Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in mallard ducks.

The highly pathogenic (HP) influenza viruses H5 and H7 are usually nonpathogenic in mallard ducks. However, the currently circulating HP H5N1 viruses acquired a different phenotype and are able to cause mortality in mallards. To establish the molecular basis of this phenotype, we cloned the human A/Vietnam/1203/04 (H5N1) influenza virus isolate that is highly pathogenic in ferrets, mice, and mallards and found it to be a heterogeneous mixture. Large-plaque isolates were highly pathogenic to ducks, mice, and ferrets, whereas small-plaque isolates were nonpathogenic in these species. Sequence analysis of the entire genome revealed that the small-plaque and the large-plaque isolates differed in the coding of five amino acids. There were two differences in the hemagglutinin (HA) gene (K52T and A544V), one in the PA gene (T515A), and two in the PB1 gene (K207R and Y436H). We inserted the amino acid changes into the wild-type reverse genetic virus construct to assess their effects on pathogenicity in vivo. The HA gene mutations and the PB1 gene K207R mutation did not alter the HP phenotype of the large-plaque virus, whereas constructs with the PA (T515A) and PB1 (Y436H) gene mutations were nonpathogenic in orally inoculated ducks. The PB1 (Y436H) construct was not efficiently transmitted in ducks, whereas the PA (T515A) construct replicated as well as the wild-type virus did and was transmitted efficiently. These results show that the PA and PB1 genes of HP H5N1 influenza viruses are associated with lethality in ducks. The mechanisms of lethality and the perpetuation of this lethal phenotype in ducks in nature remain to be determined.

Changing epidemiology and ecology of highly pathogenic avian H5N1 influenza viruses.

The H5N1 virus currently circulating is continuing to evolve, and it has already resulted in the extension of its host and geographical range. It is likely that H5N1 will become a global problem for the poultry industry. How many of the recent H5N1 changes observed have been induced by changing patterns in poultry raising? A change in attitude on the use of high-quality vaccines is a change that would drastically help in the control of the current epidemic in the poultry industry. This article provides an overview of the changing properties that have been observed during the current H5N1 outbreaks.

Acute rejection risk in kidney transplant recipients on steroid-avoidance immunosuppression receiving induction with either antithymocyte globulin or basiliximab.

Immunosuppression with rapid discontinuation of corticosteroids, usually with induction therapy, is safe in kidney transplant recipients. In 89 patients, we induced immunosuppression with basiliximab or rabbit antithymocyte globulin (17 and 72 patients, respectively). Selection criteria for basiliximab were age (>or=65 years), history (malignancy; chronic infection), and type 1 diabetes mellitus (eligible for pancreas transplant). Steroids were administered through posttransplantation day 4 (five doses); maintenance immunosuppression was with tacrolimus and mycophenolate mofetil. At last follow-up (average, 286 days), most patients were steroid-free (antithymocyte globulin, 90%; basiliximab, 88%). Protocol biopsies were performed at 1, 4, and 12 months posttransplantation. The overall risk of biopsy-proven acute rejection was 12%. At 6 months posttransplantation, acute rejection-free survival was 93% for antithymocyte globulin, 65% for basiliximab (P<.001). Median time to biopsy-proven acute rejection was 27 and 71 days, respectively. The low incidence of biopsy-proven acute rejection with steroid-avoidance immunosuppression may be further reduced with antithymocyte globulin.

Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia?

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia.

Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the "Trojan horse" of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase.

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of the mouse alpha 1-acid glycoprotein genes (AGP-1 and AGP-2) during inflammation and aging.

In this study we investigated the expression of the Balb/c mouse alpha 1-acid glycoprotein genes. Mice, like humans, have two distinct alpha 1-acid glycoprotein mRNAs. As in humans and rats, mouse alpha 1-acid glycoprotein is a strong acute-phase reactant and its expression can be induced by acute-phase stimulatory agents such as bacterial lipopolysaccharides. Southern analysis and partial sequencing of different alpha 1-acid glycoprotein genomic clones indicated the existence of three distinct alpha 1-acid glycoprotein genes in the Balb/c genome. Using oligonucleotide hybridization, we showed that two of the three genes were expressed while the third gene was either not expressed or expressed at extremely low levels. The mRNA levels for the two expressed genes, alpha 1-acid glycoprotein-1 and alpha 1-acid glycoprotein-2, were both induced during the acute-phase response. However, alpha 1-acid glycoprotein-2 mRNA was present in at least 10-fold higher levels in both induced and uninduced mice. There were also differences in the developmental patterns of the two mRNAs in that the constitutive alpha 1-acid glycoprotein-1 mRNA levels increased 20-fold between 2 and 7 months, while alpha 1-acid glycoprotein-2 mRNA pools remained constant. During the acute-phase response in aged animals, there was an increase in the time required for both mRNAs to respond, and the maximum induced level of both mRNAs decreased. These studies set the stage for future experiments to determine the mechanisms by which the different alpha 1-acid glycoprotein genes are regulated during the acute-phase response and how aging affects these regulatory processes.

The effect of aging on constitutive mRNA levels and lipopolysaccharide inducibility of acute phase genes.

Eukaryotic organisms possess natural defense processes associated with their response to injury, inflammation and pollutants. One of these, the acute phase (AP) host response, is characterized by a series of hepatic physiological reactions triggered by factors released as a result of bacterial infection, inflammation or tissue injury and is believed to be the mechanism by which cells and tissues are protected against further damage and injury. The capacity to respond to these physiological insults is known to be affected by aging. We propose that the AP response represents a series of intrinsic processes and interactions that may be affected by aging. Furthermore, we propose that this may be due to the progressive failure of the acute phase response. In this study we examine the relationship between aging and the expression of both positive and negative acute phase reactants, i.e., acute phase serum proteins whose levels are increased or decreased in response to systemic injury and infection. The mRNA levels of the positive acute phase reactants, alpha 1-acid glycoprotein (AGP), alpha 1-antitrypsin (AT), and the negative acute phase reactant, albumin were measured in both normal and inflammation-induced mice of ages 2, 7, 12, and 24 months. A significant decrease in the constitutive levels of AT and albumin mRNAs occurred as a function of increased age. Furthermore, aging decreased the ability of the AGP and albumin genes to respond to inflammation. Our studies indicate that aging may affect the transcription of these genes, processing of their mRNA or stability of the mRNA levels.

Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis.

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.