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An epidemic of Severe Acute Respiratory Syndrome (SARS) led to the identification of an associated coronavirus, SARS-CoV. This virus evades the host innate immune response in part through the expression of its non-structural protein (nsp) 1, which inhibits both host gene expression and virus- and interferon (IFN)-dependent signaling. Thus, nsp1 is a promising target for drugs, as inhibition of nsp1 would make SARS-CoV more susceptible to the host antiviral defenses. To gain a better understanding of nsp1 mode of action, we generated and analyzed 38 mutants of the SARS-CoV nsp1, targeting 62 solvent exposed residues out of the 180 amino acid protein. From this work, we identified six classes of mutants that abolished, attenuated or increased nsp1 inhibition of host gene expression and/or antiviral signaling. Each class of mutants clustered on SARS-CoV nsp1 surface and suggested nsp1 interacts with distinct host factors to exert its inhibitory activities. Identification of the nsp1 residues critical for its activities and the pathways involved in these activities should help in the design of drugs targeting nsp1. Significantly, several point mutants increased the inhibitory activity of nsp1, suggesting that coronaviruses could evolve a greater ability to evade the host response through mutations of such residues.

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Heterophils from two pure lines (A and B) of commercial broiler chickens were isolated on days 1, 4, and 7 post-hatch to evaluate their ability to (1) phagocytose Salmonella enteritidis (SE) (2) degranulate when exposed to immune-IgG opsonized SE, and (3) produce an oxidative burst. On days 1 and 4, heterophils from line A were functionally more efficient compared to heterophils from line B (p<0.05). By 7 days post hatch, heterophil functions for both lines were comparable. To further study the inheritance of heterophil functional efficiency, F1 reciprocal crosses (line C=male Bxfemale A; line D=male Axfemale B) were evaluated for functional activity and compared with the immunologically efficient (A) and non-efficient (B) parent lines. Heterophils from line D had a more efficient heterophil function (p<0.05) when compared to heterophils from C. These results suggest that heterophil function and efficiency can be genetically transferred to progeny. Moreover they indicate that heterophil function is sex-associated and genetically controlled by the rooster since progeny of line A males maintained immunologically efficient characteristics whereas heterophils from the progeny of line B roosters remained immunologically inefficient. To our knowledge, this is the first report to describe a functional relationship between pure and F1 reciprocal crosses of broiler chickens with regard to heterophils and the innate immune response.

The activation of signal transduction pathways is required for the expression of functional enhancement of cellular activities. In the present studies, initial attempts were made to identify the signal transduction factors involved in activating phagocytosis, generation of an oxidative burst, and degranulation by heterophils isolated from neonatal chickens in response to opsonized Salmonella enteritidis (opsonized SE). Peripheral blood heterophils were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (staurosporin, genistein, or verapamil) or 120 min (pertussis toxin) at 39 degrees C. The cells were then stimulated for 30 min at 39 degrees C with opsonized SE. Phagocytosis, luminol-dependent chemoluminescence (LDCL), and beta-D glucuronidase release were then evaluated in vitro. The G-protein inhibitor pertussin toxin markedly inhibited (>80%) phagocytosis of opsonized SE. Both the protein kinase inhibitor (staurosporin) and calcium channel inhibitor (verapamil) reduced phagocytosis in a dose response manner. Genistein, a tyrosine kinase inhibitor, had no effect on phagocytosis. Staurosporin had a marked inhibitory effect on LDCL (>90%) while genistein had a dose responsive inhibition on LDCL. Both verapamil (40-45%) and pertussin toxin (50-55%) had a statistically significant, but less biologically significant effect on LDCL. Genistein significantly reduced the degranulation (78-81%) of heterophils by opsonized SE. Staurosporin also reduced degranulation by 43-50%, but neither verapamil nor pertussis toxin had a significant effect on degranulation. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of the functional activities of avian heterophils. Pertussin toxin-sensitive, Ca++-dependent G-proteins appear to regulate phagocytosis of opsonized SE, protein kinase C-dependent, tyrosine kinase-dependent protein phosphorylation plays a major role in LDCL, and tyrosine kinase(s)-dependent phosphorylation regulates primary granule release.