Showing papers by "Erica Spackman published in 2006"

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

Abstract: We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR.

Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus.

Abstract: High-pathogenicity (HP) avian influenza (AI) virus of the H5N1 subtype has caused an unprecedented epizootic in birds within nine Asian countries/regions since it was first reported in 1996. Vaccination has emerged as a tool for use in managing the infection in view of future eradication. This study was undertaken to determine whether two divergent H5N2 commercial vaccine strains, one based on a European and the other a North American low-pathogenicity AI virus, could protect chickens against a recent Asian H5N1 HPAI virus. The North American and European vaccine viruses had 84 and 91% deduced amino acid sequence similarity to the HA1 segment of haemagglutinin protein of Indonesia H5N1 HPAI challenge virus, respectively. Both vaccine strains provided complete protection from clinical signs and death. The vaccines reduced the number of chickens infected and shedding virus from the respiratory and intestinal tracts at the peak of virus replication. In addition, the quantity of virus shed was reduced by 10(4) to 10(5) median embryo infectious doses. The use of specific neuraminidase inhibition tests allowed identification of infected chickens within the vaccinated groups. These data indicate that the currently available H5 vaccines of European and North American lineages will protect chickens against the Asian H5N1 HPAI virus and reduce environmental contamination by the H5N1 HPAI virus. They will be an adjunct to biosecurity measures to reduce virus transmission.

Molecular characterization and typing of chicken and turkey astroviruses circulating in the United States: implications for diagnostics.

Abstract: Avian astroviruses were detected by reverse transcriptase and polymerase chain reaction in intestinal contents collected from commercial chickens and turkeys from throughout the United States from 2003 through 2005. Astroviruses were detected in birds from both healthy and poorly performing flocks with or without enteric disease. Phylogenetic analysis was performed with sequence data from the polymerase (ORF-1b) genes of 41 turkey-origin astroviruses and 23 chicken-origin astroviruses. All currently available avian astrovirus sequence data and selected mammalian astrovirus sequence data were included in the analysis. Four groups of avian astroviruses were observed by phylogenetic analysis: turkey astrovirus type 1 (TAstV-1)-like viruses, turkey astrovirus type 2 (TAstV-2)-like viruses, both detected in turkeys; avian nephritis virus (ANV)-like viruses, detected in both chickens and turkeys; and a novel group of chicken-origin astroviruses (CAstV). Among these four groups, amino acid identity was between 50.1% and 73.8%, and was a maximum of 49.4% for all avian isolates when compared with the mammalian astroviruses. There were multiple phylogenetic subgroups within the TAstV-2, ANV, and CAstV groups based on 9% nucleotide sequence divergence. Phylogenetic analysis revealed no clear assortment by geographic region or isolation date. Furthermore, no correlation was observed between the detection of a particular astrovirus and the presence of enteric disease or poor performance. Based on these data, a revision of the present taxonomic classification for avian astroviruses within the genus Avastrovirus is warranted.

H7N3 Avian Influenza Virus Found in a South American Wild Duck Is Related to the Chilean 2002 Poultry Outbreak, Contains Genes from Equine and North American Wild Bird Lineages, and Is Adapted to Domestic Turkeys

Abstract: An H7N3 avian influenza virus (AIV) was isolated from a Cinnamon Teal (Anas cyanoptera) (A/ CinnamonTeal/Bolivia/4537/01) during a survey of wild waterfowl in Bolivia in 2001. The NA and M genes had the greatest identity with North American wild bird isolates, the NS was most closely related to an equine virus, and the remaining genes were most closely related to isolates from an outbreak of H7N3 in commercial poultry in Chile in 2002. The HA protein cleavage site and the results of pathogenesis studies in chickens were consistent with a low-pathogenicity virus, and the infective dose was 10 5 times higher for chickens than turkeys. Prior to an outbreak of avian influenza virus (AIV) in commercial chickens and turkeys in Chile in 2002 (7), the detection of AIV had not been reported in South America. Importantly, commercial poultry is routinely monitored for AIV to comply with export regulations; however, surveillance of wild aquatic birds for AIV in South America has historically been minimal. Prior to the outbreak in Chile, a study was conducted in Bolivia in 2001. A total of 93 samples (24 from Cinnamon Teal) were collected from 11 species, including ducks, sheldgeese, and doves. One AIV was isolated from a Cinnamon Teal (Anas cyanoptera), collected on 27 October 2001 (UAM 19,003: Bolivia, Dpto. La Paz, Lake Titicaca [16°1145S, 68°3728W]; elevation, 3,808 m). This isolate represents the first report and earliest isolate of an AIV from a wild bird in South America. Here we evaluate the genetics and pathobiology of this isolate. Sample collection and initial screening for AIV. Cloacal swabs were collected from wild waterfowl, placed in brain heart infusion broth, frozen in liquid nitrogen, and shipped frozen to SEPRL for processing (samples were not processed immediately for logistical reasons). RNA was extracted from cloacal swab material with TRIzol LS reagent (Invitrogen, Inc., Carlsbad, CA) in accordance with the manufacturer’s instructions. RNA was tested for AIV by real-time reverse transcription-PCR (rRT-PCR) directed to the matrix (M) gene, which detects all type A influenza viruses as previously reported (4). Virus isolation was performed in embryonated chicken eggs by using standard procedures (9) with swab material from rRTPCR-positive samples. Genetic analysis. The entire coding sequences of all eight viral gene segments were amplified by RT-PCR as previously reported (6) and directly sequenced with the BigDye termina

Phylogenetic analysis of Turkey astroviruses reveals evidence of recombination.

Abstract: Sequence data was obtained from the capsid (ORF-2) and the polymerase (ORF-lb) genes of 23 turkey astrovirus (TAstV) isolates collected from commercial turkey flocks around the United States between 2003 and 2004. A high level of genetic variation was observed among the isolates, particularly in the capsid gene, where nucleotide sequence identity among them was as low as 69%. Isolates collected on the same farm, on the same day, but from different houses could have as little as 72% identity between their capsid gene sequences when compared. Phylogenetic analysis of the capsid gene revealed no clear assortment by geographic region or isolation date. The polymerase gene was more conserved with between 86 and 99% nucleotide identity and did assort in a geographic manner. Based on differing topologies of the capsid and polymerase gene phylogenetic trees, TAstV appears to undergo recombination.

Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Abstract: Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.