Structural and functional homology among chicken, duck, goose, turkey and pigeon interleukin-8 proteins.

Interleukin (IL)-8-encoding regions of five avian species were cloned, sequenced and characterized. Each IL-8-encoding region is 312 nucleotides long and encodes IL-8 which is 103 amino acids. Pairwise sequence analysis showed that sequence identities of IL-8-encoding regions ranged from 87% to 100%. The IL-8 protein identities varied from 84% to 100%. Phylogenetic analysis indicated that IL-8-encoding regions and encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from binding reactivities of antiserum against each recombinant IL-8 (rIL-8) protein to homologous or heterologous rIL-8 proteins, chemotactic activities of each rIL-8 protein or reduction levels of the chemotactic activity of rIL-8 protein which was pretreated with homologous or heterlogous antiserum have suggested that all five IL-8 proteins were functionally active, and shared structural and functional identity with each other.

Characterization of interleukin-1beta mRNA expression in chicken macrophages in response to avian reovirus.

Inhibitors of viral disassembly or RNA and protein synthesis, viral disassembly intermediates (infectious subviral particles, ISVP), binary ethylenimine-inactivated virions, and viral particles lacking genomic double-stranded (ds) RNA (empty particles) were used to assess the expression of interleukin-1beta (IL-1beta) mRNA in chicken (chIL-1beta) macrophages in response to avian reovirus. The results demonstrate that two distinct expression patterns of chIL-1beta mRNA mediated by different steps in viral replication were found. Viral disassembly was required for the induction of a rapid, transient expression pattern of chIL-1beta mRNA that was rapidly induced at 30 min, with maximal levels reached by 2 h, and fell to a low level within 6 h post-inoculation, while viral RNA synthesis rather than protein translation, which was subsequent to membrane penetration, was required to induce a stable, sustained expression pattern of chIL-1beta mRNA that occurred at and after 6 h post-inoculation. In addition, the induction of chIL-1beta mRNA expression by the empty particles and ISVP was extremely weak, compared with the active dsRNA(+) virions or binary ethylenimine-inactivated virions, suggesting that the presence of dsRNA, even if transcriptionally inactive, may be an important factor in this response.

Avian reovirus core protein muA expressed in Escherichia coli possesses both NTPase and RTPase activities.

Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.

The sequence and phylogenetic analysis of avian reovirus genome segments M1, M2, and M3 encoding the minor core protein muA, the major outer capsid protein muB, and the nonstructural protein muNS.

The sequences and phylogenetic analyses of the M-class genome segments of 12 avian reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein muA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 avian reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein muB or muNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5' GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5' GCUUUUU terminal motif which is common to other known avian reovirus genome segments. The UCAUC 3'-terminal sequences of the M-class genome segments are shared by both avian and mammalian reoviruses. Noncoding regions of both 5'- and 3'-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced mu-class protein sequence identities indicated that the M2 genes and the muB proteins have the greatest level of sequence divergence. Computer searches revealed that the muA possesses a sequence motif (NH(2)-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458-464) indicative of N-6 adenine-specific DNA methylase. Examination of the muB amino acid sequences indicated that the cleavage site of muB into muBN and muBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment delta and the C-terminal fragment phi were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of muB by determining the amino-terminal sequences of phi proteins: between arginine 582 and glycine 583. The predicted length of delta generated from muBC is very similar to that of delta generated from mammalian reovirus mu1C. Taken together, protein muB is structurally, and probably functionally, similar to its mammalian homolog, mu1. In addition, two regions near the C-terminal and with a propensity to form alpha-helical coiled-coil structures as previously indicated are observed for each protein muB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I-V consist of a mixture of viruses from the M1 and M3 genes of lineages I-III, reflecting frequent reassortment of these genes among virus strains.

Antibody responses against avian reovirus nonstructural protein sigmaNS in experimentally virus-infected chickens monitored by a monoclonal antibody capture enzyme-linked immunosorbent assay.

Crude antigen preparations from avian reovirus (ARV)-infected chicken embryo fibroblasts (sigmaNS) or from bacterially expressed protein sigmaNS (esigmaNS) were captured by monoclonal antibody 1E1(MAb 1E1) against ARV nonstructural protein sigmaNS immobilized on the ELISA plates and were used as the MAb capture ELISA for antibody detection. Sixty one-week-old specific pathogenic free (SPF) chickens were divided into six groups and were vaccinated with live or inactivated ARV vaccine preparations in different combinations or inoculated with a virulent ARV strain. Sera collected from the birds were tested for their antibody responses to ARV nonstructural protein sigmaNS. Using the MAb capture ELISAs, the level of nonspecific binding reactions was tested on the serum samples obtained weekly from mock-infected SPF chickens from 1 to 25 weeks and compared to the results tested by the conventional ELISA. The results indicated that both MAb capture ELISAs had lower nonspecific bindings than those in the conventional ELISA, even in older birds. Antibody responses against ARV sigmaNS of the birds which received the inactivated vaccine twice (group I), inactivated vaccine followed by a live vaccine (group II), or a live vaccine followed by boosting with an inactivated vaccine (group III) were detected by MAb captured ELISA with sigmaNS crude antigens. The absorbance values increased rapidly at 1-2 weeks after boosting, approximated a peak at 5-6 weeks of age, and maintained this throughout the length of the experiment. The absorbance values of the MAb capture ELISA showed a good correlation to the SN titers ( r value > 0.85). On the other hand, serum samples from the birds which received the live vaccine twice (group IV) or were inoculated with a virulent ARV (group V) did not show antibody responses to sigmaNS, similar to those from the mock-infected birds (group VI), as the absorbance values maintained at a low level (below 0.5) throughout the length of the experiment. Similar results were obtained in the sera detected by MAb capture ELISA with crude esigmaNS antigens, except that the absorbance values in the sera from the birds in group III were gradually increased and later approximated a peak at 11 weeks of age and maintained this throughout the length of the experiments. The results suggest that MAb capture ELISAs can be readily used to detect antibody responses of the birds against ARV nonstructural protein sigmaNS which may reflect an immune status of a chicken flock, receiving ARV vaccine as long as including an inactivated vaccine.

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli.

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.