 AIDS denialism, responding to the Perth Group's arguments. Some of you viewing this video might wonder why I am making it. There's a group of people called HIV AIDS Denialists. They deny that AIDS is caused by HIV. Their videos and websites are all over the internet and YouTube. If they are wrong, and I think they are, they are spreading dangerous misinformation. I am responding to a challenge to answer the following questions from the Perth Group. A few people in Australia who have never done HIV research but demand that their questions be answered by qualified scientists. Most have ignored them as crackpots. I will show why. Here's from their website. The view of the Perth Group is that the HIV AIDS experts have not proven that the virus exists, that the antibody tests are specific for HIV, that HIV could cause AIDS, that the HIV genome originates in a unique, exogenously acquired infectious retroviral particle. They believe that HIV AIDS is not infectious, or that it's not possible to transmit the virus from mother to child. They instead argue that it's impossible for hemophiliacs to acquire HIV following transfusion, that HIV AIDS and all of the phenomena inferred as HIV are induced by changes in, and here's the fun one. Remember this odd phrase for our later video. Cellular redox brought about by the oxidative nature of substances and exposures common to all the AIDS risk group and to the cells used in the culture and isolation of HIV. The age would never spread outside the original risk groups, it's an interesting one, that the cessation of exposure to oxidants and the use of antioxidants will improve the outcome of AIDS patients, that the pharmacological data prove AZT cannot kill HIV, and that AZT is toxic to all cells and may cause some cases of AIDS. Let's deal with this argument point by point. I will start with their argument that HIV experts have never demonstrated the following, the existence of a unique exogenously acquired retrovirus called HIV. First, let's confirm the presence of viral RNA. We've confirmed the presence of viral RNA millions of times every day for 20 years. The CDC, FDA and NIH are all satisfied that this virus exists. There are FDA approved tests for viral RNA in plasma and serum, and we can detect them by the ways that RNA is often detected in a research setting, RTPCR, real-time RTPCR, fish probes, northerns and others. I've grabbed some recent random papers just to show how trivial it is to show viral RNA. Note that none of the papers is using this data to prove the existence of HIV because that has already been done so often. This is an F-I-S-H that's fluorescence and situ hybridization, fish, image, taken under a microscope. An oligonucleotide probe was developed with complementarity to HIV-1 RNA. And there it is in the nucleolus of a COS or cost cell that was experimentally infected. Here's an RTPCR product run out on an agarose gel. Three viral products are being detected over 32 hours post infection and compared to a cellular protein beta-globulin. Here's another fish image showing the localization of some recombinant HIV in a primary cell line. What's being shown in the middle image is the recruitment of an RNA polymerase to make viral RNA. I've chosen graphic-friendly techniques here. Microscopy mostly because looking at real-time PCR curves is a bit boring. All right. Part B, presence of proviral DNA. Now one of the processes in the HIV life cycle is the reverse transcription and integration of a DNA form of the RNA genome into the host DNA. So can we detect the presence of this proviral DNA? Yes, by all methodologies capable of detecting DNA sequences. Southerns, PCR, fish and sequencing or pyro sequencing. The Los Alamos National Labs maintain the official HIV database for all known sequences, which is accessible to the public at the following link. Again, I've chosen some random papers with cool graphics to show how easy this is to identify. Here, the PCR process is made specific by primary sequences and in this case they are amplifying the proviral LTR and detecting the products on an agarose gel. The numbers above the gel indicate the number of provirus sequences present. Note that the sensitivity goes as low as 10 copies in a tube. Below that you run into stochastic sampling error. Here's a meta-analysis of real-time PCR data. They are showing how linear the response is between the real-time proviral assay and the number of provirus sequences. Now this technique is even more sensitive than the previous data. Here's an old-fashioned southern blot. You don't see these much. It's named for Ed Southern, where a radio-labeled probe is annealed to the proviral HIV sequence. So lots of techniques all giving the same result. In science we call that concordance, and it's very strong for the presence of viral RNA and DNA. Now what about the presence of viral proteins? Well proteins are kind of the hardest to detect in some ways, but multiple techniques confirm the presence of HIV proteins in primary and transformed cells. The most common techniques in a research lab to detect proteins are westerns, mass spec, cell D, Malditov, and surface plasma and resonance or SPR. I can define any of these terms if anyone's truly interested in Malditov, something I've done in the past. Proteins are encoded in the DNA and RNA of the provirus and virus, respectively. So we should find that they correlate between the DNA, RNA, and protein, and they do. So we know what to look for here. People have also taken whole virus, cloned it into bacteria, and expressed the proteins to sequence. Everything adds up. Again, here are some random papers. Okay, this is a protein FISH fish against the P24 viral nucleocapsid. The same samples were also stained for alpha smooth muscle actin to verify the staining. So there it is, HIV infected tissue expressing viral proteins. Notice there is no confusion over P24 nucleocapsid and P23 smooth muscle actin. They are antigenically distinct and non-overlapping. That's something the Perth group have argued about in the past, apparently because they don't read enough papers. Here's a pretty image showing a transgenic reporter, green fluorescent protein, which has been inserted into the HIV genome. It's being expressed in infected cells. The presence of the virus and its activity can be clearly seen. Here's a fuzzy western blot for TAT, the HIV Transactivator Protein. They've also made a form that has transgenic with a tag on the end that makes it slightly larger and much easier to purify. So there's a great deal more of it on the second lane. Okay, issue two, the HIV antibody tests are specific for HIV detection. They're challenging that fact. And look, this one is annoying to have to answer. The tests today are highly specific. A 10-year-old study in Journal of the American Medical Association showed that in a super large population of donors, the chances of a false positive is one in 250,000 people. You have about as much chance of being a false positive as being struck by lightning. Now, this is not to trivialize a real risk. And for this reason, patients are repeatedly retested on a positive result. But the long list of conditions that result in a false positive fail to mention how unlikely an event that is. The other accusation that is just factually wrong is that the clinical tests do not use a reference standard. That has never been true. However, until 2006, the WHO, World Health Organization, had not established an international reference standard. Prior to that, different groups were using different reference standards. In the U.S., it was the CDC reference standard, and many countries used the British working standard. All of these standards are maintained by the National Institute of Biological Standards and Controls. It's a British group who act as the official repository for all reference standards. The HIV-1 reference standard is composed of a pool of actual patient plasma samples, and is well characterized. As I mentioned, HIV testing is not a single step. This diagram shows a flow chart of how diagnosis of HIV infection is supposed to be handled. You will notice that the patient history, ELISA, CD4 counts, and confirmatory tests are all used to confirm a diagnosis. For this reason, no single assay is approved for complete diagnosis. The FDA has approved the HIV home test system for over-the-counter use as a confirmatory diagnosis. But even then, a doctor reviews the evidence to use a little human judgment before confronting a patient with a serious diagnosis. Point three, the HIV theory of AIDS. That is, that HIV causes acquired immune deficiency, or that AID leads to the development of the clinical syndrome AIDS. This is a straw man argument. AIDS, like all syndromes, is a complex interaction. In this case between genetics, physiology, environment, and the pathogen. While CD4 T-cell depletion is a useful marker for defining immunocompromise, it is not the only symptom of the syndrome. In my next video, I will cover two points. Mechanisms of HIV pathogenesis and CD4 positive T-cell depletion and its role in immunocompromise.