July 18, 2013 — Designing an effective HIV/AIDS vaccine is something of a paradox: a good vaccine would be safe and look enough like HIV to kick-start the immune system into neutralizing the virus — but the problem is that this is exactly what the human immune system has trouble doing even when it’s exposed to the real thing.Now a team of researchers led by scientists at The Scripps Research Institute in La Jolla, CA has developed a strategy for inducing a key part of an effective immune response to HIV. By tracing the evolution of HIV-recognizing molecules called antibodies taken from the blood of rare individuals whose immune systems are naturally able to target and neutralize the virus, they may have found a way to replicate this for everybody.At a talk next week at the American Crystallographic Association meeting in Hawaii, the team will present multiple crystal structures, which like detailed architectural blueprints show how the virus interacts with components of the immune system. Examining these structures has allowed them to reverse engineer molecules that specifically activate the precursors of effective, neutralizing antibodies against the virus — molecules that may be components of a future vaccine against HIV.”What we tried to do was to learn how those [effective] antibodies developed over the course of natural infection and attempt to guide the immune response in the direction of what we know works in certain HIV-infected individuals,” said structural biologist Jean-Philippe Julien, who is presenting the work in Hawaii.He conducted the research under the direction of Professors Ian Wilson and William Schief of The Scripps Research Institute. The work was funded by the International AIDS Vaccine Initiative Neutralizing Antibody Center, the Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery and the National Institute of Allergy and Infectious Diseases (one of the National Institutes of Health). Additional support was provided through a Canadian Institutes of Health Research fellowship.Julien cautioned that the work might not, by itself, be the final answer that shows how to make an effective HIV/AIDS vaccine — but it is a step in the right direction. Most likely, Julien said, any future HIV/AIDS vaccine would combine multiple biological components in order to give the broadest possible protection against the virus.He added that their candidate molecule was able to achieve the desired immune reactions in the test tube, and they are currently testing it in animals to see if it is able to kick start the desired immune response. If those experiments go well, he said, further studies will examine whether it can protect animals against infection, and human trials for safety and vaccine efficacy would be next — though it may be years before those results are known.While designing a vaccine against any pathogen is a long, hard process, HIV has been particularly difficult, and despite decades of efforts and hundreds of millions of dollars spent in the process, we still do not yet have an effective vaccine that can prevent infection.Read more
July 19, 2013 — The rapid molecular evolution of hepatitis C virus (HCV) has been used to help incriminate the source of an outbreak in two Spanish hospitals in the late nineties. The evolutionary techniques used, described in BioMed Central’s open access journal BMC Biology, also helped separate those who were infected by the person in question from those infected elsewhere during the same time period.In the days before deep sequencing became a cheap option scientists used partial sequencing of HCV to help convict an anesthetist of infecting 275 patients with this virus. Back in 1998 an anesthetist was alleged to have injected himself with opioid painkillers, using some of the dose meant for his patients, before giving them the rest using the same needle and syringe. It is only now, after the experts’ testimony and appeals, that the science used to track the outbreak and the spread of the virus is being made public.The main difficulty in establishing a link between the source and the infected patients is that the virus continues to evolve in its host. Also unlike HIV, HCV can remain silent in an infected patient for years even though it is still capable of being transmitted.Prof Fernando González Candelas from the Universidad de Valencia, who led this multicentre study explained, “We sequenced 322 patients who were suspected to have been infected by the donor and 44 local, unrelated controls. Our analysis of over 4000 sequences from the E1-E2 region of the viral genome allowed us to exclude 47 patients as having been infected elsewhere. Because we knew the dates of infection for some patients we were able to use their data to validate a molecular clock and construct an estimated date of infection for each patient and of the source.”The patients were all infected between 1988 and 1998 shortly after the estimated date of infection of the source.Improvements in sequencing techniques and computing now make it easier to obtain whole viral genomes and the phylogenetic analysis of RNA viruses, especially the molecular clock technique, is increasingly used today to analyze disease outbreaks in order to help plan control measures.Prof González Candelas continued, “Naturally, there are very limited possibilities for a single infected patient of infecting so many recipients, but the recent case of a medical technician in New Hampshire (USA), and eight other states, shows that more such events might be revealed.”Read more
July 16, 2013 — Some strains of the avian H7N9 influenza that emerged in China this year have developed resistance to the only antiviral drugs available to treat the infection, but testing for antiviral resistance can give misleading results, helping hasten the spread of resistant strains.The authors of a study published in mBio®, the online open-access journal of the American Society for Microbiology, characterized viruses taken from the first person known to be stricken with H7N9 influenza and found that 35% of those viruses are resistant to oseltamivir (commercially known as Tamiflu) and zanamivir (Relenza), front line drugs used for treating H7N9 infections. However, lab testing of the viruses, which detects the activity of a viral enzyme, fails to detect that these strains are resistant, so monitoring for the development of resistance using this technique would prove futile.”If H7N9 does acquire human-to-human transmissibility, what do we have to treat it with until we have a vaccine? Oseltamivir. We would be in big trouble,” says corresponding author Robert Webster of St. Jude Children’s Research Hospital in Memphis, Tennessee. Resistant strains of H7N9 can flourish in patients treated with oseltamivir or zanamivir, he says, inadvertently leading to the spread of resistant infections.In the mBio study, the authors tested antiviral susceptibility of an H7N9 strain isolated from the first confirmed human case of avian H7N9 influenza using a method that tests the activity of the neuraminidase enzyme. The reassuring results were, unfortunately, misleading: the enzyme-based test indicated that the flu strain was susceptible to NA inhibiting antiviral drugs, but it is not.A closer look at the viral isolate revealed it is actually made up of two distinct types of H7N9 viruses. Roughly 35% of the viruses carry the R292K mutation, making them resistant to NA inhibitors, and 65% are sensitive to these same drugs. The enzyme-based testing gave misleading results, says Webster, because the functioning wild-type enzymes masked the presence of the non-functioning mutant enzymes.Using NA inhibitors to treat a patient infected with a resistant strain of H7N9 only encourages the virus to proliferate and can lead to enhanced spread of the resistant strain. The authors write that these results prove that it is crucial to use a gene-based surveillance technique that can detect these resistant influenza strains in a mixed infection.H7N9 first emerged in China in early 2013, in some cases infecting individuals who had been in contact with poultry or with places where poultry are housed. …Read more
July 16, 2013 — UT Southwestern Medical Center researchers have unlocked some of the mysteries of West Nile virus outbreaks and shown that use of a mosquito vector-index rating system works well to identify the best time for early intervention.West Nile infections in humans can cause long-term neurological damage and even death. The investigation analyzed a decade of West Nile infections, weather, and housing data. The 2012 data — from the nation’s largest West Nile outbreak that occurred in Dallas County, Texas — revealed that the best way to avoid an outbreak and stave off the resultant rise in human infections was to determine a mosquito vector index. The vector index value is calculated from the abundance of mosquitoes and the percentage of mosquitoes infected with West Nile virus.”When the vector index goes above 0.5 early — in June or July — large numbers of people are silently infected, and this is the best time to intervene,” said Dr. Robert Haley, chief of epidemiology and professor of internal medicine at UT Southwestern and senior author of the study published in the July 17 edition of the Journal of the American Medical Association. “In years when the vector index did not rise until late July or August, impending outbreaks just sputtered — in late summer mosquito abundance declines, and mosquitoes become less active and stop biting as much.”The study also showed that determining the number of West Nile virus infections in people is a poor way to determine how to respond to an outbreak.”After the infecting mosquito bite, it takes a week for the first symptoms to develop, a week to see people turning up at hospitals, and a week for laboratory confirmation of the diagnosis and reporting to health officials,” Dr. Haley said. “That three-week time period is crucial. Acting early from the vector index rather than after human case reports and deaths mount up can nip an outbreak in the bud. However, if mosquito data are unavailable or a decision to intervene takes longer, later intervention may still be important to terminate the outbreak.”The analysis also found that milder winters and unusually warm spring temperatures contributed to epidemic years for West Nile, a major concern as global temperatures continue to warm, Dr. …Read more
July 11, 2013 — There are 170 established HPV types. Cancerous human papillomavirus (HPV) viruses are the main cause of cervical cancer, and are found in close to 100% of cervical tumors.Cervical cancer and genital warts are caused by HPV. However, testing for the virus using standard techniques can sometimes give a negative result — in these cases, the condylomas are called ‘virus-negative’ warts.In a new study published in Virology, researchers assessed the DNA found in samples taken from 40 patients with ‘virus-negative’ genital warts. Through a general DNA sequencing approach, the researchers showed that several of the negative samples did in fact contain HPV DNA.This means that virus-negative warts can harbor small amounts of more distantly related viruses that escaped previous detection. According to the research, there is a diverse pool of previously unknown HPV types that infect humans and are detectable on genital warts.The findings have implications for the knowledge of diversity of HPV types, as these viruses are currently undetectable using traditional testing methods.Ten pools of four samples taken from virus-negative warts were tested using genetic material straight from the patient — including viral, bacterial and human DNA. Five of the pools contained HPV, and three of these contained new strains of the virus.Altogether, 1337 pieces of HPV-related DNA were detected, representing 23 new types of HPV, 10 established types of HPV and two known HPV DNA sequences.This new style of testing has highlighted previously unknown forms of the virus. As such, we learn more about the evolution of different HPV types. It is possible that the previously unknown forms of the virus do not cause condyloma but may be secondary invaders of condyloma.Read more
July 9, 2013 — University of British Columbia researchers have developed a vaccine that may halt the spread of West Nile Virus (WNV) among common and endangered bird species.WNV, a mosquito borne pathogen, arrived in North America in 1999 and is now endemic across the continent. In 2012 alone, WNV killed 286 people in the United States, and 42 people have died from the virus in Canada since 2002. There is currently no effective vaccine against WNV infection in humans or birds.Common birds such as crows, ravens and jays, and endangered species such as the Greater Sage-Grouse and the Eastern Loggerhead Shrike, are also susceptible to WNV infection, with mortality rates in some species and populations as high as 100 per cent.”West Nile Virus has been identified as a threat contributing to the extinction of some rare bird species and its presence in common birds facilitates the spread of the disease,” says Joanne Young , lead author of a study recently published in PLOS ONE and a PhD student in UBC’s Michael Smith Laboratories and Department of Zoology. “A bird vaccine would go a long way to helping combat these adverse effects.”Young and Prof. Wilfred Jefferies developed and tested a vaccine made from components of WNV and found it generated an effective immune response in birds. This may protect against the spread of virus not only among birds but also to other species. The team will now study the vaccine’s effectiveness in protecting birds against mortality caused by the disease.Read more
June 27, 2013 — A new study reveals that all children with Crohn’s disease that were examined had a commonly occurring virus — an enterovirus — in their intestines. This link has previously not been shown for this chronic inflammatory intestinal disorder.The findings are being published in the latest issue of the international journal Clinical and Translational Gastroenterology.These findings need to be confirmed in larger studies, but they are important, as this connection has never been pointed out before. This paves the way for a better understanding of what might be involved in causing the disease, says Alkwin Wanders, one of the scientists behind the study at Uppsala University and Uppsala University Hospital.In Sweden several thousand adults live with Crohn’s disease, and each year about 100 children and adolescents develop the disorder. The disease affects various parts of the gastrointestinal system and causes symptoms such as stomach aches, diarrhea, and weight loss — in severe cases fistulas, or strictures in the intestines.The cause of Crohn’s disease is not known. Mutations in more than 140 genes have been shown to be associated with the disorder, but this genetic connection is not a sufficient explanation. Many of these genes play key roles in the immune defence, which has prompted theories that the disease might be caused by impaired immune defence against various microorganisms. In that case, the disease would be a consequence of interplay between heredity and environment.Recent research has shown that some of the genes that are strongly linked to the disorder are important for the immune defence against a certain type of viruses that have their genetic material in the form of RNA, so-called RNA viruses. Using this as a point of departure, an interdisciplinary research team was established in Sweden to investigate what role this type of virus plays in the disease.The research team includes the paediatrician Niklas Nyström, the pathologist Alkwin Wanders, virus researchers Gun Frisk and Oskar Skog, the molecular biologist Mats Nilsson, and the geneticist Ulf Gyllensten at Uppsala University and Uppsala University Hospital, along with cell biologists Jonas Fuxe and Tove Berg the paediatrician Yigael Finkel at Karolinska Institutet in Stockholm.This unique composition, with complementary clinical and scientific expertise, has been extremely fruitful for our studies, says Alkwin Wanders.In the present study the researchers investigated whether the RNA virus were present in children with Crohn´s disease. They focused in particular on the prevalence of enteroviruses, a group of RNA viruses that are known to infect the intestinal mucous lining.The results showed significant amounts of enteroviruses in the intestines of all of the children with Crohn´s disease, whereas the control group had no or only minimal amounts of enteroviruses in their intestines. Similar results were obtained using two different methods. …Read more
June 28, 2013 — The development of an easy to use, low cost method of detecting dengue virus in mosquitoes based on gold nanoparticles is reported in BioMed Central’s open access journal Virology Journal. The assay is able to detect lower levels of the virus than current tests, and is easy to transport and use in remote regions.Half the world’s population is at risk of Dengue virus infection — it infects 50-100 million people per year, approximately half a million of these require hospitalization and 2.5% (most of which are children) will die. It is one of the most dangerous viruses in the world with no vaccine, and it does not respond to antiviral therapy. The main method of controlling infection remains destruction of the standing water where the mosquitoes, which transmit the virus to people, breed.It is consequently vitally important to have a way of determining if mosquitoes are carrying Dengue virus, which can be used on site and that does not require specialist equipment.Researchers from the University of Notre Dame, USA, used a DNAzyme linked to gold nanoparticles which recognises a short sequence of the viral RNA genome common to all four types of Dengue. Once bound, adding magnesium and heating to 37C causes the DNAZyme to cut the RNA leaving the gold nanoparticles free to clump together. This aggregation can be easily seen as a red to clear/colourless colour change.The components of this test are stable at temperatures above 30C which means that they are easy to store and transport and the assay is able to detect as little as 10 viruses in each sample containing 10-20 mosquitoes.The ultimate goal is to detect virus infection in just a single infected mosquito or cell. Dr James Carter, the lead author of this study explained, “Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.”Read more
June 27, 2013 — In what researchers say is the first public health study of the aerial mosquito spraying method to prevent West Nile virus, a UC Davis study analyzed emergency department records from Sacramento area hospitals during and immediately after aerial sprayings in the summer of 2005. Physicians and scientists from the university and from the California Department of Public Health found no increase in specific diagnoses that are considered most likely to be associated with pesticide exposure, including respiratory, gastrointestinal, skin, eye and neurological conditions.The study appears in the May-June 2013 issue of Public Health Reports.This week, mosquito control officials said the region’s recent rainstorms and warming temperatures have increased stagnant water and favorable conditions for mosquitoes, which will likely magnify the incidence West Nile virus and the risks of human transmission. The mosquito-borne disease first appeared in the state about 10 years ago. It already has been detected in dead birds and mosquitoes in at least 10 counties in recent weeks, including Sacramento and Yolo. However, the adult mosquito population has yet to increase to levels that require aerial spraying over heavily urbanized areas as was done in the Sacramento region in previous years.”Unfortunately, West Nile virus is endemic in California and the United States, and the controversy of mosquito management will likely arise every summer,” said Estella Geraghty, associate professor of clinical internal medicine at UC Davis and lead author of the study. “Findings from studies such as this one help public health and mosquito control agencies better understand the risks and benefits of their practices.”West Nile virus has become an increasingly serious problem throughout the United States and may become more of a threat as the climate warms. According to the Centers for Disease Control and Prevention, West Nile virus is the leading cause of viral encephalitis in the United States. The virus is transmitted to humans and animals through the bite of an infected mosquito. Mosquitoes become infected with the virus when they feed on infected birds.In California around the time of the study ― 2004 and 2005 ― hundreds of people were sickened by West Nile virus and 48 died. Most people exposed to the disease do not have symptoms, but in about 1-in-150 people it can be fatal or result in permanent neurological effects.The study evaluated emergency room visits in Sacramento County hospitals on days that pesticides were sprayed as well as the three days following spraying. …Read more
June 25, 2013 — Scientists in the US have developed a novel vaccination method that uses tiny gold particles to mimic a virus and carry specific proteins to the body’s specialist immune cells.The technique differs from the traditional approach of using dead or inactive viruses as a vaccine and was demonstrated in the lab using a specific protein that sits on the surface of the respiratory syncytial virus (RSV).The results have been published today, 26 June, in IOP Publishing’s journal Nanotechnology by a team of researchers from Vanderbilt University.RSV is the leading viral cause of lower respiration tract infections, causing several hundred thousand deaths and an estimated 65 million infections a year, mainly in children and the elderly.The detrimental effects of RSV come, in part, from a specific protein, called the F protein, which coats the surface of the virus. The protein enables the virus to enter into the cytoplasm of cells and also causes cells to stick together, making the virus harder to eliminate.The body’s natural defence to RSV is therefore directed at the F protein; however, up until now, researchers have had difficulty creating a vaccine that delivers the F protein to the specialised immune cells in the body. If successful, the F protein could trigger an immune response which the body could ‘remember’ if a subject became infected with the real virus.In this study the researchers created exceptionally small gold nanorods, just 21 nanometres wide and 57 nanometres long, which were almost exactly the same shape and size as the virus itself. The gold nanorods were successfully coated with the RSV F proteins and were bonded strongly thanks to the unique physical and chemical properties of the nanorods themselves.The researchers then tested the ability of the gold nanorods to deliver the F protein to specific immune cells, known as dendritic cells, which were taken from adult blood samples.Dendritic cells function as processing cells in the immune system, taking the important information from a virus, such as the F protein, and presenting it to cells that can perform an action against them―the T cells are just one example of a cell that can take action.Once the F protein-coated nanorods were added to a sample of dendritic cells, the researchers analysed the proliferation of T cells as a proxy for an immune response. They found that the protein-coated nanorods caused the T cells to proliferate significantly more compared to non-coated nanorods and just the F protein alone.Not only did this prove that the coated-nanorods were capable of mimicking the virus and stimulating an immune response, it also showed that they were not toxic to human cells, offering significant safety advantages and increasing their potential as a real-life human vaccine.Lead author of the study, Professor James Crowe, said: “A vaccine for RSV, which is the major cause of viral pneumonia in children, is sorely needed. This study shows that we have developed methods for putting RSV F protein into exceptionally small particles and presenting it to immune cells in a format that physically mimics the virus. Furthermore, the particles themselves are not infectious.”Due to the versatility of the gold nanorods, Professor Crowe believes that their potential use is not limited to RSV.”This platform could be used to develop experimental vaccines for virtually any virus, and in fact other larger microbes such as bacteria and fungi.”The studies we performed showed that the candidate vaccines stimulated human immune cells when they were interacted in the lab. The next steps to testing would be to test whether or not the vaccines work in vivo” Professor Crowe continued.Read more
June 25, 2013 — New research out of St. Michael’s Hospital has found that despite popular belief, the flu shot is effective in preventing the flu, even if the virus going around does not match the vaccine.”It’s quite common for people to say they are not going to get the flu shot this year because they’ve heard it does not match the strain of flu going around,” said Dr. Andrea Tricco, the lead author of the paper and a scientist at the Li Ka Shing Knowledge Institute of St. Michael’s Hospital. “However, we’ve found that individuals will be protected regardless of whether the flu strain is a match or not.”The review of the literature analyzed more than 40 years of data, from 1971 to 2011, and included 47 influenza seasons and almost 95,000 healthy people.Dr. Tricco and colleagues were particularly interested in flu seasons when the flu vaccines were not matched well to circulating strains. They wanted to understand whether the flu vaccines would still be effective when the strains were not a match.Vaccines work by giving the body an inactive, or non-infective, form of the flu virus so that the body can produce antibodies. When an individual comes into contact with the virus in the future, the body can use the natural antibodies it has created to fight it off.The study looked at the two most popular vaccine formulations in Canada — Trivalent inactive vaccine for adults and live-attenuated influenza vaccine for children. They found that both vaccines provided significant protection against matched (ranging from 65 per cent to 83 per cent effectiveness) and mismatched (ranging from 52 per cent to 54 per cent effectiveness) flu strains.The paper was published online in the journal BMC Medicine today.”Looking at matches and mismatches can be a difficult process because it’s not a yes or no variable,” Dr. Tricco said. …Read more
June 20, 2013 — Chlamydia trachomatis is a human pathogen that is the leading cause of bacterial sexually transmitted disease worldwide with more than 90 million new cases of genital infections occurring each year. About 70 percent of women infected with Chlamydia remain asymptomatic and these bacteria can establish chronic infections for months, or even years. Even when it causes no symptoms, Chlamydia can damage a woman’s reproductive organs. In addition, standard antibacterial drugs are proving increasingly ineffective in complete eradication, as Chlamydia goes in to persistent mode, leading to asymptomatic chronic infection.Share This:Researchers at the Max Planck Institute for Infection Biology in Berlin (MPIIB) now show that Chlamydia infections can cause mutations in the host DNA by overriding the normal mechanisms by which their host prevents unregulated growth of genetically damaged cells that pave the way for the development of cancer.Owing to their intracellular lifestyle Chlamydia depend on various host cell functions for their survival. Chlamydia manipulates the host cell mechanism to favour its growth, however the consequences of such alterations on the fate of host cells remains enigmatic. Even more worrying is mounting epidemiological evidence which links Chlamydia infections with the development of cervical and ovarian cancer. Cindrilla Chumduri, Rajendra Kumar Gurumurthy and Thomas F. Meyer, researchers at the Max Planck Institute for Infection Biology in Berlin, have now discovered that Chlamydia induces long-lasting effects on the genome and epi-genome of their host cells. Such changes are increasingly implicated in the development of a range of cancers.The team found increased levels of DNA breaks in Chlamydia-infected cells. In normal cells, depending on the extent of damage, cells either “commit suicide” or activate repair by special protein complexes in a process called the DNA Damage Response, which reseals the broken strands of DNA and makes sure the sequence of the genetic code has not been changed. …Read more
June 20, 2013 — A combination of the myxoma virus and the immune suppressant rapamycin can kill glioblastoma multiforme, the most common and deadliest malignant brain tumor, according to Moffitt Cancer Center research. Peter A. Forsyth, M.D., of Moffitt’s Neuro-Oncology Program, says the combination has been shown to infect and kill both brain cancer stem cells and differentiated compartments of glioblastoma multiforme.The finding means that barriers to treating the disease, such as resistance to the drug temozolomide, may be overcome. The study, by Forsyth and colleagues in Canada, Texas and Florida, appeared in a recent issue of Neuro-Oncology.”Although temozolomide improves survival for patients with glioblastoma multiforme, drug resistance is a significant obstacle,” said Forsyth, the study lead author. “Oncolytic viruses that infect and break down cancer cells offer promising possibilities for overcoming resistance to targeted therapies.”The authors note that oncolytic viruses have the potential to provoke a multipronged attack on a tumor, with the potential to kill cancer cells directly through viral infection and possibly through inducing the immune system to attack the tumor. The multipronged approach might get around some of the classical resistance mechanisms that have plagued both targeted therapies and conventional chemotherapies.Several oncolytic viruses, both alone or in combination with small molecule inhibitors, have been tested and show promise for malignant gliomas. However, most have not been effective in killing cancer cells. Two likely obstacles may be the patient’s own anti-viral immune response and limited virus distribution.”Based on our previous work with myxoma virus, we considered it to be an excellent oncolytic virus candidate against brain cancer stem cells,” explained Forsyth.The researchers found that brain cancer stem cells were susceptible to myxoma virus in the laboratory cultures (in vitro) and in animal models (in vivo), including in temozolomide-resistant cell lines.”We also found that myxoma virus with rapamycin is a potentially useful combination. The idea that cancer cells can be killed by a harmless virus is an exciting prospect for therapy,” Forsyth said.The precise mechanism rapamycin uses to enhance infection in brain cancer stem cells is unknown, and the combination therapy does not result in cures. However, researchers are investigating other drugs that may improve the effectiveness of myxoma virus when used in combination, and they are evaluating the use of other strains of myxoma virus that might be more effective.”Although our study adds myxoma virus to the list of oncolytic viruses capable of infecting and killing these cells, which strengthens its candidacy for clinical application, our model will need clinical application to determine its safety for patients,” concluded the authors. …Read more
June 19, 2013 — In fall 2012, the European Medicines Agency (EMA) approved the modified adeno-associated virus AAV-LPL S447X as the first ever gene therapy for clinical use in the Western world. uniQure, a Dutch biotech company, had developed AAV-LPL S447X for the treatment of a rare inherited metabolic disease called lipoprotein lipase deficiency (LPLD) which affects approximately one or two out of one million people. The disease causes severe, life-threatening inflammations of the pancreas. Afflicted individuals carry a defect in the gene coding for the lipoprotein lipase enzyme which is necessary for breakdown of fatty acids. AAV-LPLS447X shall be used as a viral vector to deliver an intact gene copy to affected cells.The viruses modified for gene therapy cannot integrate their DNA into the host cell genome, because they lack a particular enzyme needed for this. Nevertheless, integration may happen occasionally. “We had to exclude that AAV-LPLS447X tends to integrate at sites in the genome where this integration might activate cancer-promoting genes. This is exactly what had been observed with a virus used for gene therapy,” says Dr. Manfred Schmidt, a molecular biologist. Schmidt leads a research group at NCT Heidelberg and DKFZ that studies the safety of gene-therapeutic methods.In collaboration with scientists from uniQure, the Heidelberg researchers analyzed the genome of five LPLD patients who had been treated with AAV-LPLS447X . …Read more
May 30, 2013 — To test the severity of a viral infection, clinicians try to gauge how many viruses are packed into a certain volume of blood or other bodily fluid. This measurement, called viral load, helps doctors diagnose or monitor chronic viral diseases such as HIV/AIDS and hepatitis. However, the standard methods used for these tests are only able to estimate the number of viruses in a given volume of fluid. Now two independent teams have developed new optics-based methods for determining the exact viral load of a sample by counting individual virus particles. These new methods are faster and cheaper than standard tests and they offer the potential to conduct the measurements in a medical office or hospital instead of a laboratory.
The teams will present their latest results at the Conference on Lasers and Electro-Optics (CLEO: 2013), to be held June 9-14, in San Jose, Calif.
One research group, led by electrical engineer and bioengineer Aydogan Ozcan of UCLA, is working to directly image single virus particles using holographic microscopy. The other, led by electrical engineer Holger Schmidt of the University of California, Santa Cruz (UCSC), is detecting single particles tagged with fluorescent labels on a microfluidic chip. Both teams expect to use their work to develop commercial instruments useful for on-site diagnosis and monitoring with rapid results and fast turnaround.
Ozcan’s UCLA team has demonstrated the ability to capture optical images of single viruses and nanoparticles over a comparatively large field of view — about the size of a postage stamp — using nanolenses that self-assemble around the virus particles like little magnifying glasses.
“Because viruses are very small–less than 100 billionths of a meter–compared to the wavelength of light, conventional light microscopy has difficulty producing an image due to weak scattering of sub-wavelength particles,” Ozcan says. When lighted, the team’s new nanolens-nanoparticle assembly projects a hologram that can be recorded using a CMOS imager chip (a type of semiconductor-based light detector) and digitally reconstructed to form an optical image of the particle. “The resulting image improves the field-of-view of a conventional optical microscope by two orders of magnitude,” says Ozcan.
This wide field of view allows the device to form images of many nanoparticles in a single photograph and provides a high-throughput platform for a direct and accurate viral load count. The instrument can be made sufficiently compact and lightweight for field applications and, attached to a cell phone, could become useful even in remote locations.
The UCSC researchers will present the results of a collaborative effort between UCSC, Liquilume Diagnostics Inc., and the groups of infectious disease clinician and virologist Charles Chiu at University of California, San Francisco, and engineer Aaron Hawkins at Brigham Young. While Ozcan’s group visually counts individual viruses, Schmidt’s counts them by detecting their nucleic acids–the genetic makeup of the viruses. The nucleic acids are labeled with a fluorescent dye, and light from the fluorescence is detected as they pass through a channel in a microfluidic chip about the size of a thumbnail.
Current tests for determining viral load generally rely on a technique called polymerase chain reaction (PCR), which amplifies a small sample of nucleic acid, such as DNA, and makes it easier to detect. “The gold standard for viral load detection is PCR, due to its sensitivity and specificity,” Schmidt says, but PCR is limited to merely estimating the number of viruses. In contrast, the new method counts real particles as they pass through the fluorescence detector on the chip. “We have demonstrated actual virus counts of specific nucleic acids in less than 30 minutes with minimal sample workup,” Schmidt says. So far, the group has collected reliable data on samples diluted to a point well within the range required for clinical detection.
Unlike direct visualization techniques, Schmidt’s chip-based method requires that the targeted virus particles be labeled. The labeling technique would allow clinicians to target specific viruses while ignoring unlabeled background material. This makes the process potentially useful in situations where clinicians already know what they are looking for — often the case for viral load tests.
The chip is currently housed in an instrument about one foot square, making the device portable. Along with rapid analysis turnaround, this portability should make the technique useful for point-of-treatment tests. In addition to detecting viruses, the device may also find uses as a sensor for cancer biomarkers, for environmental analyses of chemicals, and even in industrial production monitoring.Read more