Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

EXtensive nucleotide sequence analysis of the 5' and the 3' terminal of the RNA segments of the genome of fowl plague virus, an avian strain of influenza virus, confirms the presence of a common sequence at the 5' terminus of each segment and a common sequence at the 3' terminus of each segment. Between the ends of each individual segment there is a complementary sequence which may be important in the control of transcription and replication of the genome. In addition, the probable sites of initiation of translation of fowl plague virus mRNA are indicated along with the corresponding NH2-terminal amino acid sequences of the virus polypeptides.

Avian influenza virus strains representing most hemagglutinin (HA) subtypes were compared with human influenza A (H1N1,H3N2) and B virus isolates, including those with no history of passaging in embryonated hen's eggs, for their ability to bind free N-acetylneuraminic acid (Neu5Ac) and sialylollgosaccharides in a competitive binding assay and to attach to gangliosides in a solid-phase adsorption assay. The avian viruses, irrespective of their HA subtype, showed a higher affinity for sialyl-3-lactose and the other Neu5Ac2-3Gal-terminated oligosaccharides and a lower affinity for sialyl-6-lactose than for free Neu5Ac, indicative of specific interactions between the HA and the 3-linked Gal and poor accommodation of 6-linked Gal in the avian receptor-binding site (RBS). Human H1 and H3 strains, by contrast, were unable to bind to 3-linked Gal, interacting instead with the asialic portion of sialyl-6-(N-acetyllactosamine). Different parts of this moiety were recognized by H3 and H1 subtype viruses (Gal and GlcNAc, respectively). Comparison of the HA amino acid sequences revealed that residues in positions. 138, 190, 194, 225, 226, and 228 are conserved in the avian RBS, while the human HAs harbor substitutions at these positions. A characteristic feature of avian viruses was their binding to Neu5Ac2-3Gal-containing gangliosides. This property of avian precursor viruses was preserved in early human H3 isolates, but was gradually lost with further circulation of the H3 HA in humans. Consequently, later human H3 isolates, as well as H1 and type B human strains, were unable to bind to short Neu5Ac2-3Gal-terminated gangliosides, an incompatibility that correlated with higher glycosylation of the HA globular head of human viruses. Our results suggest that the RBS is highly conserved among HA subtypes of avian influenza virus, while that of human viruses displays distinctive genotypic and phenotypic variability.

Antigenic changes are often induced in the haemagglutinin (HA) of human isolates of influenza A(H1N1) and B viruses during their isolation and propagation in embryonated hens' eggs. In this report we describe molecular changes in the HA of an A(H1N1) virus which accompany egg adaptation. The HA1 amino acid substitutions of egg-adapted variants are located in the vicinity of the receptor binding site. This suggests that egg-adapted variants are selected on the basis of altered receptor site specificity with concomitant effects on the antigenic configuration of the HA molecule. In this study, at least three antigenically distinct groups of egg-adapted variants were observed. These observations have implications for the indiscriminate use of egg-adapted viruses in sero-epidemiological studies and vaccine production.

Polyadenylated transcripts of influenza virus RNA are incomplete copies of the individual genome segments, lacking sequences complementary to the 5'-terminal nucleotides of the virion RNA. By using a procedure which depends on the polyadenylic acid tail of the mRNA being encoded in part by the genome, we have determined that the common tract of uridine residues, approximately 17 to 22 nucleotides from the 5' end of each segment, is the site of polyadenylation of influenza virus mRNA.

In 1943 Burnet reported on changes in the hemagglutinating properties of human influenza virus which occurred during adaptation of the virus to growth in chicken eggs. Only recently has direct evidence been presented that these changes affect the antigenic properties of the virus. Schild et al.(G. C. Schild, J. S. Oxford, J. C. deJong, and R. G. Webster (1983), Nature (London) 303, 706-709) demonstrated that egg adaptation of influenza B virus selects variants which are antigenically distinct from virus grown from the same source in mammalian cells. The molecular changes in the hemagglutinin (HA) of influenza B virus associated with adaptation to growth in eggs have now been identified. A specific glycosylation site at the distal tip of the HA of influenza B virus grown exclusively in mammalian cell culture is lost or altered during egg adaptation. Since the HA functions in adsorption of virus to cells, it is concluded that removal or modification of an oligosaccharide structure at this position is required for influenza B virus to attach to and infect the allantois cells of the egg and that this has important implications for the antigenic configuration of the molecule.

Synthetic sialylglycoconjugates bearing 3'-sialyllactose, 6'-sialyllactose, or 6'-sialyl(N-acetyllactosamine) moieties attached to the polyacrylic acid carrier (P-3-SL, P-6-SL, and P-6-SLN, respectively) were prepared and tested for their ability to bind to influenza virus isolates from different hosts in a competitive solid phase assay. The virus panel included egg-grown avian and porcine strains, as well as human viruses isolated and propagated solely in mammalian (MDCK) cells and their egg-adapted variants. A clear correlation was observed between the pattern of virus binding of two glycopolymers, P-3-SL and P-6-SLN, and the host species from which the virus was derived. Avian isolates displayed a high binding affinity for P-3-SL and a two to three orders of magnitude lower affinity for P-6-SLN. By contrast, all non-egg-adapted human A and B viruses bound P-6-SLN strongly but did not bind P-3-SL. Unlike the "authentic" human strains, their egg-adapted counterparts acquired an ability to bind P-3-SL, indicative of a shift in the receptor-binding phenotype toward the recognition of Neu5Ac2-3Gal-terminated sugar sequences. Among the porcine viruses and human isolates with porcine hemagglutinin, few displayed an avian-like binding phenotype, while others differed from both avian and human strains by a reduced ability to discriminate between P-3-SL and P-6-SLN. Our data show that sialylglycopolymers may become a useful tool in studies on molecular mechanisms of interspecies transfer, tissue specificity, and other structure-function relationships of the influenza virus hemagglutinin.

This report describes recommended techniques for radiopharmaceutical biodistribution data acquisition and analysis in human subjects to estimate radiation absorbed dose using the Medical Internal Radiation Dose (MIRD) schema. The document has been prepared in a format to address two audiences: individuals with a primary interest in designing clinical trials who are not experts in dosimetry and individuals with extensive experience with dosimetry-based protocols and calculational methodology. For the first group, the general concepts involved in biodistribution data acquisition are presented, with guidance provided for the number of measurements (data points) required. For those with expertise in dosimetry, highlighted sections, examples and appendices have been included to provide calculational details, as well as references, for the techniques involved. This document is intended also to serve as a guide for the investigator in choosing the appropriate methodologies when acquiring and preparing product data for review by national regulatory agencies. The emphasis is on planar imaging techniques commonly available in most nuclear medicine departments and laboratories. The measurement of the biodistribution of radiopharmaceuticals is an important aspect in calculating absorbed dose from internally deposited radionuclides. Three phases are presented: data collection, data analysis and data processing. In the first phase, data collection, the identification of source regions, the determination of their appropriate temporal sampling and the acquisition of data are discussed. In the second phase, quantitative measurement techniques involving imaging by planar scintillation camera, SPECT and PET for the calculation of activity in source regions as a function of time are discussed. In addition, nonimaging measurement techniques, including external radiation monitoring, tissue-sample counting (blood and biopsy) and excreta counting are also considered. The third phase, data processing, involves curve-fitting techniques to integrate the source time-activity curves (determining the area under these curves). For some applications, compartmental modeling procedures may be used. Last, appendices are included that provide a table of symbols and definitions, a checklist for study protocol design, example formats for quantitative imaging protocols, temporal sampling error analysis techniques and selected calculational examples. The utilization of the presented approach should aid in the standardization of protocol design for collecting kinetic data and in the calculation of absorbed dose estimates.