Tuesday, November 27, 2012

A Rival for HIV


   In my previous blog post, I hosted 3 relatively new terminologies of interest. Today, I want to chat about a GB virus that has raised its interest in Clinical Virology, not because it causes a disease, but probably it is a virophage against HIV!!!

    GB virus (or more preferentially known as GBV-C or Hepatitis G virus) is a Flaviviridae member. The virus name comes from G. Barker, a surgeon, who fell ill in 1966 with a non-A non-B hepatitis which at the time was thought to have been caused by a new, infectious hepatic virus. The virus was first reported isolated in 1955 (Reference). It is not currently assigned to any particular genus, but proposed to be put under the Pegivirus. GBV-C has 7 genotypes in it. (Reference). GB virus is a lymphotrophic, non pathogenic virus found in a small set of human populations. The Fig 1 shows the distribution of GBV-C distribution.

Fig 1: Distribution of GB virus C/hepatitis G virus genotypes. Source
   The term "non pathogenic virus", that i have come across in literature is questionable in this context. If there is a virus that can be seen in humans and can infect lymphocytes do you call it non pathogenic. I do agree that healthy individuals, clear the virus from their system and cause no observable effects in many. Evidence of infection has been demonstrated by HGV RNA detection and serum anti-HGV-E2 antibody among children with histories of transfusions. So that means they have been in and out of cell. And to call a virus non pathogenic, is not easily digestible to me. The virus is transmitted efficiently through sexual contact, mother to infant, and blood products.

    If the GBV-C virus is such a non interesting case where the infection is blown below the carpet, then why is medical virology taking such a large interest in this virus? The answer comes from a remarkable concept, "Retro positive patients co-infected with GBVC tend to show a slower disease progression". That is something science can bank upon.

Photo 1: Press conference with Jack T. Stapleton, MD, University of Iowa.
   In a press conference in 49th Annual meeting of infectious disease society of America, JT Stapleton posed a question "Every study that shows persistent [GBV-C] infection over time has shown a survival benefit to HIV co-infected patients. How does this work? Why does this virus improve survival among people with HIV? Can we use it therapeutically?” “If we put this peptide on top of cells it does not inhibit, but if we introduce it inside the cell it does inhibit HIV". I read over it again and again. Are we dealing with concept of virophage here?. For source of Photo 1 and information go here.
   There are several theories as to what is the mechanism. But most of theory revolves around 4 important players- RANTES, MIP-1α, MIP-1β, and SDF-1.
Every study that shows persistent [GBV-C] infection over time has shown a survival benefit to HIV co-infected patients. How does this work? Why does this virus improve survival among people with HIV? Can we use it therapeutically?”

Read more: http://sciencespeaksblog.org/2011/10/21/gb-virus-c-hiv%e2%80%99s-viral-enemy/#ixzz2DPdNs8Fu
49th Annual Meeting of the Infectious Diseases Society of America in Boston

Read more: http://sciencespeaksblog.org/2011/10/21/gb-virus-c-hiv%e2%80%99s-viral-enemy/#ixzz2DPd2UbX6
Fig 2: In vivo effects of GB virus C (GBV-C) infection that can potentially interfere with HIV replication. Source
     RANTES or CCL5, stands for "Regulated on Activation, Normal T Cell Expressed and Secreted". RANTES is known to compete with HIV envelope for co-receptor binding, which alters activation of the HIV fusogenic machinery. RANTES is also known to produce a steric hindrance in HIV binding.  MIP-1α (CCL3) and MIP-1β (CCL4) represents Macrophage Inflammatory Protein are known to be involved in suppression of HIV infection in a dose dependent pattern.

     SDF-1 (Stromal cell derived factor-1; CXCL12) can bind to CXCR4 and inhibit viral entry, but also increased susceptibility to CCR5. This provides an interesting case where effects of SDF-1 on the HIV-1 life cycle is multiple and often a paradox, with inhibition of viral entry and a stimulation of proviral gene expression (Reference). That to some extent does explain why X4 strains are predominant in early infection and later an increase is seen in CCR5 tropic strains.

    With the Co-infection of GBV-C, it is speculated that the interfering virus causes an increased expression of RANTES, MIP-1α, MIP-1β, and SDF-1 which by said mechanisms above can inhibit the HIV to a observable extent. This brings me a question. Can I achieve the same with any other virus? The most obvious answer is "Yes". For example human herpesvirus type 6 inhibits R5 strains of HIV-1 in vitro by inducing RANTES (Reference) and Influenza virus can inhibit HIV through a type 1 interferon action (Reference). T. cruzi produces a substance called as cruzipain (Secreted by trypomastigote form) has also been shown to inhibit HIV-1 replication (Reference). But then its known that, clinically significant HIV suppression doesn't occur with HHV 6 or Influenza. Its plausible that the apropos factors are not the only and deeper mechanism should exist.

Fig 3: In vitro effects of GB virus C (GBV-C) proteins E2 and NS5A on CD4+ T cells. Source
      So when I had a detailed look at the above figure (from a review by JT Stapleton), it made sense. The protein E2 and NS5A (Nonstructural Phosphoprotein 5A) had a direct impact on the HIV replication system rather than just the entry system. The exact mechanism of action is not known but then there is a impact. Possibly the GBV-C is a competitor of HIV. And how does that make sense, in evolutionary terms? As I can see it, GBV-C and HIV, both can be sexually transmitted, passed through blood products etc. Possibly, their co infection induced rivalry. I have no paper to cite this or support my saying but i don't see why not!!!

    Take home message is simple.GBV-C and HIV are antagonistic with significant effects. Probably we can think of probiotic with this virus.

ResearchBlogging.orgBhattarai N, & Stapleton JT (2012). GB virus C: the good boy virus? Trends in microbiology, 20 (3), 124-30 PMID: 22325031

Further Reading:

1. Mohr EL, Murthy KK, McLinden JH, Xiang J, Stapleton JT. The natural history of non-human GB virus C in captive chimpanzees. J Gen Virol. 2011 Jan;92(Pt 1):91-100. Link

2. Sánchez-Martín MJ, Urbán P, Pujol M, Haro I, Alsina MA, Busquets MA. Biophysical investigations of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide. Chemphyschem. 2011 Oct 24;12(15):2816-22. Link

Monday, November 19, 2012

Virophage, Viroporins and Trogocytosis

Time for some microbiology,

        I'm simply amazed at the set of terminologies that keep arriving in microbiology. For last few days, I spent some time listening to some talks which i could grab hold of online. When i completed listening to the talks, i came to the conclusion that i could have a whole blog post just talking about some terminologies that have slowly managed to squeak into the Medical literature in terms of microbiology and immunology. One of them is vomocytosis, about which i have already written. More to come include- Viroporins, Virophage, Trogocytosis, NETosis, Paleovirology, Fungivore etc.


Photo 1: Giant mamavirus particles (red) and
 satellite viruses of mamavirus
called Sputnik (green). Source
    So what is this virophage? The name would imply to a reader almost immediately that these viruses that infect other viruses. But, the truth is this isn't true. Baffling!! Virophage is actually a misnomer. When the first giant Mamavirus was discovered (inside an Acanthameoba), a smaller co infecting virus referred as Sputnik was seen as a companion. Scientists soon realized that the sputnik was taking advantage of Mamavirus replication factory for its own replication. Moreover, presence of Sputnik reduced the turnover of Mamavirus!!

     This observation went as far as to say that viruses can be infected by other viruses and hence virus should be considered to be a living entity. The comment by Jean-Michel Claverie, a virologist reads so “There’s no doubt this is a living organism. The fact that it can get sick makes it more alive”. The argument has taken a long way of impressive research. With current set of understanding, virophages looks more like, commonly known helper viruses. However, the exception is that in this case there is a actual viral interference. But i still have enough doubts to say one virus parasitize another, simply because the first and the second virus both use the same host resource, rather than the later using earlier virus entirely. This probably means the two virus competing with each other for the same niche.


Fig 1: Viroporins, Source
      Viroporins are small proteins (typically 50- 120 amino acids long), produced by virus encoding a function of ion channel. The most important feature is that they contain at least one hydrophobic transmembrane, to form an amphipathic alpha helix. Based on the structure the viroporins are classified into 2 class- Class I (have a single membrane-spanning domain) and Class II (form helix–turn–helix hairpin motifs that span the membrane). 

     Some of the best studied viroporins include- Poliovirus 3A, Poliovirus 2B, Coxsackievirus B3 2B, HIV-1 Vpu, Influenza A virus M2, Influenza B virus NB, HCV p7, BEFV alpha-1 protein etc. A wide variety of functions are implicated to be caused by viroporins. This inculdes- Alteration of plasma membrane potential, Alteration of cellular Ca2+ homeostasis, induce intracellular membrane remodelling to generate viroplasm and essential part in assembly, budding and release of the viral progeny.


Fig 2: Trogocytosis. Source
   When a cell, lends its membrane lipids and membrane bound proteins, directly from one cell to other, the phenomenon is called as Trogocytosis. This basically is a synapse between two cells (as shown in Fig 2 to the left) that leads to transfer of materials. The mechanism of extreme importance in immunology and occurs commonly than actually thought.

     Trogocytosis has been shown to be important in transfer of antigenic material from macrophages to lymphocytes, uptake of macrophage Fc receptors and MHC molecules by T cells, acquisition of recipient
MHC class I and II molecules on donor thymocytes in bone-marrow chimaeras, transfer of MHC class II
proteins from splenic cells to allogenic T-cell clones, capture of B-cell surface immunoglobulin by T cells and more (Taken from Ahmed etal). More recently it has also been shown to be important in sustaining Intracellular Signaling in CD4 + T Cells

Desnues C, Boyer M, & Raoult D (2012). Sputnik, a virophage infecting the viral domain of life. Advances in virus research, 82, 63-89 PMID: 22420851

Nieva JL, Madan V, & Carrasco L (2012). Viroporins: structure and biological functions. Nature reviews. Microbiology, 10 (8), 563-74 PMID: 22751485

Osborne DG, & Wetzel SA (2012). Trogocytosis Results in Sustained Intracellular Signaling in CD4+ T Cells. Journal of immunology (Baltimore, Md. : 1950), 189 (10), 4728-39 PMID: 23066151

Further reading

1. Fischer MG, Suttle CA. A virophage at the origin of large DNA transposons. Science. 2011 Apr 8;332 (6026): 231-4. Link

2. Gonzalez ME, Carrasco L. Viroporins. FEBS Lett. 2003 Sep 18; 552 (1): 28-34. Link

3. Ahmed KA, Munegowda MA, Xie Y, Xiang J. Intercellular trogocytosis plays an important role in modulation of immune responses. Cell Mol Immunol. 2008 Aug;5(4):261-9. Link

Monday, November 12, 2012

Dynamic Gut Microbiome


    Let me begin with a question. What is the most common bacteria in the gut flora? Before reading on, give a small thought and come up with your own answer. If you answered it E coli, you are wrong. You are not the only one to come up with this answer. Often when I ask this question to my students, they come up with the same answer. Reason, almost always when you attempt bacterial culture your routine culture plates grows E coli. So let me explain why it is not the case.

Photo 1: Lactose fermenting pink colonies
E coli. Image source
    Culture media is designed to allow growth of certain group of bacteria. No matter what culture media you have, it will support the growth of limited set of organisms. Suppose, I use a specific culture media, say for example McConkey's agar which supports growth of enteric organisms and my growth conditions is aerobic, near neutral pH so on and so forth. The bacteria that can grow most rapidly in the plate under predefined conditions is at a competitive advantage of growth. The bacteria that fits very easily on to this criterion is E coli. However, this doesn't imply that the species is most predominant there.

McConkey's Agar contains

Enzymatic Digest of Gelatin ................................................. 17 g
Enzymatic Digest of Casein ................................................. 1.5 g
Enzymatic Digest of Animal Tissue....................................... 1.5 g
Lactose .............................................................................. 10 g
Bile Salts Mixture ............................................................... 1.5 g
Sodium Chloride ................................................................. 5 g
Neutral Red ........................................................................ 0.03 g
Crystal Violet ..................................................................... 0.001 g
Agar .................................................................................. 13.5 g

Final pH: 7.1 ± 0.2 at 25 C

   The E coli growth is abundant in this case since it can tolerate the bile salt mixture well and is suited to grow in the conditions. For argument sake, had I tried to grow Staphylococcus here, it simply wouldn't grow no matter how much much quantity of Staphylococcus, I try to add. This is for the simple reason that the Staphylococcus cannot tolerate bile salts.

    So, now that i have impressed you the fact that E coli is not the most common organism in gastro-intestinal tract am obliged to answer the who? The answer now doesn't seem to be so simple. The most accepted fact is that bacterial species making up the human gut microbiota are strict anaerobes, of which predominant organisms are members of the genus Bacteroides. Several species has been documented to inhabit the human gut flora. The list includes- Bacteroides fragilis group, Bacteroides plebeius, Bacteroides coprocola, Bacteroides helcogenes, Bacteroides intestinalis, Bacteroides finegoldii, Bacteroides dorei, Bacteroides cellulosilyticus sp. nov, Bacteroides xylanisolvens sp. nov and Bacteroides thetaiotaomicron.

Photo 2: Intestinal section from a gnotobiotic mouse model inoculated with selected bacterial species found in the human gut. Blue=Bacteroides WH2, green=Bacteroides thetaiotamicron, pink=Bacteroides vulgatus, yellow= Collinsella aerofaciens, red=Ruminococcus torques. Credit: Yuko Hasegawa/MBL Woods Hole. Source
    Other more common bacteria found in the gut flora include Prevotella speciesa and Fusobacterium species. However to quote one particular species or group as normal abundant bacterial community in the human microbiome is misleading. Let me quote from Huse , a researcher in Human Microbiome Project (HMP). “The more closely we look, the more bacterial diversity we find, We can’t even name all these kinds of bacteria we are discovering in human and environmental habitats. It’s like trying to name all the stars. What this means is, there is not just one way to be healthy, There doesn’t have to be one or two ‘just right’ gut communities, but rather a range of ‘just fine’ communities”. Source. As predictable, the gut flora is probably dynamic and most influenced by the diet. As a matter of fact, the type of diet (Vegetarian or Non vegetarian diet decides the percentage count of Bacteroides spp., Bifidobacterium spp etc in the gut flora. (Reference)

ResearchBlogging.orgHuse, S., Ye, Y., Zhou, Y., & Fodor, A. (2012). A Core Human Microbiome as Viewed through 16S rRNA Sequence Clusters PLoS ONE, 7 (6) DOI: 10.1371/journal.pone.0034242

Further Reading

1. Celine Robert, Christophe Chassard,  Paul A. Lawson and Annick Bernalier-Donadille. Bacteroides cellulosilyticus sp. nov., a cellulolytic bacterium from the human gut microbial community. IJSEM (July 2007); 57 (7): 1516-1520. Link

Sunday, November 11, 2012

The puzzling Vomocytosis


      Late, late, late.... The post is now out at the last moment of the week. I know, i was supposed to be posting earlier. I was out this week, on a conference and have kept busy for a few days. But then, i just have thinks to talk about. Cause posting things here, at least in part helps me to analyze and remember concepts. Thats the way, it works for me.

Table 1: First Results of Phase 3 Trial of RTS,S/AS01 Malaria Vaccine
        Well, i don't have much to say as a breaking news of the week. But then the RTS,S vaccine seems to be failing from what was expected from it. 4 species P. falciparum, P. vivax, P. ovale and P. malariae are well known pathogens of malaria. P. knowlesi is a recent discovery in the list of pathogens and seen commonly in the South Asian regions. RTS is a hybrid polypeptide consisting of a portion of the circumsporozoite protein (CS), a sporozoite surface antigen of the malaria parasite P. falciparum strain NF54, fused to the amino-terminal end of the hepatitis B virus S protein. AS01 adjuvant System consists of a liquid suspension of liposomes with two immunostimulant components: 3’-O-desacyl-4’-monophosphoryl lipid A (MPL) and Quillaja saponaria 21 (QS21). The first results of the vaccine results was published in NEJM. The preliminary results was encouraging (See Table 1). As can be seen, the vaccine efficacy against clinical and severe malaria of nearly 50% among children 5 to 17 months of age.

      However the second end point published on November 2012 in NEJM, show that the vaccine efficacy in the second year, has dropped further. The study is a US$200 million project funded by the Bill gates foundation. Bill gates said "The efficacy came back lower than we had hoped, but developing a vaccine against a parasite is a very hard thing to do, The trial is continuing and we look forward to getting more data to help determine whether and how to deploy this vaccine". David Kaslow has a defensive statement to make "We always want to see high levels of protection, but I think we have shown again that RTS,S has an acceptable safety profile, and helps to provide protection against clinical malaria in the population". The completed data will be obtained only by 2014, which means much remains to be seen. As Kaslow says "To jump to conclusions is a disservice to the public". For source of information see here and here.

     The second highly discussed article of the week is by Joel V. Weinstock in Nature. The article discussed the approach of administering helminthic parasites (Preferentially as microscopic eggs) to people who have autoimmune disorders. Autoimmunity is a problem posed by the immune system chiefly because the cells of the immune system are improperly regulated. The infection by the worm can somehow dampens the extra immune response possibly by training Treg cells. The method has been found to be safe with very little side effects. That means i could think about administering a simple helminthic egg and treat people with conditions like asthma and Inflammatory bowel disease. Oh yes, a lot of work has been done before this on the subject. The most famous of this series include the hookworm administration to fight asthma. But retrospectively i could find many articles that argue against the hypothesis (Reference here and here). Probably this article will stimulate the search for a ground solid evidence.

     The last part of my post is Vomocytosis. You have most possibly heard of terms such as phagocytosis, pinocytosis, Endocytosis, exocytosis and more. But then this is most probably a new term for you. The term is not as famous as the others that i just mentioned. I just looked up into the Pubmed and found that there are just 3 articles (As of today on Nov 11, 2012) that talk about it. This pertains to a special feature exhibited by Cryptococcus neoformans, called as "Non-lytic exocytosis" or "Vomocytosis". I have previously written a post on a special set of cells called Titan cells, where you can give a background reading.

Fig 1: Vomocytosis
      A wide set of earlier laboratory investigations have pointed to a phenomenon where the phagosome containing C. neoformans undergoes acidification, fusion with lysosomes, and maturation. The punch line is almost any pathogen that wants to survive inside a phagocytic cell will interfere at least with one of the process there. Instead Cryptococcus enjoys sitting inside a fully mature vacuole without being effected. The secret lies in ability to produce highly powerful anti-oxidants that probably deconstructs the long time acidification. And when the fungus is interested in getting out of the vacuole at will. The mechanism of coming out without lysing the cell or interruption with cellular phagosome is called as Non lytic exocytosis or vomocytosis. This mechanism allows direct lateral spread of the fungus from one cell to another, first shown by Ma etal. This lateral transfer is not effected by cytochalasin D. That means the process is independent of actin based movement unlike other lateral pathogen transfer. The paper (from BMC) also shows 2 movie files on the lateral fungal transfer. You really need to give a look.

      Not much is known about the mechanism of vomocytosis. Its an open research question. What we know is, the phagosome containing the pathogen slowly swells due to secretion of capsular polysaccharide by the yeast. This swelling is followed by expulsion approximately after six hours, with a sudden expulsion. The pH of phagosome is thought to play an influential role. Neutralization of pH of vacuole can change the osmotic conditions which may in turn explain a sudden burst. A very detailed molecular study by Chayakulkeeree etal showed that cryptococcal expulsion from macrophages is CnPlb1 (Secreted phospholipase B1) -dependent (CnSEC14-1-dependent CnPlb1 secretion pathway).

     So i thought, what exactly does this CnPlb1 do? But then i couldn't find much data on its enzymatic activity. Nor am aware if the gene has been cloned and studied. But what i could find was CnPlb1 can reacylate lysophospholipids and reverse the damage caused to neutrophils by other secreted cryptococcal components (Djordjevic). Thats just interesting.

   The take home message is simple. Cryptococcus can get out of a macrophage by the mechansim called as vomocytosis, which is important in lateral transfer and dispersion of fungus within host. Is vomocytosis blocked by our immune system. Otherwise how do you explain Cryptococcal infection in Immunocompromised hosts only. Perhaps, much remains to be learnt.

Nicola AM, Robertson EJ, Albuquerque P, Derengowski Lda S, & Casadevall A (2011). Nonlytic exocytosis of Cryptococcus neoformans from macrophages occurs in vivo and is influenced by phagosomal pH. mBio, 2 (4) PMID: 21828219

Chayakulkeeree M, Johnston SA, Oei JB, Lev S, Williamson PR, Wilson CF, Zuo X, Leal AL, Vainstein MH, Meyer W, Sorrell TC, May RC, & Djordjevic JT (2011). SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans. Molecular microbiology, 80 (4), 1088-101 PMID: 21453402

Further reading:

1. Hansong Ma, Joanne E Croudace, David A Lammas and Robin C May. Direct cell-to-cell spread of a pathogenic yeast. BMC Immunology (2007), 8:15. Link

2. Sabiiti W, May RC. Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans. Future Microbiology. (2012 Nov) ;7 (11): 1297-313. Link

3. Julianne Teresa Djordjevic. Role of phospholipases in fungal fitness, pathogenicity, and drug development – lessons from Cryptococcus neoformans. Front. Microbio., 11 November 2010. Link

Thursday, November 01, 2012

New interest in Hecolin


    First, i must agree that i have been busy working on various aspects and just haven't got sufficient time to post microbiology. But then, i have lots to say. I have been thinking if i could post more than once a week just to cover up what i want to say. But then, time makes a factor. Learning new exciting things takes time and i believe you agree. Also I'm glad to see that my blog rank has significantly increased as per the technorati ranking. That motivates me to write more anyway.

Photo 1: Hepatitis E virus
   I have so many stories to talk about. However, i have been intending to write on this or long. Hepatitis E virus (HEV), is an enterically-transmitted acute viral hepatitis. The infection was first documented in 1955, is a member of the genus hepevirus in the family Hepeviridae. This is a an icosahedral, non-enveloped with single stranded positive-sense RNA genome. This virus has been sequenced and there appears to be at least 4 genotypes. Genotype 1 and 2 are human specific, but genotype 3 and 4 infect humans and animals. Genotype 3 has been linked to least virulence. Read more here. The infections have an incubation period of nearly 6 weeks (2- 9 weeks), and becomes clinically apparent in about 10% of the cases. The infection is usually self limiting, but has devastating effects in pregnant women (Reference).

Photo 2: Hecolin Vaccine
      There is no FDA licensed vaccine available for HEV. Hecolin was a prototype vaccine that had successfully entered clinical trials. It showed promising early results. The contents of vaccine was called as HEV 239 (30 microg of purified recombinant hepatitis E antigen adsorbed to 0.8 mg aluminium hydroxide suspended in 0.5 mL buffered saline) (Reference). This has happened in 2010.

     So, why am i pulling this out now? Its because of renewed interest, since the vaccine has been approved in China in 2011 by SFDA (China’s State Food and Drug Administration). Hecolin will be marketed by Xiamen Innovax Biotech Co. Ltd. Zhang, an investigator in the process says “Many people — including representatives of multinational pharmaceutical companies, venture capitalists, Chinese local government officials and Chinese entrepreneurs — think this is a worthy example of biotechnology investment,” he says". (Source).

    I wouldn't be surprised if the WHO decides to join hands with the Innovax and start vaccinating people around the world. 

Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Wang H, Yang CL, Jiang HM, Cai JP, Wang YJ, Ai X, Hu YM, Tang Q, Yao X, Yan Q, Xian YL, Wu T, Li YM, Miao J, Ng MH, Shih JW, & Xia NS (2010). Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet, 376 (9744), 895-902 PMID: 20728932
Park, S. (2012). Hepatitis E vaccine debuts Nature, 491 (7422), 21-22 DOI: 10.1038/491021a

Further Reading:

1.  Pathogenesis and Treatment of Hepatitis E Virus Infection. Gastroenterology. Volume 142, Issue 6 , Pages 1388-1397.e1, May 2012. doi:10.1053/j.gastro.2012.02.014. Link

2. Hepatitis E Vaccine Debuts. Scientific American; Oct 30-2012. Link