Expert Elicitation Provides a Rapid Alternative to Formal Case-Control Study of an H7N9 Avian Influenza Outbreak in the United States.

An expert elicitation was staged to rapidly decipher plausible routes and risks of pathogen transmission in the 2017 H7N9 avian influenza (AI) outbreak in the four-state region of Tennessee, Alabama, Georgia, and Kentucky. The process included the identification of risk factors found in a preponderance of commercial broiler breeder case farms over matched controls and an opinion-based weighting of risks and mitigations perceived influential to this outbreak. Although the two highly pathogenic AI case farms had general location and company ownership in common, obvious connections were lacking for the remainder of H7N9-infected (all low pathogenicity) commercial farms. Expert elicitation of differences between known cases and controls suggested a key role for environmental rather than lateral (business network) pathways in the distribution of low pathogenicity AI across commercial broiler breeder operations. Factors with greatest strength as predictors of disease, whether or not they were causal, included mesopredator or rodent incursions, enclosure defects, and habitat disturbance that might attract wildlife to the farm (e.g., feed spills and vacating of neighboring properties). Business affiliations that may have facilitated farm-to-farm transfer, in contrast, were limited. Biosecurity standards varied across this study group but were no more or less stringent among cases over controls. However, results from a parallel hypothetical scenario staged to address field data gaps suggest that uniformity and consistency in the implementation of biosecurity practices may impact risk of disease introduction. Importantly, this study was conducted within a few weeks and with little disruption to emergency response activities. As such, the approach offers an alternative model for interim field investigation of new or emerging high-consequence diseases with immediate decision support needs.

Hypoxic-ischemic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.

Monocyte chemoattractant protein-1 (MCP-1) regulates monocyte accumulation in several macrophage-dependent experimental disease models. In the neonatal brain, activated microglia accumulate rapidly after hypoxic-ischemic injury. These cells produce potentially neurotoxic factors that may contribute to the progression of injury. To determine whether MCP-1 could be one of the molecular signals that influences the microglial response to hypoxic-ischemic injury in the neonatal brain, we examined the impact of acute hypoxic-ischemic injury on MCP-1 mRNA and protein expression. Seven-day-old rats underwent right carotid artery ligation, followed by 3 hours of 8% oxygen exposure, to elicit ipsilateral forebrain hypoxic-ischemic injury. To detect MCP-1 mRNA in situ hybridization assays were performed using 35S-labeled antisense riboprobes generated from rat MCP-1 cDNA. Animals were evaluated 0, 1, 2, 4, 8, 16, 24, 48, and 120 hours after hypoxic exposure (N > or = 3/group). Immunocytochemistry (with a polyclonal rabbit antirat MCP-1 antibody) was used to determine the anatomic and temporal distribution of MCP-1, in samples obtained 10 minutes to 5 days after hypoxic exposure (N > or = 3/group). Monocyte chemoattractant protein-1 mRNA was first detected in periventricular regions of the lesioned hemisphere 1 hour after hypoxia-ischemia; periependymal and intraparenchymal MCP-1 mRNA expression were detected at 4 hours; hybridization signal peaked at 8 to 24 hours; and no MCP-1 mRNA was detected at 48 and 120 hours. In lesioned forebrain, MCP-1 protein expression were consistently detected at 2.5 to 48 hours after hypoxia-ischemia. Many immunoreactive cells appeared to be neurons. These results suggest that in the developing brain, MCP-1 could represent a functionally important molecular signal for the microglial response to hypoxic-ischemic injury.

Hypoxic-ischemic injury acutely disrupts microtubule-associated protein 2 immunostaining in neonatal rat brain.

We evaluated the influence of an acute hypoxic-ischemic insult on the neuronal dendritic cytoskeletal protein microtubule-associated protein 2 (MAP2) in the immature rat brain. Studies were performed using a well-characterized perinatal rodent stroke model, in which unilateral ischemic forebrain injury was induced in 7-day-old rats by right carotid artery ligation, followed by 3 h exposure to 8% oxygen. Changes in the neuroanatomic distribution of MAP2 in the first 48 h after injury were evaluated by immunocytochemistry with a monoclonal mouse anti-MAP2 antibody. The distribution of MAP2 immunoreactivity was markedly disrupted in the lesioned hippocampus; prominent reductions in MAP2 immunostaining evolved concurrently in lesioned cortex, caudate and thalamus. These data demonstrate that at this developmental stage, MAP2 immunocytochemistry provides a sensitive indicator of the distribution and severity of hypoxic-ischemic neuronal injury.

PCR amplification and DNA sequencing of SRV-2 from archived tumor tissues.

Fibroproliferative tumors, known as retroperitoneal fibromatosis (RF) are predominantly associated with type D simian retrovirus serotype 2 (SRV-2) infections in pigtailed macaques (Macaca nemestrina) at the University of Washington Regional Primate Research Center (UWRPRC). In this report we examined genetic changes that occurred in SRV-2/Washington (SRV-2/WA) from archived RF tissues and from fresh tissue and lymphocytes. DNA was extracted from paraffin-embedded RF tumor tissues and was used to amplify a portion of the SRV-2/WA genome encoding an open reading frame, 3'orf. Since 3'orf is encoded in an alternate reading frame within the outer membrane glycoprotein (gp70) env gene, sequences of 3'orf also provide information on a portion of env gp70. To define the changes that have occurred in this portion of the viral genome, we compared the nucleotide and deduced amino acid sequences amplified from the various tumor tissues. The paraffin section PCR revealed that all samples containing amplifiable DNA harbored SRV-2/WA genomes. DNA sequence analysis showed that the 3'orf/gp70 portion of the SRV-2/WA genome was nearly identical in all tissues but was distinguishable from the prototype SRV-2/OR isolate from Celebes black macaques (M. nigra). A T-cell activating domain (Env residues 233-249) was 100% conserved in the SRV-2/WA isolates.

gp120, an HIV-1 protein, increases susceptibility to hypoglycemic and ischemic brain injury in perinatal rats.

Recent data suggest that gp120, a glycoprotein secreted by HIV-1-infected macrophages, is neurotoxic, and that toxicity is mediated, at least in part, by overactivation of NMDA-type excitatory amino acid receptors. In experimental animals, considerable evidence indicates that hypoglycemic and ischemic neuronal injury are mediated by endogenous excitatory amino acids. We hypothesized that in the presence of gp120 the severity of brain injury resulting from hypoglycemia and cerebral ischemia would increase. To test this hypothesis in vivo, we evaluated the influence of gp120 on the extent of brain injury resulting from these two clinically relevant pathophysiological insults in 7-day-old (P7) rats, the developmental stage of peak susceptibility to NMDA neurotoxicity. We compared the severity of hippocampal injury resulting from right intrahippocampal injections of gp120 (50 ng) in P7 rats rendered markedly hypoglycemic (n = 10) and in controls (n = 12). We also determined the influence of gp120 administration on the severity of hypoxic-ischemic injury, using a perinatal rat stroke model. P7 rats received intrahippocampal injections of gp120 (50 ng) (n = 23) or saline (n = 18) and then underwent right carotid ligation, followed by 2 h exposure to 8% oxygen. Brain injury was evaluated 5 days later, based on neuropathology evaluation and measurements of bilateral regional cross-sectional areas. The severity of hippocampal injury, based on cross-sectional area measurements, was considerably greater in animals from the hypoglycemic group than in litter-mate gp120-injected controls. Among the animals that underwent hypoxic-ischemic lesioning, the severity of injury, based on histopathology scoring and regional volume measurements, was considerably greater in animals that received gp120 than in those that received saline. These results provide support for the hypothesis that locally secreted HIV peptides, such as gp120, may potentiate the neurotoxicity of endogenous excitatory amino acid neurotransmitters in HIV-infected brain.

Characterization of infectious type D retrovirus from baboons.

Infectious virus resembling type D simian retrovirus (SRV) was isolated from Ethiopian baboons (Papio cynocephalus) (SRV-Pc) housed at the University of Washington Regional Primate Research Center. When baboon peripheral blood mononuclear cells (PBMC) or tissues were cocultured with the H-9 human T-cell line or the Raji human B-cell line, large multinucleated syncytia positive for SRV-2 antigens were observed microscopically. Immunoblot analysis of purified SRV-Pc from cell culture supernatants demonstrated that the viral core and envelope proteins reacted with rabbit anti-SRV-2 serum. Fresh PBMC and cocultured cells were positive by polymerase chain reaction using two different sets of SRV-2 primers. Preliminary sequence analysis of two separate isolates from portions of the SRV-Pc p27 and gp20 regions revealed homology with SRV-1, SRV-2, and Mason-Pfizer monkey virus. The homologies in the p27 segment were 91-94% and the homologies in the gp20 segment were 72-75%.