Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.     

Population dynamics of Walia ibex (Capra walie) at Simien Mountains National Park,Ethiopia

Intensive total direct counts of Walia ibex (Capra walie) population were performed at Simien Mountains National Park (SMNP) in 2009. Historical data were collected from SMNP and literature reviews. Different models were suited to determine population growth rates and intrinsic rate of increase. The population size estimated was 745 animals. The correlation between the two repeated counts was significant (r = 0.99 and P < 0.01). Mean instantaneous growth rate (r), growth rate per capita (λ) and population annual growth rate (Λ) were 2.6 ± 2.6, 0.03 ± 0.18 and 19.5 ± 50.4, respectively. Instantaneous growth rate and growth rate per capita were positively correlated (r = 0.958, P < 0.01). Average growth rate (rΛ) and intrinsic rate of increase (rr) under ideal (r = 0.950, P < 0.01) and random environments (r = 0.810, P < 0.01) were positively correlated. The population grows by 2.5% under ideal environments with an intrinsic increase of 0.04 (0.006%) and by 0.13% under random environments with intrinsic rate of decrease of ?0.184 or ?0.025% per year, respectively. The mean rank of the flock structure of whole population was 3.13, 3.88, 2.00 and 1.00 for males, females, juveniles and unidentified, respectively.     

Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1

2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase.     

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection

Background

Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication in vitro. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although in vivo evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15.

Methods

Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIV_SF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure.

Results

Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID₅₀ (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models).

Conclusions

Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the in vivo evaluation of lubricants with regards to HIV transmission.     

H-NS regulation of IraD and IraM antiadaptors for control of RpoS degradation

RpoS, the master sigma factor during stationary phase and under a variety of stress conditions, is regulated at multiple levels, including regulated degradation. Degradation is dependent upon ClpXP and the RssB adaptor protein. H-NS, a nucleoid-associated protein, affects the regulated degradation of RpoS; in the absence of H-NS, RpoS is stable. The mechanisms involved in this regulation were not known. We have found that H-NS inhibits the expression of iraD and iraM, the genes coding for two antiadaptor proteins that stabilize RpoS when overexpressed. The regulation by H-NS of iraM is independent from the previously demonstrated regulation by the PhoP/PhoQ two-component system. Moreover, differences in the behavior of several hns alleles are explained by a role for StpA, an H-NS-like protein, in the regulation of RpoS stability. This finding parallels recent observations for a role of StpA in regulation of RpoS stability in Salmonella.     

Genetic diversity and differentiation of cultivated amochi (Arisaema schimperianum Schott) in Ethiopia as revealed by AFLP markers

Amochi (Arisaema schimperianum Schott) is an off-season crop plant in southern Ethiopia, grown during the dry season on residual moisture, for its edible tubers. It has gained importance as a "security crop" especially during the years of moisture stress and food shortage. Amochi is irritating in contact to the skin. Removal of this effect is an important question for breeding. As the first step, however we attempt to establish base line information of its breeding system and genetic variability using AFLPs. The extent of genetic differentiation among 11 populations (96 individuals) of amochi sampled along altitudinal gradients that varied from 1700 to 3200 m a.s.l. was investigated. The populations were classified in to three altitudinal groups: lowland (1700 to 2200 m a.s.l.), central-highland (2201 to 2600 m a.s.l.) and highland (2601 to 3200 m a.s.l.). Polymorphic loci (167) scored from four primer pair combinations, were used for principal component analysis (PCA), and analysis of molecular variance (AMOVA). Both PCA and unweighed pair group with arithmetic mean (UPGMA) clearly differentiated populations into their respective altitude groups, with large genetic distances. AMOVA analysis revealed 70.5%, 16.7% and 12.8% variability between altitude groups, between populations and within populations respectively. Average diversity indices within populations were also low. Since the largest proportion of variation is located between altitude groups, rather than within populations, we suggest future studies on the chemical composition, low irritation, and other desirable traits should consider populations from different altitude ranges.     

Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

Background

Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis.

Methods

Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival.

Results

In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-X_L ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice.

Conclusions

Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.     

Community Health Seeking Behavior for Suspected Human and Animal Rabies Cases,Gomma District,Southwest Ethiopia

Background

Timely presentation to appropriate health service provider of sick animals/humans from zoonotic diseases like rabies is important for early case/outbreak detection and management. However, data on community’s health seeking practice for rabies in Ethiopia is limited. Therefore the objective of this study was to determine community’s health seeking behavior on rabies, Southwest Ethiopia.

Methods

A cross-sectional survey was conducted from January 16-February 14, 2015 to collect data from 808 respondents where the respondents were selected using multistage sampling technique. Data were collected using interviewer administered structured questionnaire by trained epidemiology graduate level students. Data were entered to Epidata version 3.1 and analyzed using SPSS version 20 for windows.

Result

Eight hundred three (99.4%) respondents participated in the study. Out of 28 respondents who reported their family members’ exposure to rabies, 8 of them replied that the exposed family members sought treatment from traditional healers. More than nine in ten respondents perceived that humans and domestic animals with rabies exposure should seek help of which 85% of them suggested modern health care facilities as the preferred management option for the sick humans and domestic animals. However, among those who reported sick domestic animals, near to 72% of them had either slaughtered for human consumption, sold immediately, visited traditional healer, given home care or did nothing for the sick domestic animals.

Conclusion

Majority of the respondents had favorable perception of seeking treatment from modern health care facilities for rabies. However, significant number of them had managed inappropriately for the sick domestic animals from rabies. Hence, raising awareness of the community about management of sick domestic animals from rabies and the need for reporting to both human and animal health service providers is needed.