MicrobiologyBytes

Mimivirus, a nucleocytoplasmic large double stranded DNA virus infecting Acanthamoeba species, is the largest virus identified to date. Its icosahedral fibrillated capsid has a diameter of 750 nm. Besides its outstanding particle size, the genome of Mimivirus is also exceptional both in size and complexity. The initial sequencing revealed a linear genome of 1,181,404 nt (roughly the size of the spirochaete bacterium Treponema pallidum genome) harboring 911 protein coding genes and 6 tRNAs. Some of these genes were observed for the first time in a virus, the most salient being those involved in protein translation and DNA repair. These unique features reawaked conceptual discussions on the nature of viruses and the frontier between viruses and cellular organisms.

Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing. (2011) Virology Journal 2011, 8:99 doi:10.1186/1743-422X-8-99
Background: Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes.FindingsWe now report a much deeper analysis using the SOLiD technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. Conclusions: This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.

Related:

• DNA Viruses – Really Big Ones
• Marvellous mimivirus
• Marine mimivirus relatives are large algal viruses

and on Amazon.com!

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common respiratory pathogens. Both viruses comprise two genetic groups, A and B, distinguishable genetically and serologically which circulate with fluctuating frequencies. This gives rise to the observation of switching of the predominantly circulating subtype between seasons. Repeat HRSV infections occur throughout life with decreasing morbidity, and increasingly evidence suggests the same is also true for HMPV. In neither case has it yet been possible to make an effective vaccine against these troublesome pathogens.

Molecular Epidemiology and Evolution of Human Respiratory Syncytial Virus and Human Metapneumovirus. (2011) PLoS ONE 6(3): e17427
Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, the Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.

Related:

• Newly discovered respiratory viruses
• Human metapneumovirus glycoprotein G is an important virulence factor

Extensively drug-resistant (XDR) tuberculosis (TB) is defined as TB that is resistant to at least rifampin and isoniazid (multidrug resistant [MDR]), as well as to any member of the quinolone family and at least one second-line anti-TB injectable drug: kanamycin, capreomycin, or amikacin. According to the World Health Organization (WHO), XDR-TB has been reported in 57 countries and is a major concern for global health. The WHO Global Task Force on XDR-TB has recommended laboratory-based surveillance to better understand the prevalence of XDR TB in developing countries. However, surveillance data on XDR-TB from China remain scarce. Shandong Province is the second largest province in China, with a population of 94 million. Shandong Provincial Chest Hospital (SPCH) is the only provincial-level hospital specializing in TB clinical service and control. In collaboration with the SPCH TB reference laboratory, researchers retrospectively analyzed the drug-resistance profiles of a group of clinical Mycobacterium tuberculosis isolates to estimate the prevalence of XDR-TB in China.

32% of isolates resistant to more than one first-line drug; 10% isolates were multidrug resistant, and 2% were XDR. XDR-TB is of major concern in China.

Laboratory-based Surveillance of Extensively Drug-Resistant Tuberculosis, China. EID 17(3) March 2011
To estimate the prevalence of extensively drug-resistant tuberculosis (XDR TB) in China, we retrospectively analyzed drug-resistance profiles of 989 clinical Mycobacterium tuberculosis isolates. We found 319 (32.3%) isolates resistant to >1 first-line drugs; 107 (10.8%) isolates were multidrug resistant, of which 20 (18.7%) were XDR. XDR TB is of major concern in China.

Related:

• Extreme drug resistant tuberculosis (XDR-TB)
• XDR tuberculosis – implications for global public health.N Engl J Med. (2007) 356: 656–659

Torque teno viruses (TTVs) are small, non-enveloped viruses that contain a circular single-stranded DNA genome of negative polarity, presently classified in the family Anelloviridae. TTVs were first detected in 1997 in a Japanese patient with post-transfusion hepatitis of unknown etiology. TTVs are not restricted to human hosts and have also been identified in a number of other species, including non-human primates, cats, dogs, pigs, chickens, cows and sheep. Do they cause any diseases? The jury is still out on that question. In fact, we really know very little about these relatively common viruses…

Torque Teno Sus Virus (TTSuV) in Cell Cultures and Trypsin. (2011) PLoS ONE 6(3): e17501. doi:10.1371/journal.pone.0017501
Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells.

It is eye-opening to search the internet for the term MRSA (methicillin-resistant Staphylococcus aureus) these days. Instead of epidemiological treatises from Morbidity and Mortality Weekly Report, or reports of decreasing surgical site infections attributed to the advent of active surveillance followed by decolonization and contact isolation procedures, one finds that the first items that are highlighted link to Michael Jackson’s nose infection following another of his minor rhinoplastic touch-ups; a web page devoted to the new best-seller wannabe, Maryn McKenna’s book Superbug, The Fatal Menace of MRSA; and recently World MRSA Day (October 2nd, in case your laboratory wishes to have an event) sponsored by the MRSA Survivors’ Network. Yes, MRSA has celebrity spokespersons, in the shape of actors Tanner Richie and Alicia Cole. How did this microbe, only a few years older than HIV, become so infamous? Actually, we have ourselves partly to blame…

MRSA: a case of pathogens, politics and penalties. Trends Microbiol. 23 Feb 2011
In the current era of public scientific ‘debate’ such as the scientific merit of climate change, it should come as no surprise that a bacterium would have its 15 minutes of political limelight. Furthermore, a few dedicated citizens can truly influence the lives of many by changing the law of the land. For microbiologists, who often complain that our contributions go unnoticed and that we have no political power, this story serves to prove otherwise.

Related:

• MRSA: Methicillin-resistant Staphylococcus aureus
• Clustering of MRSA strains across Europe
• Staphylococcus aureus: superbug

“NASA astrobiologist Richard Hoover thinks he’s found fossilized alien bacteria inside a meteorite. If he’s right, it’s world-shaking news. But that’s a very, very big if.
The claims have already set off an internet tempest of commentary, some of it noting Hoover’s relatively unusual announcement venue. The Journal of Cosmology is produced by a community of colorful astrobiologists who are sometimes zealously evangelical about galactic panspermia, or the notion that Earth was seeded by life arriving from space.”

Read more: Alien Microbe Claim Starts Fight Over Meteorite | Wired Science

Personally, I remain to be convinced. Ever heard of Occam’s Razor? And we’ve been here before.

Viroids are the smallest known infectious agents and induce disease in a wide variety of plant hosts, including many crop species. Ranging in size from ca. 250–400 nucleotides (nt) replication of their single-stranded, circular, non-coding RNA genomes is entirely dependent on transcriptional and processing machinery supplied by the host. Their small size and unique molecular structure makes these molecules an attractive system with which to analyze many different aspects of host–pathogen interaction.

Although replication occurs in different subcellular compartments, members of both families of viroids induce RNA silencing and the accumulation of viroid-specific small RNAs following infection. Post-transcriptional gene silencing (RNA silencing) provides a multi-layer defense system which protects plants from invasion by exogenous RNA replicons such as viruses and viroids. Silencing is triggered by conversion of double-stranded or hairpin RNAs to small RNAs whose sizes ranging between 18 and 26 nucleotides. Infected plants contain high levels of viroid-specific small RNAs, but the circular genomic RNAs themselves appear relatively resistant to RNA silencing – raising the possibility that viroid replication may also be resistant. The mechanism underlying this resistance/tolerance is not yet understood, but certain transgenic tomato lines expressing high levels of hairpin RNA-derived small viroid RNAs are resistant to infection. This paper looks at the accumulation pattern and size distribution of viroid-specific small RNAs in infected plants and identifies several potential targets for RNA silencing mediated by small RNAs.

Accumulation of Potato spindle tuber viroid-specific small RNAs is accompanied by specific changes in gene expression in two tomato cultivars. Virology. 24 Feb 2011
To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in potato spindle tuber viroid (PSTVd)-infected tomato plants. Large-scale sequence analysis of viroid-specific small RNAs revealed active production from the upper portion of the pathogenicity and central domains, two regions previously thought to be underrepresented. Profiles of small RNA populations derived from PSTVd antigenomic RNA were more variable, with differences between infected Rutgers (severe symptoms) and Moneymaker (mild symptoms) plants pointing to possible cultivar-specific differences in small RNA synthesis and/or stability. Using microarray analysis, we monitored the effects of PSTVd infection on the expression levels of >100 tomato genes containing potential binding sites for PSTVd small RNAs. Of 18 such genes down-regulated early in infection, two genes involved in gibberellin or jasmonic acid biosynthesis contain binding sites for PSTVd small RNAs in their respective ORFs.

Related:

• The Biology of Viroid-Host Interactions
• Resistance to Plant Viruses

One of my New Year resolutions this year was to try to lay off the “omics” papers here on MicrobiologyBytes. Most of them are not very good and even those that are get indigestible fairly quickly. I’m making an exception for this one as it’s reasonably easy to read and highly relevant to chronic obstructive pulmonary disease (COPD), a progressive and eventually fatal lung disease that is projected to be responsible for the fifth largest burden of disease worldwide by 2020.

The results show that the lungs of “healthy” smokers contain a bacterial microbiome that is quantitatively significant, diverse (but of limited membership), and quite distinct from that reported for the oral cavity or nasopharynx. The diversity of the lung bacterial microbiome is lower in subjects with decreased lung function, most commonly associated with dominance by Pseudomonas spp. This is the first study to describe that the numerous microanatomic sites within the lung can give rise to significant differences in bacterial community structure. By demonstrating that one person’s lungs can harbor both generalized areas of “healthy” microbiome and a single site containing a “pathogenic” community, the results suggest a mechanism by which the interaction of lung pathogens and host immunity might contribute to localized disease progression, even in the absence of overt symptoms.

Even if you feel OK, stop smoking, stupid!

Analysis of the Lung Microbiome in the “Healthy” Smoker and in COPD. (2011) PLoS ONE 6(2): e16384. doi:10.1371/journal.pone.0016384
Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry (“heathy smokers”, HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Related:

• Human Microbiome Project (HMP)
• Circumcision alters penis microbiome