MicrobiologyBytes

There is intense interest in developing a cure for HIV. How such a cure will be quantified and defined is not known. Researchers applied a series of measurements of HIV persistence to the study of an HIV+ adult who has exhibited evidence of cure after a stem cell transplant.

Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV was detected in blood cells, spinal fluid, lymph node, or small intestine, and no infectious virus was recovered from blood. However, HIV was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in antiretroviral treated patients. The occasional, low-level HIV signals might be due to persistent HIV or might reflect false positives. The sensitivity of the current generation of assays to detect HIV RNA, HIV DNA, and infectious virus are close to the limits of detection. Improvements in these tests will be needed for future curative studies.

The lack of rebounding virus after five years without therapy, the failure to isolate infectious virus, and the waning HIV-specific immune responses all indicate that the Berlin Patient has been effectively cured.

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient. (2013) PLoS Pathog 9(5): e1003347. doi:10.1371/journal.ppat.1003347
There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

Over the last few years, many microbiomes (all of the microbobes found in a particular enviromnet) have been published. Most of these long list of bugs have been purely descriptive, but this knowledge is now starting to pay off by finding out which microbes are associated with particular diseases. In many cases, it’s not as simple as a single organism causing a disease.

For example, take chronic rhinosinusitis, a disease with a significant societal burden, but despite extensive research efforts, an unknown pathophysiology. There is emerging evidence that microorganisms play an important role in the exacerbation and perpetuation of mucosal inflammation:

The microbiome of chronic rhinosinusitis: culture, molecular diagnostics and biofilm detection. (2013) BMC Infectious Diseases, 13:210 doi:10.1186/1471-2334-13-210
Bacteria and fungi are believed to influence mucosal inflammation in chronic rhinosinusitis (CRS). However their presence and relationship to disease is debated. This study used multiple detection methods to compare microbial diversity and microbial abundance in healthy and diseased sinonasal mucosa. The utility of contemporary detection methods is also examined. Sinonasal mucosa was analyzed from 38 CRS and 6 controls. Bacterial and fungal analysis was performed using conventional culture, molecular diagnostics (polymerase chain reaction coupled with electrospray ionization time-of-flight mass spectrometry) and fluorescence in situ hybridization. Microbes were detected in all samples, including controls, and were often polymicrobial. 33 different bacterial species were detected in CRS, 5 in control patients, with frequent recovery of anaerobes. Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS and controls, respectively. Using a model organism, FISH had a sensitivity of 78%, and a specificity of 93%. Many species were detected in both CRS and controls however, microbial abundance was associated with disease manifestation. This study highlights some cornerstones of microbial variations in healthy and diseased paranasal sinuses. Whilst the healthy sinus is clearly not sterile, it appears prevalence and abundance of organisms is critical in determining disease. Evidence from high-sensitivity techniques, limits the role of fungi in CRS to a small group of patients. Comparison with molecular analysis suggests that the detection threshold of FISH and culture is related to organism abundance and, furthermore, culture tends to select for rapidly growing organisms.

Or consider the news that antibiotics may ease chronic back pain:

Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. (2013) European Spine Journal, 1-11.
The study was a double-blind RCT with 162 patients whose only known illness was chronic LBP of greater than 6 months duration occurring after a previous disc herniation and who also had bone edema demonstrated as Modic type 1 changes in the vertebrae adjacent to the previous herniation. Patients were randomized to either 100 days of antibiotic treatment (Bioclavid) or placebo and were blindly evaluated at baseline, end of treatment and at 1-year follow-up. Primary outcome, disease-specific disability, lumbar pain. Secondary outcome leg pain, number of hours with pain last 4 weeks, global perceived health, EQ-5D thermometer, days with sick leave, bothersomeness, constant pain, magnetic resonance image (MRI). 144 of the 162 original patients were evaluated at 1-year follow-up. The two groups were similar at baseline. The antibiotic group improved highly statistically significantly on all outcome measures and improvement continued from 100 days follow-up until 1-year follow-up. At baseline, 100 days follow-up, 1-year follow-up the disease-specific disability-RMDQ changed: antibiotic 15, 11, 5.7; placebo 15, 14, 14. Leg pain: antibiotics 5.3, 3.0, 1.4; placebo 4.0, 4.3, 4.3. Lumbar pain: antibiotics 6.7, 5.0, 3.7; placebo 6.3, 6.3, 6.3. For the outcome measures, where a clinically important effect size was defined, improvements exceeded the thresholds, and a trend towards a dose–response relationship with double dose antibiotics being more efficacious. The antibiotic protocol in this study was significantly more effective for this group of patients (CLBP associated with Modic I) than placebo in all the primary and secondary outcomes.

In treating chronic diseases such as these, those long lists of organisms associated with certain diseases states is going to start paying off over the next few years.

Polyphosphate (polyP) is a linear polymer of a few to many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. This ubiquitous polymer is found in bacteria, protists, and mammalian cells, and probably predates living cells. In bacteria, polyP accumulates in volutin or metachromatic granules, which are equivalent to acidocalcisomes. In eukaryotic cells, polyP is present in different compartments, including the cytosol, nucleus, lysosomes, and mitochondria, but is preferentially accumulated in acidic vacuoles such as the yeast vacuole and acidocalcisomes.

Acidocalcisomes were first described in trypanosomes and later found in Apicomplexan parasites, algae, slime molds, fungi, eggs of different origins, and human cells. These organelles were originally described as acidic compartments storing high concentrations of calcium, and later work found that they are highly enriched in polyP. As the description of acidocalcisomes progressed over the years, it was found that they are similar to the volutin or metachromic granules described in bacteria and are now considered to be the only organelles maintained over evolutionary time from bacteria to human cells.

The function of polyP has been studied mainly in prokaryotes: as a Pi store, an energy source to replace ATP, in cation sequestration and storage, in cell membrane formation and function, in gene transcription control, in regulation of enzyme activities, in response to stress and stationary phase, and in the structure of channels and pumps. PolyP is also important in the physiological adjustments of bacteria to growth, development, stress, and deprivation; its role in biofilm development, quorum sensing, and virulence, as well as in long-term survival and expression of virulence factors.

PolyP, which in bacteria is mainly of long-chain type (>300 and up to 1,000 Pi residues), has been reported to be important for virulence of different bacteria, such as Salmonella spp., Shigella flexneri, Vibrio cholerae, Neisseria meningitidis, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, but the mechanism involved is not known. It has also been reported that conditions that decrease the levels of polyP in parasites such as T. brucei, T. gondii, or L. major reduce their pathogenicity. Whether this is due to osmotic fragility of the parasites as a result of changes in polyP levels that impact their ability to grow in vivo, making the immune response against them more successful, or to a role of polyP in modulating the immune response is not yet known.

PolyP has been found in bacterial to human cells and has been reported to be important for virulence of different bacteria and a number of parasites, including those that cause toxoplasmosis, African trypanosomiasis, and leishmaniasis. Even more exciting are the findings about the role of polyP in cancer metastasis, blood coagulation, inflammation, and innate immunity. For example, a significant finding is that enzymes involved in polyP metabolism could be excellent targets for drug design not only against bacteria and parasites but also for regulation of important physiological and pathological processes such as coagulation, inflammation, innate immunity, and thrombosis.

Polyphosphate and Its Diverse Functions in Host Cells and Pathogens. (2013) PLoS Pathog 9(5): e1003230. doi:10.1371/journal.ppat.1003230

If there is one truism about picornaviruses, it is that the entire replication cycle of these simple positive-strand RNA viruses takes place in the cytosol. This statement is usually made to directly contrast picornaviruses with retroviruses or DNA viruses that require transport to the nucleus. However, the statement is meant quite literally. Some enveloped RNA viruses enter organelles to bud from the cellular secretion pathway, while other RNA viruses replicate their genomes in tightly controlled organelle invaginations. In contrast, every step in the replication of picornaviruses, once the genome has entered the cytosol, has long been thought to take place directly in the cytoplasm or on the cytoplasmic face of membranous structures

The role of autophagosomes in poliovirus replication has long been controversial. Some believe the cytoplasmic face of these vesicles to be a site of virus RNA replication. This was primarily due to the localization of multiple virus-encoded RNA replication proteins to the autophagosome membrane. A competing hypothesis emerged observes that viral RNA replication proteins localized to single-membraned vesicles containing components of the cellular COPII machinery.

But the COPII and autophagy hypotheses might not be mutually exclusive. Single-membraned vesicles predominate in the first few hours of poliovirus infection. Later, convoluted invaginations of the single-membraned vesicles are observed. This results in structures morphologically similar to the crescent-shaped phagophore, which is the precursor to the double-membraned autophagosome. By 6 hours post-infection, double-membraned vesicles predominate. Viral proteins and active RNA replication is associated with both types of structure. However, the exponential phase of RNA replication occurs when predominantly single-membraned vesicles are present. The authors proposed a model in which single-membraned vesicles morph into double-membraned vesicles, and suggested that the single-membraned vesicles are the primary sites of viral genome replication.

Picornaviruses are among the simplest human viruses, physically consisting of a positive-sense RNA genome and a capsid. The current model for exit of picornaviruses from cells is disruption of the plasma membrane resulting in a lysis event that releases waiting cytoplasmic virions. However, if a cell full of virus-containing double-membraned vesicles lyses, releasing the vesicles, then two lipid bilayers remain between the virions and the receptors on the surface of the next cell.

This new model leaves us with a new paradigm for picornavirus replication. These viruses, for so long thought to be cytoplasmic, may in fact be more infectious if engulfed in an organelle lumen. These so-called “naked viruses,” thought to be bare in the cytoplasm, may in fact swaddle themselves in multiple layers of membranes prior to cell lysis. This work may reveal a replication strategy that can provide a mechanistic evolutionary link between the enveloped and nonenveloped viruses.

Behind Closed Membranes: The Secret Lives of Picornaviruses? (2013) PLoS Pathog 9(5): e1003262. doi:10.1371/journal.ppat.1003262

See: The Mystery of the Extra “Envelope”

The failure of poisons affecting the cytoskeleton to inhibit the replication of a diverse set of viruses strongly suggests that viruses do not require a functional cytoskeletal system for replication, either because they do not utilize it or are able to utilize alternate pathways when it is not available.

Do viruses require the cytoskeleton? Virology Journal 2013, 10: 121 doi:10.1186/1743-422X-10-121
It is generally thought that viruses require the cytoskeleton during their replication cycle. However, recent experiments in our laboratory with rubella virus, a member of the family Togaviridae (genus rubivirus), revealed that replication proceeded in the presence of drugs that inhibit microtubules. This study was done to expand on this observation. The replication of three diverse viruses, Sindbis virus (SINV; family Togaviridae family), vesicular stomatitis virus (VSV; family Rhabdoviridae), and Herpes simplex virus (family Herpesviridae), was quantified by the titer (plaque forming units/ml; pfu/ml) produced in cells treated with one of three anti-microtubule drugs (colchicine, noscapine, or paclitaxel) or the anti-actin filament drug, cytochalasin D. None of these drugs affected the replication these viruses. Specific steps in the SINV infection cycle were examined during drug treatment to determine if alterations in specific steps in the virus replication cycle in the absence of a functional cytoskeletal system could be detected, i.e. redistribution of viral proteins and replication complexes or increases/decreases in their abundance. These investigations revealed that the observable impacts were a colchicine-mediated fragmentation of the Golgi apparatus and concomitant intracellular redistribution of the virion structural proteins, along with a reduction in viral genome and sub-genome RNA levels, but not double-stranded RNA or protein levels.

“Scholars have argued that in risk communication a dilemma exists between the media functions of informing the media audience about rational risk behavior, and providing an arena for public deliberation about risk. Optimizing the information function would suggest that media provide clear, unanimous advice without creating confusion by reporting uncertainty and controversy. Optimizing the deliberative function, in contrast, would require media to include different (even contradictory) voices. A similar dilemma exists between incompatible expectations of different fractions of the audience. Part of the audience may trust the media to provide the best available advice. These audience members may be prepared to take the mediated advice at face value, not wanting to be unsettled by controversy and uncertainty. But another part of the audience may prefer to learn about the full spectrum of opinions, including outsider views, and may want to develop their own conclusions on whom to trust and whose advice to follow. Presenting only the mainstream view may motivate members of that part of the audience to seek information in alternative channels – such as blogs or rumors.”

Similar challenges but different responses: Media coverage of measles vaccination in the UK and China. (2012) Public Understanding of Science. doi: 10.1177/0963662512445012
For several decades scholars have studied media reporting on scientific issues that involve controversy. Most studies so far have focused on the western world. This article tries to broaden the perspective by considering China and comparing it to a western country. A content analysis of newspaper coverage of vaccination issues in the UK and China shows, first, that the government-supported ‘mainstream position’ dominates the Chinese coverage while the British media frequently refer to criticism and controversy. Second, scientific expertise in the British coverage is represented by experts from the health and science sector but by experts from health agencies in the Chinese coverage. These results are discussed with respect to implications for risk communication and scientists’ involvement in public communication.

The use of animal virus-like particles (VLPs) as vectors for the delivery of genes to mammalian cells has been explored for many years. Plant viruses are almost without exception “just” genetic material (DNA or RNA) surrounded by a shell composed of the capsid protein (CP) – with no membrane envelope. Plant-derived VLPs have not been used for direct gene delivery and expression. There have been no attempts to use a spherical plant viral capsid to deliver heterologous genes for expression in mammalian cells, even though there are several independent demonstrations of the internalization of plant virus by cells.

In light of there being no direct demonstration of spherical plant viruses disassembling and thereby releasing their contents in animal cells, this paper asks the question: Can heterologous genes in spherical plant VLPs be made available to a mammalian cell and their protein products synthesized?

Getting RNA into cells is a major barrier to future theraputic approaches, so robust systems are urgently needed.

Reconstituted plant viral capsids can release genes to mammalian cells. Virology. 19 April 2013 doi: 10.1016/j.virol.2013.03.001
The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. In this work we demonstrate that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells. This result establishes the first step in the use of plant viral capsids as vectors for gene delivery and expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting.

Actinic keratosis is no laughing matter – untreated, up to 20% of cases may pregress to skin cancer. At the same time, if this paper had been published on 1st April (it wasn’t) I would have had my doubts. I’ve seen some interesting virus sampling protocols in my time (bear in mind we sequenced the horse faeces virome), but this one is pretty unique. As a side benefit, also reduces the risk of monobrow.

Eyebrow hairs from actinic keratosis patients harbor the highest number of cutaneous human papillomaviruses. BMC Infectious Diseases 2013, 13: 186 doi:10.1186/1471-2334-13-186
Cutaneous human papillomavirus (HPV) infections seem to be associated with the onset of actinic keratosis (AK). This study compares the presence of cutaneous HPV types in eyebrow hairs to those in tissues of normal skin and skin lesions of 75 immunocompetent AK patients. Biopsies from AK lesions, normal skin and plucked eyebrow hairs were collected from each patient. DNA from these specimens was tested for the presence of 28 cutaneous HPV (betaPV and gammaPV) by a PCR based method. The highest number of HPV prevalence was detected in 84% of the eyebrow hairs (63/75, median 6 types) compared to 47% of AK lesions (35/75, median 3 types) (p< 0.001) and 37% of normal skin (28/75, median 4 types) (p< 0.001), respectively. A total of 228 HPV infections were found in eyebrow hairs compared to only 92 HPV infections in AK and 69 in normal skin. In all three specimens HPV20, HPV23 and/or HPV37 were the most prevalent types. The highest number of multiple types of HPV positive specimens was found in 76% of the eyebrow hairs compared to 60% in AK and 57% in normal skin. The concordance of at least one HPV type in virus positive specimens was 81% (three specimens) and 88-93% of all three combinations with two specimens. Thus, eyebrow hairs revealed the highest number of cutaneous HPV infections, are easy to collect and are an appropriate screening tool in order to identify a possible association of HPV and AK.

Welcome to the Weekend!

World Malaria Day recognises global efforts being made in the fight against malaria. First established in 2007 by the World Health Assembly, it falls on 25 April every year.

Measles outbreak: In graphics
Great use of data by BBC News – students take note – this is how you do it.

Strain of measles possible cause of dolphin deaths [Audio]
Scientists think that more than a hundred dead dolphins found washed up on the Italian coast, may have been infected with a killer strain of “measles”. [Human measles, or another Paramyxovirus?]

Mapping the H7N9 avian flu outbreaks
Where are the 104 human cases reported so far and where might the virus go next?

Promising Treatment for New Human Coronavirus
A new report says that two antiviral drugs, ribavirin and interferon-alpha 2b, will stop nCoV from replicating in cells grown in the lab.

Radioactive Listeria cures cancer – or does it?
This report just appeared in PNAS. Let’s hope it’s a generalisable method, but for the present this is one small scale study – still a long way to go before this is a routine treatment.

The Truth About Why Microbes Make You Sick
Between fevers, congestion and diarrhea, there are numerous ways that microbes can make us feel sick. But just how do microorganisms cause these symptoms?

Gonorrhea could be untreatable by 2015 [Audio]
There’s been a sharp increase in the number of cases of the sexually transmitted infection gonorrhoea – up 25% in 2011. It is also becoming harder to find antibiotics that treat it, which has raised the prospect that it could soon become untreatable.

Holy Virus Treasure Trove, Batman!
Think about the type of animal that would make an ideal host for a virus. It would gather in large dense groups, making it easier for the virus to jump into fresh hosts. It should have a relatively long lifespan, so any single individual has many chances of becoming infected. It would certainly travel over long distances to spread the infection far and wide. Humans certainly fit the bill. So do bats.