What if you do a IGRA for detecting TB in HIv infected patient? Just think about it before you read on. Its a immunocompromised situation and the assay depends on sound immunity. And mind you TB infection is more common in HIV infected patients at least as far as i know. The paper states that the performance of QFT-IT is Immuno-dependent and T spot is not. (I don't know the explanation in this case).

Table 1: Interpretation of QFT (Source: ECDC guidance for IGRA)
 
     "The aim of this study was to evaluate predictors of indeterminate gamma interferon (IFN- ) responses, particularly in HIV infected persons". In this paper the main finding was confirming an association between low CD4 counts in HIV-coinfected persons and higher odds of indeterminate IFN- results. A previous BCG exposure also accounted for some of the intermediate results. My interpretation of this paper is that if I'm going to use this assay then, i will preferable retest the intermediate results or declare it as negative. I urge the readers to read this paper for more information as it is open access. Another paper that i want to discuss here is that by Nels C. Elde and others on poxvirus. Poxviruses are DNA viruses, the most famous members among them include small pox and vaccinia. For readers interested in reading some history and eradication of Small pox please go here. Coming to the point of paper, DNA viruses mutate slowly in comparison to RNA viruses. Yet, they are equally comparable to how they adapt and evolve to challenges in a cell. A theory in evolution referred as Red queen Hypothesis states, "In reference to an evolutionary system, continuing adaptation is needed in order for a species to maintain its relative fitnessamongst the systems being co-evolved with" (Taken from Wikipedia).

Fig 5: Protein kinase R activation in defense (Source)
     One of the cellular defending strategy on sensing a ds-RNA is to activate Protein Kinase R (PKR; see fig on the left). This PKR phosphorylates the translation initiation factor eIF2a to inhibit protein production This interferes with viral replication.

     I need to take a step back here. I was talking about dsDNA virus and now am telling that sensing RNA is important in defense. If you seem to miss the point, the dsRNA is found as an intermediate in the life cycle of poxvirus.

      Coming back to the point. If the host evolves to produce a resistance as per the red queen hypothesis the virus should also evolve a bypass strategy. So what does the virus have? In case of vaccinia 2 antagonists seems to be important- K3L and E3L. They simply inhibit the PKR form activation.

     The researchers now tried to answer the question "How the poxvirus can mutate quickly with a DNA genome, that is specified to have a lower mutation rate". To understand this they passaged the vaccinia virus  nullified for E3L from Copenhagen strain thus creating an artificial selection pressure on K3L to inhibit PKR. The mutant virus (E3L-), replicated poorly for first few rounds in comparison to its wild counterpart. The method was to inoculate new batch of HeLa cells from previous growth. By 6th round of replication they were able to see a 10 fold increase in each subsequent passage. In short, the virus was getting better and better. Now thats what is capability. You put too much pressure, yet the virus emerges successful. Boy, thats impressive.

      Looking for answers to this phenomenon in genome of the virus what they found is truly smashing. They found only two non-synonymous and one synonymous polymorphism in the genomes of passage 10 virus replicates at a frequency higher than 1%. Mean to say mutation was low. They had also carried out southern blot of viral DNA at various points of passage and found that the copy number of K3L was increasing slowly. Here's the explanation. the increased number can sustain increased fitness and so better replication. Despite the fact that K3L is weak antagonist of PKR the mere concentration overcame the problem of not having the better counterpart E3L. And yes they also did a bunch of experiments to prove that K3L was involved here.

     A specific mutation was noted in high K3L containing viruses. (Note that, all viruses that had gained fitness didn't possess this change). There was a codon change- H47R. Mean to say, Histidine changed at amino acid number 47 to Arginine. Though the paper doesn't address if the specific change has produced any specific action, the authors did comment that somehow it produced a gain of function. My impression was that possibly the specific site had increased some kind of slippery replication to cause increased copy numbers (I don't know if thats the case, but thats a possibly worthy explanation from my side).
Fig 6: Propose mechanism for rapid evolution (Source)
      Nels C. Elde, the lead author of this paper commented "Our studies show that increasing K3L copy number leads to increased expression of K3L and enhanced viral replication, providing an immediate evolutionary advantage for those viruses that can quickly expand their genome.” and “Our observations reveal that, while poxviruses do undergo gene mutation, their first response to a new, hostile host environment can be rapid gene expansion. We also found evidence that the virus genome can contract after acquiring an adaptive mutation, thus alleviating the potential trade-off of having a larger genome, while leaving a beneficial mutation in place.” (Source)

      This is not a new phenomenon. various bacteria simply increase their products, such as efflux proteins that can throw the drug out of bacterial cell. so if u increase drug concentration, the enemy increases his efflux.

      All said, i have a take on this paper other than what the author proposes. The first take for me if a virus can simply increase gene copies to overcome inhibition, this is a difficult situation. If we are to make drugs that are inhibitory in action, this strategy can easily overcome the pharmacological defense. The virus can simply increase its product. But my doubt is how much. I mean after all its a virus how much copy number can it increase? The genome needs to be accommodated after all. So i just do some wild math here. If the virus can increase its gene by even a 10% which can be packed in, then the drug dose is required to sustain this is somewhere in 100 times. At that concentration any antiviral is toxic or non usable. So we don' stand a chance at least theoretically.


ResearchBlogging.org
Tolu Oni, Hannah P. Gideon, Nonzwakazi Bangani Relebohile Tsekela, Ronnett Seldon, Kathryn Wood, Katalin A. Wilkinson, Rene T. Goliath, Tom H. M. Ottenhoff and Robert J. Wilkinson (2012). Risk Factors Associated with Indeterminate Gamma Interferon Responses in the Assessment of Latent Tuberculosis Infection in a High-Incidence Environment Clinical and vaccine immunology, 19 (8), 1243-1247 DOI: 10.1128/CVI.00166-12

Nels C. Elde, Stephanie J. Child, Michael T. Eickbush, Jacob O. Kitzman, Kelsey S. Rogers, Jay Shendure, Adam P. Geballe, & Harmit S. Malik (2012). Poxviruses Deploy Genomic Accordions to Adapt Rapidly against Host Antiviral Defenses Cell Host & Microbe, 150 (4), 831-841 DOI: 10.1016/j.cell.2012.05.049

Further reading:
  1. Rutherford M, Alisjahbana B, Maharani W, Sampurno H, van Crevel R, et al. (2010) Sensitivity of the Quantiferon-Gold In-Tube Assay in Sputum Smear Positive TB Cases in Indonesia. PLoS ONE 5(8): e12020. doi:10.1371/journal.pone.0012020. Link
  2. H Ludwig etal. Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara. J Gen Virol May 2006 vol. 87 no. 5 1145-1155. Link
  3. M V Davies, H W Chang, B L Jacobs, and R J Kaufman. The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms. J Virol. 1993 March; 67(3): 1688–1692. Link

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