Engineered female-specific lethality for control of pest Lepidoptera.

The sterile insect technique (SIT) is a pest control strategy involving the mass release of radiation-sterilized insects, which reduce the target population through nonviable matings. In Lepidoptera, SIT could be more broadly applicable if the deleterious effects of sterilization by irradiation could be avoided. Moreover, male-only release can improve the efficacy of SIT. Adequate methods of male-only production in Lepidoptera are currently lacking, in contrast to some Diptera. We describe a synthetic genetic system that allows male-only moth production for SIT and also replaces radiation sterilization with inherited female-specific lethality. We sequenced and characterized the doublesex (dsx) gene from the pink bollworm (Pectinophora gossypiella). Sex-alternate splicing from dsx was used to develop a conditional lethal genetic sexing system in two pest moths: the diamondback moth (Plutella xylostella) and pink bollworm. This system shows promise for enhancing existing pink bollworm SIT, as well as broadening SIT-type control to diamondback moth and other Lepidoptera.

Multiplex detection and SNP genotyping in a single fluorescence channel.

Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR.

Practical high pathogenicity avian influenza first-response training exercises.

The response to an avian influenza outbreak, especially highly pathogenic avian influenza (HPAI), should focus on four basic principles: 1) protect humans first, 2) protect animals 3) contain the virus, and 4) ensure that the outbreak remains a single event, by preparing response teams to work together effectively through advance training. The Stamping Out Pandemic and Avian Influenza (STOP AI) project is a U.S. Agency for International Development-funded global activity. STOP AI has designed and conducted practical, experiential training exercises that engage participants in simulated experiences that enhance their confidence and ability to apply these principles during a real HPAI outbreak. This article describes three specific exercises: 1) wearing and removing personal protective equipment (PPE) in a controlled environment, 2) site zoning, and 3) a planning and resource mapping exercise staged in Poultopia--a fictional region in a developing country. The PPE activity emphasizes the physical challenges of working in full PPE and the importance of proper equipment removal. In the zoning exercise, response teams focus on the areas and the tasks required by setting up a clean area, transit corridor, infected/culling area, and nontransit areas at a farm, village, or other location. In Poultopia, participants must determine where surveillance should occur, decide where roadblocks should be placed during an outbreak, choose which birds to cull first and determine how to dispose of them safely, ascertain the types of personnel and equipment needed, and assess timing issues. The Poultopia scenarios are adapted to the conditions of the region where the training takes place, thus adding to their realism and utility. The practical techniques described here have been taught successfully through STOP AI in more than 30 countries in Europe, Asia, and Africa.

Antigenic and genomic characterization of turkey enterovirus-like virus (North Carolina, 1988 isolate): identification of the virus as turkey astrovirus 2.

A small round virus (SRV) was isolated in 1988 from droppings of enteritis-affected turkeys in North Carolina and tentatively identified as an enterovirus on the basis of size (18-24 nm in diameter), intracytoplasmic morphogenesis, and a single-stranded RNA genome of approximately 7.5 kb. Additional characterization studies based on antigenic and genomic analyses were done to determine the relationship of this turkey enterovirus-like virus (TELV) to turkey astrovirus 2 (TAstV2), a recently characterized SRV of turkeys. Cross-immunofluorescence studies with TELV- and TAstV2-specific antisera indicated a close antigenic relationship between these viruses. TELV RNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with oligonucleotide primers specific for TAstV2 polymerase gene (open reading frame [ORF] 1b) and capsid protein gene (ORF 2). Subsequent sequence analyses of these TELV-derived RT-PCR products indicated a high degree of similarity with polymerase gene (98.8%) and capsid gene (96.9%) of TAstV2. These studies definitively identify TELV (North Carolina, 1988 isolate) as TAstV2.