Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

UNLABELLED: The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ßA strand region under the icosahedral 2-fold axis rather than antiparallel to the ßB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

Impact of livestock-associated MRSA in a hospital setting.

OBJECTIVES: The Netherlands is known for a stringent search and destroy policy to prevent spread of MRSA. In the hospital setting, livestock-associated MRSA (LA-MRSA) is frequently found in patients coming from the high density farming area in the south of the Netherlands. The aim of the study was to determine the contribution of LA-MRSA in the epidemiology of MRSA in cases found following the Dutch search and destroy policy. PATIENTS AND METHODS: From two hospitals serving a population of 550,000 persons all data on MRSA cultures and subsequent control measures from 2008 and 2009 were retrospectively collected and analyzed. RESULTS: A total of 3856 potential index patients were screened for MRSA, 373 (9.7%) were found to be positive, 292 ( 78%) LA-MRSA and 81 (22%) non-LA-MRSA respectively. No secondary cases were found among contact research in persons exposed to LA-MRSA (0/416), whereas similar contact research for non-LA-MRSA resulted in 83 (2.5%) secondary cases. LA-MRSA were rarely found to cause infections. CONCLUSIONS: LA-MRSA is more prevalent than non-LA-MRSA in Dutch Hospitals in the South of the Netherlands. However, retrospectively studied cases show that the transmission rate for LA-MRSA was much lower than for non-LA-MRSA. This suggest that infection control practices for LA-MRSA may possibly be less stringent than for non-LA-MRSA.

What prevents young adolescents from smoking? Self-reported motives of 12-15-year-old non-smokers.

OBJECTIVES: This study investigated the real motives of German non-smokers. METHODS: In the German SToP ("Sources of Tobacco for Pupils") Study 707 non-smoking pupils were asked to write down their motives for being non-smokers. A total of 1324 partially very elaborate free text statements (mean/range: 1.9/1-7 distinguishable motives) were evaluated in a qualitative content analysis. RESULTS: The most important and frequently mentioned motives for not smoking were health-related arguments (78.1%). Except for cancer significant main health risks of tobacco consumption like cardio- and cerebrovascular diseases or COPD were hardly perceived. Further important reasons were an aesthetic aversion (38.6%), missing perception of a benefit (25.2%), and economic motives (20.8%). Girls and pupils from academic high schools named aesthetic motives significantly more often than boys and pupils from secondary schools respectively. A historical comparison shows that the motive "lacking benefit" reached a higher rank in our study than in the 1990s. CONCLUSIONS: When non-smokers are asked directly, extrinsic reasons (restrictions, smoking bans) do not seem to be relevant for them. Reasons concerning health, good physical shape, and beauty should be a central argument in the medical practise with young smokers.

Methicillin-resistant Staphylococcus aureus in a beauty salon, the Netherlands.

An outbreak of community-associated USA300 methicillin-resistant Staphylococcus aureus occurred in a beautician and 2 of her customers. Eight other persons, who were either infected (n = 5) or colonized (n = 3), were linked to this outbreak, including a family member, a household contact, and partners of customers.

Transmission of hepatitis B virus from a surgeon to his patients during high-risk and low-risk surgical procedures during 4 years.

OBJECTIVE: We investigated cases of acute hepatitis B in The Netherlands that were linked to the same general surgeon who was infected with hepatitis B virus (HBV). DESIGN: A retrospective cohort study was conducted of 1,564 patients operated on by the surgeon. Patients were tested for serologic HBV markers. A case-control study was performed to identify risk factors. RESULTS: The surgeon tested positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) with a high viral load. He was a known nonresponder after HBV vaccination and had apparently been infected for more than 10 years. Forty-nine patients (3.1%) were positive for HBV markers. Transmission of HBV from the surgeon was confirmed in 8 patients, probable in 2, and possible in 18. In the remaining 21 patients, the surgeon was not implicated. Two patients had a chronic HBV infection. One case of secondary transmission from a patient to his wife was identified. HBV DNA sequences from the surgeon were completely identical to sequences from 7 of the 28 patients and from the case of secondary transmission. The duration of the operation and the occurrence of complications during or after surgery were identified as independent risk factors. Although the risk of HBV infection during high-risk procedures was 7 times higher than that during low-risk procedures, at least 8 (28.6%) of the 28 patients were infected during low-risk procedures. CONCLUSIONS: Transmission of HBV from surgeons to patients at a low rate can remain unnoticed for a long period of time. Prevention requires a more stringent strategy for vaccination and testing of surgeons and optimization of infectious disease surveillance. Policies allowing HBV-infected surgeons to perform presumably low-risk procedures should be reconsidered.