A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection

We report the isolation and characterization of products from a subtractive cDNA library from the haemolymph of Penaeus monodon experimentally infected with white spot syndrome virus (WSSV). One cDNA derived from up-regulated mRNA was identified. A homology search indicated similarity to the putative protein syntenin (TE8). The nearly complete nucleotide sequence of TE8 was obtained by rapid amplification of cDNA (RACE). Its putative protein product contained a tandem repeat of PDZ domains (postsynaptic density protein or PSD-95, DlgA and ZO-1). We propose that TE8 may function as an adapter that couples PDZ-binding protein(s) in a signaling pathway involved in the shrimp response to WSSV.     

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.     

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.     

On the sensitivity of forward scattering patterns from bacterial colonies to media composition

Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition –0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Morphology and Mitochondrial Genome of Fischoederius sp. 1 in Thailand

A rumen fluke Fischoederius elongatus is assigned to the type species of genus Fischoederius, family Gastrothylacidae. However, the mitochondrial sequences recently published are thought to be of inconsistent species, suggesting that several morphologically similar but genetically distinct species might be classified as Fischoederius elongatus. Thus, mentions of F. elongatus from South, Southeast, and East Asia might unintentionally refer to different species. The present work describes morphology and a full mitochondrial genome sequence of one of these species. The fluke specimens were collected from 2 infected cattle in Thailand. An interesting finding was the presence of a second tRNA-Asp gene next to a partial ND1 gene. It is suggested that these duplicated sequences are the remnants of non-reciprocal recombination events caused by inverted repeats located between ND2 and ND1 mitochondrial genes.     

Directly observed therapy and improved tuberculosis treatment outcomes in Thailand

Background

The World Health Organization (WHO) recommends that tuberculosis (TB) patients receive directly observed therapy (DOT). Randomized controlled trials have not consistently shown that this practice improves TB treatment success rates. In Thailand, one of 22 WHO-designated high burden TB countries, patients may have TB treatment observed by a health care worker (HCW), family member, or no one. We studied whether DOT improved TB treatment outcomes in a prospective, observational cohort.

Methods and Findings

We prospectively collected epidemiologic data about TB patients treated at public and private facilities in four provinces in Thailand and the national infectious diseases hospital from 2004–2006. Public health staff recorded the type of observed therapy that patients received during the first two months of TB treatment. We limited our analysis to pulmonary TB patients never previously treated for TB and not known to have multidrug-resistant TB. We analyzed the proportion of patients still on treatment at the end of two months and with treatment success at the end of treatment according to DOT type. We used propensity score analysis to control for factors associated with DOT and treatment outcome. Of 8,031 patients eligible for analysis, 24% received HCW DOT, 59% family DOT, and 18% self-administered therapy (SAT). Smear-positive TB was diagnosed in 63%, and 21% were HIV-infected. Of patients either on treatment or that defaulted at two months, 1601/1636 (98%) patients that received HCW DOT remained on treatment at two months compared with 1096/1268 (86%) patients that received SAT (adjusted OR [aOR] 3.8; 95% confidence interval [CI] 2.4–6.0) and 3782/3987 (95%) patients that received family DOT (aOR 2.1; CI, 1.4–3.1). Of patients that had treatment success or that defaulted at the end of treatment, 1369/1477 (93%) patients that received HCW DOT completed treatment compared with 744/1074 (69%) patients that received SAT (aOR 3.3; CI, 2.4–4.5) and 3130/3529 (89%) patients that received family DOT (aOR 1.5; 1.2–1.9). The benefit of HCW DOT compared with SAT was similar, but smaller, when comparing patients with treatment success to those with death, default, or failure.

Conclusions

In Thailand, two months of DOT was associated with lower odds of default during treatment. The magnitude of benefit was greater for DOT provided by a HCW compared with a family member. Thailand should consider increasing its use of HCW DOT during TB treatment.