Another creationist doesn't understand science. Who knew?!

Nephilimfree, Youtube creationist extraordinaire and posterboy for the giant headphone lobby, has posted a new video entitled "Overlapping and Embedded Genes". Unfortunately for him, I am a lover of any and all things genetic. Also unfortunately for him, he doesn't have a clue what he's talking about. Fortunately for me, this provides me an opportunity to tear his video to shreds.

A little background may be necessary. Nephilimfree (who I will henceforth refer to as "Nephy", as I am an avid believer of making infuriatingly inane things tolerable by giving them cute names) made this video in response to a conversation he had with Sofiarune. He does not mention it, but I was also involved, as I am Sofiarune's go-to guy for molecular biology. I guess you can say I have a personal interest in this video, but I'd pick apart Nephy's claims in any event since they are, as usual, stupid on a Goats On Fire level.

First, the video. Be warned, it is rather long, running at just over 14 minutes. If you don't have the time (or the patience) to watch the whole thing, I'll give you the tl;dr (...or is that tl;dw...) version below. Go grab some coffee, because this post is gonna be a long one.



Nephy's argument is basically this: (i) the existence of overlapping genes is an obstacle to evolution because a single mutation will be deleterious to multiple genes and (ii) these gene pairs could not have possibly evolved and are thus evidence that they have been created. As we will see below, both of these points are patently false, and they demonstrate the extent to which Nephy is ignorant of both evolution and genetics.

Nephy starts the video off by mentioning his discussion with Sofiarune, and his insistence that genes overlap and that this is somehow proof that evolution isn't true. He states that "being the evolutionist that she is, she said 'No, no, no. Genes are read linear and they are not embedded'." Nephy is only telling a half truth here. In their original conversation, he presented "overlapping genes" in a very different manner than he does in this video, as the figure below demonstrates:

 

So Nephy broached the subject with a cryptic sentence about overlapping sequences "causing entropy" to "the code". Understandably, Sofia asked for clarification. In response, he talked about overlapping sequences in shotgun sequencing. This is entirely different. Overlapping sequences in shotgun sequencing and overlapping genes in the genome are not the same thing - one is a bioinformatic technique used to align sequence data in the proper order and the other is the idea that sequences can have multiple open reading frames (ORFs) coding for different gene products. He followed this up with a link to an article [not shown] which further obfuscated what he had meant. It talked about how, in the early days of molecular biology, there was debate over how the genetic code was read; were sequences read in one long linear manner, or were all three possible reading frames of a sequence read at once, resulting in overlapping sequences? It was determined early on that the correct answer was the former - codons were read one at a time in a linear fashion. In this context, Sofia was entirely justified in saying that genes did not overlap. Nephy seemed to have gotten all these concepts confused, but in his video, he presents it as if he gave Sofia a concrete definition of "overlapping genes". Leave it to a creationist to be vague, only to misrepresent your response.

Nevertheless, Nephy now has a concrete definition: overlapping genes are multiple coding ORFs contained within a sequence. Such overlapping genes do exist, primarily in RNA viruses. A study done by Chirico et al¹ (which I will go into some detail below) found that 75%  of the 2000 or so known species of virus have some extent of gene overlap. This does not mean that it is a widespread phenomenon as Nephy seems to believe. He points to an article entitled Mammalian Overlapping Genes: The Comparative Perspective² and is seemingly impressed by the numbers. It shows a total of 774 total overlapping genes in the human genome, and this might be impressive to Nephy, but it isn't to anyone who actually took the time to read the paper. This 774 is out of the 34,604 annotated gene sequences posted to the NCBI human genome assembly (build 33). That's a whopping 2.2% of genes.  So overlapping genes in humans are more the exception than they are the rule. This is the same for pretty much all eukaryotic organisms; overlapping gene sequences do exist, but they are pretty rare. They are mostly seen in viruses (indeed, overlapping sequences were first discovered in he late 1970s by researchers working with the phage φX174). Nephy talks about overlapping genes as if they are as numerous as the grains of sand on the beach, but they are actually quite infrequent. 

But Nephy isn't only concerned with the existence of overlapping genes. He claims that overlapping genes are a problem to evolutionists. He thinks this is because a mutation in the overlapping sequence would be deleterious to both genes and evolution could never favour such a situation. This is demonstrably false, and the evolution of overlapping genetic sequences can, and has, been explained. A paper by Rancurel et al.³ (2009) did just that. According to the authors, there are two characteristics of overlapping genes that alleviate evolutionary constraints on them: (i) overlapping proteins are full of amino acids with a high level of codon degeneracy and (ii) the regions of proteins encoded by overlapping sequences have a tendency towards structural disorder. It is worth going into some detail about these.

(i) To understand what the first point means, you will need to understand what is meant by "codon degeneracy". There are 64 different codons which can be constructed from the four nucleotides, but only 20 main amino acids which comprise any polypeptide. This means that there will be some amino acids that are encoded by multiple codons. Arginine, for example, has 6 different codons - CGU, CGC, CGA, CGG, AGA, and AGG. You'll notice that these codons are pretty similar to each other, and that is pretty handy for the cell. If, for instance, a mutation changes a CGU codon into CGC, CGG or CGA, it will still encode for arginine. The sequence has changed, but the output of the sequence remains the same. The mutation will have no effect on the protein sequence. This is what is meant by codon degeneracy. Amino acids with codon degeneracy will be able to tolerate some degree of mutation. This is important to overlapping genes, because the proteins produced by overlapping genes are high in amino acids that have codon degeneracy. What this means is that mutations to the sequence can be tolerated and won't have a deleterious effect. This is quite the opposite of what Nephy claims. He feels that a mutation will be doubly deleterious to overlapping genes, and he would be right if it weren't for the high frequency of codon degeneracy found in these proteins. In many cases, a mutation will have no effect at all on either protein in the pair.

ii) Structural disorder describes the extent to which the 3D structure of a protein is defined. Those proteins with a high degree of order have a strictly defined 3D secondary structure, while those with a high degree of structural disorder do not. They tend to rapidly change from one structural form to another - their secondary structure is not strictly defined. Since it is the amino acid sequence of a polypeptide that largely determines it's 3D shape and thus function, those with a high degree of structural disorder can tolerate some degree of mutation. Mutations will not affect the protein's 3D shape much because it doesn't have a definite shape to begin with. As the authors put it, "[d]isordered proteins are generally subject to less structural constraint than ordered ones". It should not be surprising, then, that the regions of proteins which are encoded by overlapping sequences tend to be structurally disordered. Those parts do not have a rigid secondary structure, so mutations will have less of a deleterious effect on these overlapping regions. Again, this is antithetical to Nephy's claim that mutations in overlapping genes are a death sentence for a cell.

As an aside, this point about structural disorder raises an interesting theological question: if overlapping genes were created by God as Nephy believes, then why did God decide to create proteins which have regions of structural disorder? If he created these proteins for a particular reason, then would he not have designed them all to have a definite 3D structure that belies their function? Did God just get lazy and figure "Eh, I'll just make some of these proteins structurally ambiguous. No one's gonna notice"? From a design perspective, it doesn't make sense, especially when the designer is perfect and all powerful. Why would he even design overlapping genes to begin with? Wouldn't he just stick to the good old "one gene = one gene product" plan? It's much simpler. It's a hallmark of poor design when things are more complicated than they need to be. How can a perfect God have an imperfect design, anyway?

So Rancurel and co. have thoroughly shown Nephy's point on mutation to be bunk. But they also give his claim that overlapping genes could not have evolved a thrashing. In fact, they do this in the very first paragraph of their paper! They state:

"Among several mechanisms, they [overlapping gene pairs] can be created by a process called "overprinting", in which a DNA sequence originally coding only one protein undergoes a genetic modification leading to the expression of a second reading frame in addition to the first one...The resulting overlap encodes and ancestral "overprinted" protein region and a protein region created de novo (i.e., not by duplication) called an "overprinting" or "novel" region" [See figure below, click to embiggen].

In other words, overlapping gene pairs can be explained easily by a natural process. Two genes exist with reading frames shifted relative to one another, with one upstream from the other. The loss of a stop codon in the upstream gene results in this gene being extended into the region of the second gene where it ends at a stop codon. The result, then, is two genes which share a region of sequence. This has an important evolutionary consequence. The novel region is free to take on novel cellular functions. For the reasons stated above, the novel region is under fewer constraints. In fact, the creation of proteins de novo in this manner has likely played a very important part in viral evolution⁴. In many viruses, the novel protein in overlapping protein pairs are virulence factors or encode enzymes that allow the virus to escape host defence mechanisms. De novo creation of novel proteins by overlapping genes represents another mechanism by which novel "information" is added to the genome, something creationists love to deny is possible. 

But not only can we explain how overlapping proteins pairs have evolved but we also have some good ideas why they would evolve. Chirico et al give four possible reasons why overlapping gene sequences may have evolved in viruses.

1.  Mutation rates: given the high mutation rates seen in viruses, it makes sense that there is pressure on viruses to keep their genomes short. Longer genomes will accrue more mutations than shorter ones. Overlapping genes are a way for a virus to expand its repertoire of genes without extending its genome.
2. Capsid size: some viruses have capsids of limited size. Increasing the size of these capsids to accommodate increased genome sizes is quite the undertaking and is likely to have a fitness cost. Thus it would be advantageous for the virus to have genes which overlap, since this takes up less space within the capsid. 
3. Gene length: larger genomes tend to have less overlap than smaller genomes. This might be because there is more room in larger genomes for the genes. Viruses may have overlapping genes simply because they have small genomes and cannot fit all the genes linearly.
4. Expression regulation: it is possible that overlapping genes evolved so that the genes in a gene pair can be regulated together.

The authors studied the sequence data from 62 different virus families to see which of the four possibilities best matched. They determined the likely cause for the evolution of overlapping genes in viruses was the constraint caused by capsid size. Nonetheless, it is entirely possible that any of the other three may have occurred in certain cases.

The reason I bring this up is because science has at least made an attempt to explain why overlapping genes exist at all. Nephy and his creationist brethren are content to claim that God created overlapping genes but they don't say a word about why he would have done so. The naturalistic explanation offered by evolution not only explains how but also why. The explanatory power of evolution is massive, while creationism offers little more than just-so stories and ad hoc justifications.

But despite all this, Nephy talks for 9 minutes about how overlapping genes are evolutionarily impossible, while saying absurd things like "Geneticists today are shying away from the use of the word 'gene' because it really doesn't describe what we observe in the genome of life. The preferred term is 'sequences' because it is difficult to say what code ends where and starts where in some cases". As someone with a degree in molecular genetics, this took me by surprise. No one sent me that memo! This statement makes little sense; genes and sequences are not equivalent, and it's actually pretty easy to determine where one sequence starts and one sequence ends. We've even been able to develop computer software that can predict such things in a matter of milliseconds. Given that Nephy has little scientific background and is definitely not part of the geneticist community, I'd like to know just where he got the idea that the term "gene" was passé. It's still very much a useful term and is used constantly in the scientific literature. 

He finishes off his video with a five minute snippet of a talk given by Hubert Yockey. I find it amusing that Nephy introduces this clip by saying "And now a few select moments from a lecture by a geneticist", followed immediately by a screen declaring "Biophysicist Hubert Yockey". Nephy, a biophysicist and a geneticist are not the same thing. Not even close. And Yockey isn't even a biophysicist to begin with; he's a physicist. He did some work on information theory and how it applies to biology, but this does not make him a biophysicist. I know these are big words for you, but do try to understand the distinction between them before you toss them around again. I won't actually go through the claims made by Yockey in the video (to be honest, I didn't even watch that part) because really, a physicist's claims about biology are about as meaningful as a butcher's claims about nuclear physics.

So once again, Nephilimfree has demonstrated that he has no idea what he is talking about. Overlapping gene sequences are NOT evidence of Divine creation - they make no sense if one assumes they were created, and they make perfect sense when interpreted in the light of evolution. We have a perfectly naturalistic explanation of their origin which conforms to experimental observation. Furthermore, mutations within overlapping genes are not always a bad thing, and have likely contributed to the evolution of pathogenicity in many species of virus. His claim that mutations in overlapping genes are always deleterious is patently nonsense. In fact, at one point in the video he claims that "all genetic mutations cause degretory[sic] effects to the genome's code", a claim that literally thousands of examples can disprove. He says that he has told evolutionists this for a long time and they "refuse to listen". There's a simple reason for that, Nephy - it's because you're flat out wrong.

Consider this a challenge, Nephy. Show me how what I have written is incorrect. Show me why the authors I have quoted are wrong - critique their work. Give me evidence taken from peer-reviewed scientific literature that backs up your claims, and not just idle speculation on your part. Give me a thorough refutation, thoroughly sourced. Because until you do your claims will be little more than verbal diarrhea.

---------------------------------------------------------------------------------

1. Nicola Chirico, Alberto Vianelli and Robert Belshaw. Why genes overlap in viruses. Proceedings of The Royal Society Biological Sciences . 2010 . 277: 3809-3817

2. Vamsi Veeramachaneni et al. Mammalian overlapping genes: the comperative perspective . Genome Research . 2004 . 14:280-286

3. Corinne Rancurel et al. Overlapping genes produce proteins with unusual sequence properties and offer insight into de novo protein creation . Journal of Virology . 2009 . 83(20): 10719-10736

4. F. Li and S. W. Ding . Virus counterdefence: diverse strageties for evading the RNA-silencing immunity. 2006 . Annual Review of Microbiology . 60:503-531

Not exactly the type of thing I thought I'd be associated with.

Going over my blog stats today I noticed something odd. I wrote a post two years ago about the interesting discovery of a virus that uses another virus as it's host. This actually seems to be the most popular thing I've written on my blog - it has about twice as many views as the next most viewed piece. In the last two days it has had quite a few views, and this is not odd, but I did notice that someone had found it by googling "cwgk.blogspot.com/2008/08". Kinda weird. I was curious what kind of search results that would turn up, so I tried it myself, and I found only four results: three were from my blog (expected) and one more from a forum called RevivalSermons.org.

Ok, so some Bible lover linked to my virus post on a religious forum. That in itself is kinda strange. But not as strange as what I found when I clicked on the link...

"Virus > could viruses and prions be Satan's attempts to create the building blocks of life - unsucessfully [sic] - or just a Satanic weapon ?" 

This was followed by four links to different sites that talked about the virus dead/alive debate, mine being the first. I have no idea how my blog post supports the poster's idea that viruses are some kind of satanic weapon. To be honest, I'm kinda creeped out.

AIDS Denialism: Deadly Ignorance Part III: Fuzzy Math and Distorted Reality

In the previous two part of this series (see Part I and Part II), I tackled the forerunners of the AIDS "skeptics" community, and addressed how Koch's Postulates support HIV as the cause of AIDS. In this part, I will assess how some of the deniers distort studies and misinterpret math to support their dangerous ideas.

If you spend any time on AIDS denialist websites, there are likely a few statistics that will show up repeatedly. This is because it is a common practice for AIDS denial websites to copy and paste the same arguments verbatim from one website to the other, no matter how old the information might be, or if the arguments have been debunked already. One such statistic that the deniers constantly toss around is that half – or more - of Africans who qualify as having AIDS test as HIV-negative¹. Specifically, they routinely cite three specific studies²:

122 patients with "AIDS": 69% test HIV-negative (Brindle, 1993)

227 patients with "AIDS": 59% test HIV-negative (Hishida, 1992)

913 patients with "AIDS": 71% test HIV-negative (Songok, 1994)

But if one were to take a critical look at each of these studies, they do not support the claims of the AIDS "skeptics" in the least. Let's take a look at each of them.

The first paper by Brindle et al. is entitled "Quantative bacillary response to treatment in HIV-associated pulmonary tuberculosis"³. In this study, the authors looked at 122 patients with "culture-proven pulmonary tuberculosis". These patients were divided into two groups, and each group had their tuberculosis treated with one of two different treatments. They then looked at how the HIV-positive individuals in each group took to their treatments compared to the HIV-negative individuals. The purpose of this was to determine if patients with HIV would respond to chemotherapeutic treatments differently than those without HIV (and they found that there was very little difference).

It should be noted that the 122 patients in the study were NOT patients with AIDS. They were patients with pulmonary tuberculosis. This is a symptom of AIDS, but of course, pulmonary tuberculosis can arise in humans in a variety of ways; it is not exclusive to AIDS patients. Of the 122 patients, only some of them had AIDS. So where did the denialists get their numbers? Of the patients that were given the first treatment, 17 were HIV-positive and 57 were HIV-negative. Of those that were given then second treatment, 20 were HIV-positive and 35 were HIV-negative. In total, 37 of the 122 patients were HIV-positive, so 85 out of 122, or 69%, were HIV-negative. But this is not the number of AIDS patients who were HIV-negative; rather it is the number of tuberculosis patients who were HIV-negative. This is a different thing altogether. The AIDS "skeptics" misrepresent this number to support their case, when in fact, it does not.

The second paper is "Clinically diagnosed AIDS cases without evident association with HIV-1 and 2 infections in Ghana" but Hishida et al⁴.The authors' aim in this paper was simple: to test patients suspected of having AIDS for HIV. It is important to remember that the patients they looked at were suspected cases of AIDS – the authors state "CD4 cells were not counted [in these patients] because of insufficient facilities". They looked at 227 cases and found the following results : 48 were positive for HIV-1, 17 were positive for HIV-2, 11 were positive for both stains, and 16 were of indeterminate HIV status. The remaining 135 patients were determined to be HIV seronegative. Those 135 of 227 patients amounts to the 59% the AIDS denialists toss about. So at a superficial glance at the numbers might be seen as giving support to the denialist arguments.

But the denialists apparently didn't read any further than the raw numbers, because the authors continue: the 135 seronegative samples were from patients that had "weight loss, prolonged diarrhea, chronic fever" – so is this AIDS? The authors didn't think so. They state "We believe that many patients of this group were perhaps improperly diagnosed or had other unidentified diseases…[t]he existence of other agents causing AIDS-like syndromes might be possible for these so-called HIV-negative cases." In other words, it's likely the HIV-negative cases were from patients who did not have AIDS to start with – remember that these were samples from suspected AIDS cases, and not confirmed cases. The denialists take a quick look at the numbers, point out the 59% HIV-negative cases and ask "How could this be?" without bothering to read further and realize that the authors have answered that very question!

I also feel that I should point out that the authors used serological techniques to detect the presence of HIV antibodies in the samples, rather than using more sensitive PCR techniques. I am confident that if this study was repeated today, using PCR and improved diagnostic techniques, the suspected AIDS cases that test HIV-negative would be quite fewer.

The third study the denialists refer to is Songok et al's paper "Low Prevalence of Human T-Lymphotrophic Virus Type 1 (HTLV-1) in HIV patients in Kenya"⁵. They claim that this study showed a huge 71% of AIDS patients were HIV-negative; such a bold claim better have a wealth of evidence supporting it. The first thing one might notice when reading it, however, is that it isn't a peer reviewed research study; it's just a letter to the editor. Nevertheless, the authors report on some original research they were doing: they wanted to check to see the prevalence of HTLV-1 infections in HIV infected individuals. They did this by collecting 913 samples from suspected AIDS cases in Kenya. Again – remember that these are not confirmed cases of AIDS. Using these samples, they tested for HIV-1 using both ELISA and western blots. They reported that 265 (or 29%) of these tested positive for HIV-1 on both tests. This is where the denialists get their statistic. However, this number represents the samples that were positive on both the ELISA and the western blot – it excludes those samples that tested positive on one or the other tests. They also tested for HIV-1 alone; they did not test for HIV-2, and this undoubtedly reduced the number of positive results. And again, the researchers did not use more sensitive PCR techniques, so it is entirely plausible that many positive cases were missed.

What all three papers have in common is that the estimates of HIV-positive individuals are underestimated. All three papers were written before sensitive PCR-based methods for detecting HIV were developed and put into common use, and before doctors were able to accurately diagnose AIDS and differentiate it from other immunodeficiency syndromes. And what deniers need to realize that it is impossible to obtain a 100% detection rate. Multiple factors exist that prevent the presence of HIV from being detected 100% of the time. If the AIDS "skeptics" really wish to show that HIV does not cause AIDS, then perhaps they should find a study to cite that isn't two decades old.

So as usual, critical examination of denialist claims shows that they don't have a leg to stand on. Better luck next time, guys. Come back when you have recent research that you can cite without twisting the statistics into misleading conclusions.

-------------------------------------------------------------------------------------------------------------------------------

1. See http://www.healtoronto.com/nih/ for an example.
   
2. These three statistics were also stated by the Youtube user who prompted this series on debunking AIDS denial nonsense.
   
3. Brindle et al. Quantative bacillary response to treatment in HIV-associated pulmonary tuberculosis. 1993. American Review of Respiratory Disease
   
4. Hishida et al. Clinically diagnosed AIDS cases without evident association with HIV-1 and 2 infections in Ghana. 1992. The Lancet
   
5. Songok et al. Low Prevalence of Human T-Lymphotrophic Virus Type 1 (HTLV-1) in HIV patients in Kenya. 1994. Journal of Acquired Immune Deficiency Syndromes

AIDS Denialism: Deadly Ignorance Part II: Koch’s Postulates, AIDS and more Wackaloonery

(Click for Part I and Part III of this series)
In my previous post, I wrote about some of the faces of AIDS denialism, and the arguments (and I use that word loosely) that these "skeptics" present. What their arguments revolve around - and indeed the arguments put forth by virtually all members of the AIDS denialism community - is that there is no evidence that AIDS is caused by HIV. Certainly, by all accounts, the medical and scientific communities have demonstrated otherwise. But how do we know that HIV is the causal agent behind AIDS? How do we know what the causal agent is behind any disease? Do we have a standard method of determining causality, and is the HIV/AIDS connection supported by this method? The answer is yes.

In 1884, a Prussian physician by the name of Robert Koch, along with is colleague, the bacteriologist Friedrich Loeffler formulated a set of criteria that they used to establish a causal relationship between an illness and a causative agent. These criteria were published in 1890 by Koch and have since been known as Koch's Postulates. Koch used this set of postulates to determine the etiology of both tuberculosis and anthrax¹, and in the 120 years since their first publication, the causes of many other diseases have been determined by successful application of Koch's Postulates. The four tenants of Koch's Postulates are as follows:

1. The causative agent must be found in all organisms exhibiting the disease, and not in healthy individuals.
   
2. The causative agent must be isolated from a diseased individual and grown in culture.
   
3. The causative agent, when introduced to a healthy individual, should cause the original disease.
   
4. The causative agent must be reisolated from the inoculated, diseased host and shown to be identical to the original agent.
   

Note that the first postulate is not universally required, as many diseases exhibit asymptomatic carriers (as is the case for HIV). The third postulate is unique in that it states "should" rather than "must" – this is because infection rates are never 100%. The fourth postulate was originally required by Koch, but fulfilling the first three postulates is generally considered to be sufficient to indicate causation. If all four postulates are fulfilled by a particular causative agent, then it can be said that the agent is the likely cause of the disease.


So, when these four postulates are applied to HIV/AIDS, does the result support the claim that HIV causes AIDS? The answer is a resounding "yes". Let's take the postulates one at a time.


The causative agent must be found in all organisms exhibiting the disease, and not in healthy individuals.

As noted above, this first postulate is not universally required, as asymptomatic carriers of HIV appear as healthy individuals. Nevertheless, those with HIV-1 and no symptoms at time of diagnosis, eventually progress to AIDS. And in 95% of cases, individuals suspected to have AIDS test positive for HIV virions, HIV antibodies or positive for HIV viral loads². This association between HIV and AIDS is supported by a wealth of evidence from countless studies, and cannot be dismissed.

The AIDS denialists, however, will insist that this first postulate has not been fulfilled. They will point out that there are cases of individuals exhibiting AIDS-defining characteristics who have not tested positive for HIV antibodies. This, they are quick to claim, is in violation of the first postulate, since these cases show the disease without the putative causative agent. There is a very straightforward answer to this, though: immune deficiencies can develop in more ways than just being induced by HIV infection. There is a virtual plethora of ways immune deficiencies can arise, and these will exhibit symptoms similar to AIDS – bacteria, genetic conditions, environmental factors, and immune suppressing drugs can all cause symptoms associated with AIDS. In such cases, it is not surprising that patients test HIV negative! But in cases where individuals present all or most of the AIDS-defining characteristics – particularly a depletion of CD4+ lymphocytes – the HIV virus is found³. Koch's first postulate remains fulfilled.


The causative agent must be isolated from a diseased individual and grown in culture.
Culturing the proposed pathogen is not only the second postulate but also, perhaps, the most important step in identifying the suspected pathogen. Viruses can be more difficult to grow in culture than bacterial strains, but this has not prevented HIV from being cultured from samples taken from AIDS patients. Over the years, it has been cultured in T-cells, macrophages as well as in HeLa cells. Furthermore, isolated and cultured strains of HIV have had their genomes sequenced and cloned, with literally thousands of unique sequences added to sequence data bases. Isolation of HIV from samples is not an issue, and there have been multiple^4,5 protocols established and published on how to accomplish this. There are even commercially available kits which allow HIV to be isolated from infected samples. Culturing HIV has become a routine practice in HIV research to the point where it is more or less a triviality. Postulate 2 has been fulfilled and continues to be filled on a daily basis.

The causative agent, when introduced to a healthy individual, should cause the original disease.
The third postulate is perhaps the most controversial of the postulates. For one, it cannot be fulfilled solely on the basis of epidemiological data but must rather rely on experimental evidence. When looking at diseases in animals or when using animal models, the third postulate is easy to demonstrate experimentally. When it comes to the transmission of diseases in human subjects, however, the third postulate is tougher to demonstrate. No ethics board would ever allow researchers to experimentally infect healthy people with cultured HIV to see if they progress to AIDS. Nevertheless, this postulate has been fulfilled due to the unfortunate accidental transmission to humans who were doing research on HIV⁶.

In this case, three lab workers were working with HIV-1, and all became accidentally infected with the virus. None of these workers belonged to any of the high-risk groups; they were not drug users, had not had blood transfusions, and did not engage in homosexual behaviour. All three of the workers exhibited depleted CD4+ cell counts (two of which were low enough to warrant an AIDS diagnosis) and one progressing to pneumonia⁷. AIDS "skeptics" who insist that HIV does not cause AIDS and that AIDS is caused by drug use are at a loss when it comes to explaining this case study. How else could the infected workers, who were not part of any risk groups, have developed AIDS unless it was due to HIV infection? How do they explain the onset of symptoms in these workers if HIV is, as they claim, pathologically asymptomatic?

Evidence from animal studies has also given support to the third postulate. Studies in which baboons have been infected with HIV-2 have shown these animals to develop AIDS-like symptoms including depletion of CD4+ lymphocytes⁸ (and I highly doubt these baboons were drug users!). Mouse strains used to model SCID which have been given human peripheral blood lymphocytes (hPBLs)⁹ also develop severe immunodeficiencies when infected with HIV¹⁰. These mice also show depleted CD4+ cell counts.

Studies like this show experimentally that cultured HIV, introduced into healthy individuals, result in the hosts progressing to AIDS. This is sufficient to fulfill the third postulate.

The causative agent must be reisolated from the inoculated, diseased host and shown to be identical to the original agent.
This fourth postulate, as noted above, is now often considered extraneous to suggesting causation by a suspected pathogen. Nevertheless, the example of the infected lab workers cited above is a good case of this postulate also being demonstrated. The virus was reisolated from the infected lab workers, and sequenced¹¹. The divergence between the sequences from the strains isolated from the workers was very small – about the same as the divergence in sequence between HIV-infected infants and their mothers. In other words, it was the same strain that they had been working with. Consider Postulate 4 demonstrated.
 
So it is plainly obvious that all of Koch's postulates are fulfilled, and HIV can be said to be the likely causative agent behind AIDS. Koch's postulates demonstrate reasonable criteria for determining causation, and provide reasonable evidence that the AIDS "skeptics" are wrong.

I will continue in Part III, where I examine the fuzzy math used by AIDS "skeptics" to distort reality and push their dangerous nonsense.

-------------------------------------------------------------------------------------------------------------------------

1. The infectious agents behind these diseases, Mycobacterium tuberculosis and Bacillus anthracis, respectively, were discovered and first isolated by Koch himself.
   
2. See the references in Part I for more on this.
   
3. There is an exception to this. A rare condition exists called hypogammaglobulinemia where individuals are unable to mount an immune response to invading pathogens. People with this disorder who are infected with HIV will not show HIV antibodies present when tested.
   
4. See http://www.miltenyibiotec.com/download/protocols_macsmolecular_en/516/SP_Iso_HIV1.pdf
   
5. See http://www.currentprotocols.com/protocol/im1202
   
6. Weiss SH, Goedert J J, Gartner S, Popovic M, Waters D, Markham P, Veronese FD, Gall MH, Barkley WE, Gibbons Jet et al: Risk of human immunodeflciency virus (HIV-1) infection among laboratory workers. Science 1988, 239:68-71.
   
7. I have been unable to find any information on these three workers more recent than the above referenced article. If anyone has updated information, I would be interested in hearing it!
   
8. Barnett SW, Murthy KK, Herndier BG, Levy JA: An AIDS-like condition induced in baboons by HIV-2. Science 1994,266:642-646.
   
9. SCID-model mice are unable to produce B and T lymphocytes. Transplanting hPBLs into these mice reconstitutes a functioning immune system.
   
10. Mosier DE, Bulizia R J, Baird SM, Wilson DB, Specter DH, Spector SA: Human immunodeficiency virus infection of human PBLSCID mice. Science 1991,251:791-794.
    
11. Reitz MS Jr, Hall L, Robert-Gurofl M, Lautenberger .I, Hahn BH,Shaw GM, Kong LI, Weiss SH, Waters D, Gallo RC,Blattner W: Viral variability and serum antibody response in a laboratory worker infected with HIV-1 (HTLV-IIIB). AIDS Res Hum Retroviruses 1994, 10:1143-1155.

Dead or Alive: The Debate on Viruses is Renewed

One of the biggest debates in biology today is over whether or not viruses count as life. Most scientists would contend that viruses are not alive and for good reasons: they cannot replicate on their own and require hijacking of their host's replication machinery in order to reproduce, they do not have any metabolism, they have no cellular structure and their replication is driven by spontaneous assembly or virus particles rather than by cellular division. Classifying viruses as "alive" would mean that cellular organization would not be included as a requirement for life (and opening the category up to things like prions). But according to a new article published in Nature, new findings are beginning to challenge the notion that viruses are not alive.

In 2003, a new family of viruses was discovered by researchers in France. From a sample taken from a cooling tower in the UK, researchers found a new type of virus that was infecting ameobae in the sample.¹ This virus was way bigger than any other viruses known at the time, both in physical size and genome size. Having about 900 protein-coding genes - 3 times that of the next biggest virus and larger than some bacterial species - the virus is a behemoth. It was called mimivrus, for "mimicing microbe".

But that isnt the interesting part.

The same research team recently isolated a new strain of this giant virus, again from a cooling tower - this time in Paris. Electron micrographs of the virus showed that this strain, called mamavirus, had a friend. They discovered another, tiny virus that was closely associated with the mamavirus. They nicknamed this one Sputnik because it seemed to be a satellite of its larger companion. The team noticed that the mamavirus infected ameobae, just like its mimivirus cousin, and turned the cells into a virus assembly line to pump out more mamaviruses. However, if the amoebae were coinfected with Sputnik, the little satellite virus would hijack - not the amoeba's machinery - but the viral factory set up by the mamavirus! The mamavirus particles would turn out all screwed up and less infectious. It would seem that Sputkik is a viral virus! The researchers had found the first known virophage.²

Just like bacteriophages prey on bacteria, virophages prey on bacteria. They hijack the virus' machinery (that the virus itself has hijacked from the host cell), and this causes the virus to become ill. And that is what has rekindled the debate over viral "life". How can a virus - something that supposedly isnt alive - become sick?

I dont belive that this really throws a monkey wrench in the current classification of viruses as "nonlife". What the virophage is doing is interfering with the mamavirus' "virus factory" for its own use, and the mamavirus progeny turn out sickly. This is not a whole lot different from mutating a step in the assembly line so the virus churns out avirulant progeny. Mutate gene A so that protein B isn't incorpreted into the viral capsid, and you get viruses that are less infectous. Does that mean that the virus is alive? Not really. In both cases, the same sort of assembly line is being interrupted. A virus falling prey to another virus makes neither of them any more "alive" than viruses that have been mutated in a lab. The only difference here is that the changes to the viral factory are being carried out by another virus instead of a mutagen. Does that mean that the virophages are "alive"? No more than bacteriophage are alive. The new findings, I feel, do not suggest that viruses are alive. They suggest that viruses can fall prey to their own devious schemes.

But, draw your own conclusions. The debate on whether or not viruses are alive will not end with these new findings, and is likely to only become more intense. But, regardless of whether you think viruses are alive or not, you have to admit: mimi/mamaviruses and virophages are pretty frickin' cool.

============================================
1. La Scola, B. et al. Science 299, 2033 (2003)
2. La Scola, B. et al. Nature doi:10.1038/nature07218 (2008)