The cellular receptors and determinants that mediate entry of WNV are unclear to date. The notable ability of WNV to infect a broad range of species (mosquitoes, reptiles, birds and mammals), and virtually every in vitro cell line is supposed to be related to cellular proteins, relevant for virus entry and replication, which are highly conserved among divergent host species.

By using integrin knock-out cell lines which lack the particular integrin subunits, this study demonstrate that the presence of αv-, β1- or β3-integrins is not required for the attachment of four different WNV strains to the cell surface. However, β1- and β3-integrin expression significantly enhances virus amplification. These findings imply that other routes are used in the absence of these integrins, or that different routes are generally used in parallel.

Integrins modulate the infection efficiency of West Nile virus into cells. J Gen Virol. 08 May 2013
The underlying mechanisms allowing West Nile virus (WNV) to replicate in a large variety of different arthropod, bird and mammal species are largely unknown but are believed to rely on highly conserved proteins relevant for viral entry and replication. Consistent with this, the integrin αvβ3 has been proposed lately to function as the cellular receptor for WNV. More recently published data, however, are not in line with this concept. Integrins are highly conserved among diverse taxa and are expressed by almost every cell type at high numbers. Our study was designed to clarify the involvement of integrins in WNV infection of cells. A cell culture model, based on wild-type and specific integrin knock-out cell lines lacking the integrin subunits αv, β1 or β3, was used to investigate the susceptibility to WNV, and to evaluate binding and replication efficiencies of four distinct strains (New York 1999, Uganda 1937, Sarafend and Dakar). Though all cell lines were permissive, clear differences in replication efficiencies were observed. Rescue of the β3-integrin subunit resulted in enhanced WNV yields of up to 90% regardless the virus strain used. Similar results were obtained for β1-expressing and non-expressing cells. Binding, however, was not affected by the expression of the integrins in question, and integrin blocking antibodies failed to have any effect. We conclude that integrins are involved in WNV infection but not at the level of binding to target cells.

The post Integrins modulate the infection efficiency of West Nile virus appeared first on MicrobiologyBytes.

Researchers investigated the response of mosquitoes infected with P. falciparum malaria parasites and uninfected to human odor collected on fabric. Mosquitoes that were infected with the parasites landed and probed significantly more than uninfected mosquitoes in response to the odor. Previous research has already shown that the malarial parasite can alter mosquito behavior in ways that increase the rate of malaria transmission. For example, malaria-infected mosquitoes also consume larger, more frequent blood meals than their uninfected counterparts.

Studies of mosquito behavior in the context of malaria transmission usually use uninfected mosquito subjects. This study suggests that such behavioral studies may not always be representative of the behavior of infected mosquitoes. They conclude that understanding the olfactory changes underlying the behavior of these infected mosquitoes may help identify new compounds that could be used to develop mosquito traps for surveillance programs.

Malaria Infected Mosquitoes Express Enhanced Attraction to Human Odor. (2013) PLoS ONE 8(5): e63602. doi:10.1371/journal.pone.0063602
There is much evidence that some pathogens manipulate the behaviour of their mosquito hosts to enhance pathogen transmission. However, it is unknown whether this phenomenon exists in the interaction of Anopheles gambiae sensu stricto with the malaria parasite, Plasmodium falciparum – one of the most important interactions in the context of humanity, with malaria causing over 200 million human cases and over 770 thousand deaths each year. Here we demonstrate, for the first time, that infection with P. falciparum causes alterations in behavioural responses to host-derived olfactory stimuli in host- seeking female An. gambiae s.s. mosquitoes. In behavioural experiments we showed that P. falciparum-infected An. gambiae mosquitoes were significantly more attracted to human odors than uninfected mosquitoes. Both P. falciparum-infected and uninfected mosquitoes landed significantly more on a substrate emanating human skin odor compared to a clean substrate. However, significantly more infected mosquitoes landed and probed on a substrate emanating human skin odor than uninfected mosquitoes. This is the first demonstration of a change of An. gambiae behaviour in response to olfactory stimuli caused by infection with P. falciparum. The results of our study provide vital information that could be used to provide better predictions of how malaria is transmitted from human being to human being by An. gambiae s.s. females. Additionally, it highlights the urgent need to investigate this interaction further to determine the olfactory mechanisms that underlie the differential behavioural responses. In doing so, new attractive compounds could be identified which could be used to develop improved mosquito traps for surveillance or trapping programmes that may even specifically target P. falciparum-infected An. gambiae s.s. females.

The post Malaria infected mosquitoes are more attracted to human odor appeared first on MicrobiologyBytes.

1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Simply finding a potentially-disease causing organism does not necessarily mean it’s up to no good!

The bacterium Mycoplasma pneumoniae is carried at high rates in the upper respiratory tracts of healthy children and usual diagnostic tests cannot differentiate between such asymptomatic carriage and actual respiratory tract infection, according to a new study. These findings are important as they suggest that clinicians may need to reconsider the clinical significance of a positive test result for the presence of this bacterium.

The researchers compared upper respiratory tract swabs and blood culture results taken from 321 children (aged 3 months to 16 years) admitted to hospital with a respiratory tract infection with those from 405 healthy children undergoing an elective surgical procedure. They found that the prevalence of M. pneumoniae (as measured using PCR tests) did not differ significantly between the asymptomatic group and the symptomatic group. There was also no difference in prevalence when diagnosed using blood tests. In addition, a high rate of other bacteria and viruses was found in both asymptomatic and symptomatic children.

This data indicates that the presence of M. pneumoniae in the upper respiratory tract is common in asymptomatic children. Current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection. Cinicians may need to readdress the clinical significance of a positive test result.

Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study. (2013) PLoS Med 10(5): e1001444. doi:10.1371/journal.pmed.1001444
Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. This study was conducted at the Erasmus MC–Sophia Children’s Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p=0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.

The post Asymptomatic carriage of Mycoplasma pneumoniae common in children appeared first on MicrobiologyBytes.

The aim of this review is to provide an overview of VOCs produced by the six most abundant and pathogenic bacteria in sepsis, including Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. Such VOCs could be used as biological markers in the diagnostic approach of critically ill patients. A systematic review of existing literature revealed 31 articles. All six bacteria of interest produce isopentanol, formaldehyde, methyl mercaptan, and trimethylamine. Since humans do not produce these VOCs, they could serve as biological markers for presence of these pathogens.

Targeted studies should be performed to identify potential sets of volatile biological markers and evaluate the diagnostic accuracy of these markers in critically ill patients.

Volatile Metabolites of Pathogens: A Systematic Review. (2013) PLoS Pathog 9(5): e1003311. doi:10.1371/journal.ppat.1003311

The post Don’t hold your breath appeared first on MicrobiologyBytes.

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.

The post Is the Berlin patient really cured? appeared first on MicrobiologyBytes.

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.

The post What have microbiomes ever done for us? appeared first on MicrobiologyBytes.

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

The post Polyphosphate in Host Cells and Pathogens appeared first on MicrobiologyBytes.

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 post Picornavirus interactions with cellular membranes and vesicles appeared first on MicrobiologyBytes.

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.

The post Do viruses require the cytoskeleton? appeared first on MicrobiologyBytes.