Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: a putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif

The genome segment B sequence of infectious pancreatic necrosis virus was determined for both the Jasper and Sp serotypes. The sequences are 2784 and 2630 bp long, respectively, and contain a single large open reading frame encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp) of IPNV. The proteins exhibit an 88% homology with each other, but only 41% with infectious bursal disease virus (IBDV) VP1, another member of the Birnaviridae. Despite the low overall homology between the IPNV and IBDV VP1 proteins, homologous regions were detected within the central portion of the proteins. The carboxy-proximal regions of the VP1, which contain very low amino acid homology, displayed evidence of conservation in structural features such as a hydrophilic, highly basic domain. Consensus sequences associated with GTP-binding proteins and RdRps were also detected in VP1. However, unlike the RdRps associated with single-stranded plus RNA viruses, the birnavirus RdRp lacks the Gly-Asp-Asp motif characteristic of this enzyme family.     

Identification of bent DNA and ARS fragments in the genome of Choristoneura fumiferana nuclear polyhedrosis virus.

We identified four CfMNPV DNA fragments with autonomously replicating sequences (ARS) functional in Saccharomyces cerevisiae. A 0.9-kb fragment which, mapped to 54.5 to 55.3 map units within EcoRI HI of the CfMNPV genome, showed the strongest ARS activity of the four. Sequence analysis of this 0.9-kb DNA segment revealed an A+T-rich region separated from a G+C-rich region by 320 bp. Although no sequence matched exactly the ARS core-consensus sequence, 13 near-matches differing by only one or two nucleotides from the core-consensus sequence, were identified. Ten near-matches were clustered within a 105-bp A+T-rich region, and were arranged as inverted repeats. A section of bent DNA structure was predicted within this region. The bent DNA, which showed temperature-dependent retardation during polyacrylamide gel electrophoresis, was unique as its sequence was arranged as a symmetrical 'tilde' (approximately) structure. The second (1.0 kb) and third (1.6 kb) ARS-bearing fragments mapped within EcoRI-E and -B fragments which contain homologous repeat sequences. The fourth (1.5 kb) fragment had the weakest ARS activity and mapped to the EcoRI-D or -B regions of the genome.     

Komplementäre Therapien in der Onkologie

Die Gynäkologie - Komplementärmedizin wird im Sinne einer integrativen Onkologie ergänzend zur konventionellen Medizin eingesetzt und grenzt sich damit ab von der sog....     

Reuse of Disposable Isolation Gowns in Rodent Facilities during a Pandemic

Reuse of disposable personal protective equipment is traditionally discouraged, yet in times of heightened medical applications such as the SARS CoV-2 pandemic, it can be difficult to obtain. In this article we examine the reuse of disposable gowns with respect to still providing personnel protection. XR7, a fluorescent powder, was used to track contamination of gowns after manipulation of rodent cages. Mouse cages were treated with XR7 prior to manipulations. Disposable gowns were labeled for single person use and hung in common procedure spaces within the vivarium between usages. A simulated rack change of 140 cages was completed using XR7-treated cages. One individual changed all cages with a break occurring after the first 70 cages, requiring the gown to be removed and reused once. To simulate research activities, 5 individuals accessed 3 XR7-treated cages daily for 5 d. Each mouse in the XR7-treated cages was manipulated at least once before returning cages to the housing room. Disposable gowns were reused 5 times per individual. Gowns, gloves, clothing, bare arms, and hands were scanned for fluorescence before and after removing PPE. Fluorescence was localized to gloves and gown sleeves in closest contact with animals and caging. No fluorescence was detected on underlying clothing, or bare arms and hands after removing PPE. Fluorescence was not detected in procedure spaces where gowns were hung. The lack of fluorescence on personnel or surfaces indicate that gowns can be reused 1 time for routine husbandry tasks and up to 5 times for research personnel. A method for decontamination of used gowns using Vaporized Hydrogen Peroxide (VHP) was also validated for use in areas where animals are considered high risk such as quarantine, or for fragile immunocompromised rodent colonies.

The development of allergies is a common risk associated with handling laboratory animals.^1,4 A report by the Division of Occupational Health and Safety (OSHA) states that 1 out of every 3 to 5 individuals who work with laboratory animals will develop allergic symptoms, and that the best approach to reducing the development of allergies is to eliminate or minimize exposure through the use of engineering controls and PPE such as masks, gowns and gloves.⁴ PPE when used in combination with engineering controls, such as the use of biosafety cabinets (BSCs), increased room ventilation rates, and individually ventilated caging (IVC) systems, helps provide an extra barrier for personnel from hazards such as allergens. When hazards such as allergens cannot be controlled by other means, PPE serves as the last means of protecting personnel from exposure to laboratory animal allergens (LAA).¹¹ According to a national survey of 198 organizations, the most commonly used PPE items to control exposure were uniform/clothing covers and gloves.¹⁵ This further solidifies the importance of PPE items such as isolation gowns, in minimizing personnel exposure to LAA and further confirms the need to maintain a supply of gowns for personnel use. When considering the reuse of disposable gowns, it is important to determine if the item can provide an effective barrier and minimize personnel exposure to LAA.Current requirements for animal care technicians at this institution include changing into scrubs prior to working in the animal vivarium, with minimum PPE requirements for entering a sterile or barrier housing room being a disposable gown, surgical facemask, hair bonnet, and gloves.⁹ Research staff are not required to change into scrubs but are held to the same minimum PPE requirements. Scrubs for animal care technicians are donned in a common use locker room outside of the animal facility. PPE is not required to enter the vivarium but is required before entering the animal housing spaces. PPE is typically donned before entering the animal housing spaces directly outside of the animal room door. Disposable PPE is required to be discarded after a single use and work uniforms worn by animal care technicians are required to be removed prior to leaving and laundered at the facility as another measure to minimize exposure to LAA. All barrier rodent housing rooms and procedure rooms are ventilated rooms with a minimum of 10 to 15 air exchanges and use engineering controls such as BSCs when handling rodents. For these studies, the air changes per hour (ACH) in procedure spaces (n = 20) were measured at 44.38 ± 0.9 ACH and at 14.3 ± 0.2 ACH in animal rooms (n = 39). In addition, 98% of rodent housing rooms use IVC systems which have been proven to minimize allergen exposure and decrease health risks among personnel.^6,7 Studies have also shown that building ventilation in conjunction with the use of IVC systems function to reduce disease spread within animal populations.⁵ The use of different engineering controls within our facilities made the reuse of PPE items such as disposable gowns a viable option without compromising personnel or animal health.Recent global impacts of the novel coronavirus SARS-CoV-2 (COVID-19) have resulted in shortages and significant price increases for single-use PPE items such as isolation gowns, surgical face masks and eye protection.¹³ Isolation gowns accounted for the largest expenditure in the PPE budget at our institution, indicating the significance of this item for working in the animal facility.⁹ A recent inhouse survey on the utilization of PPE by facility staff, performed prior to the COVID-19 pandemic and this study, found that each animal care technician (n = 26) used an average of 5.7 gowns per day, while each veterinary technician (n = 5) used an average of 4.9 gowns per day. This further supports the importance and need to maintain a supply of isolation gowns for staff use. Prior to COVID-19, gown expenditure was $63.90 per day for staff including both animal care and veterinary technicians. Current expenditure for disposable gowns using the average gown usage for facility staff prior to COVID-19 and current prices for isolation gowns, equates to $369.58 per day, a 4-fold increase. The high rate of use for disposable gowns, in combination with current shortages and high prices, prompted the decision to reevaluate gown use requirements in the rodent vivarium. The goal of this study was to develop practices to prevent a lapse in supply while continuing to protect personnel from contaminants such as LAA. Current CDC guidelines for optimizing supplies of isolation gowns recommend strategies such as alternatives, reusable cloth, and reuse of disposable gowns.¹²This study was designed to evaluate the disposable isolation gown’s ability to provide protection to personnel from exposure to contaminants, such as LAA, after multiple uses. This process requires an assessment of the level and distribution of contamination after use, and during storage between uses. XR7 contamination powder, a nontoxic, inexpensive, fluorescent powder was used to simulate possible contaminants such as LAA. Fluorescent dyes and powders, such as XR7, are used frequently in hospital and medical settings as a means of demonstrating environmental contamination due to its ease of use and ability to simulate contaminants on different surfaces.⁹Finally, in collaboration with another institution, this study tested a method to decontaminate isolation gowns using Vaporized Hydrogen Peroxide (VHP) and thereby to extend the life of single use gowns used in areas for restricted animal populations. Decontamination of used isolation gowns can be considered for those facilities that wish to extend the reuse period in areas where animals are considered high risk, such as quarantine, or for fragile immunocompromised rodent colonies. Decontamination may also allow the reuse of gowns in specific situations working with particular “restricted” mouse populations such as Corynebacterium bovis, and murine norovirus negative animals.Given a strict barrier facility, with increased air exchanges, ventilated caging, and the use of BSCs when performing animal use activities, we hypothesized that extending the use of disposable isolation gowns would have minimal impact on exposure of personnel to particulates generated during manipulation of rodent cages. We also hypothesize that extending the use of disposable isolation gowns will have a substantial impact on PPE expenditure, which will help facilities to continue to purchase PPE items such as disposable isolation gowns, on limited budgets.