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SUMMARY. The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens (Ckn-LBM and Ckn-Australia) and wild (Anhinga) and exotic (YN parrot, pheasant, and dove) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of 10 4-wk-old specific-pathogen-free white leghorn chickens were inoculated intraconjunctivally with each one of the isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hr to 14 days postinoculation or at death. Tissues were examined by histopathology, by immunohistochemistry (IHC) to detect viral nucleoprotein (IHC/NP), and by in situ hybridization to detect viral mRNA and were double labeled for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ([TUNEL] or IHC/caspase-3) and viral nucleoprotein (IHC/NP). Birds infected with the three low virulence viruses (Ckn-LBM, YN parrot, and Ckn-Australia) did not develop clinical disease. Microscopic lesions were observed only at the inoculation site and in organs of the respiratory system. The detection of viral nucleoprotein (N) was restricted to the inoculation site. The pheasant and dove isolates were highly virulent for chickens with marked tropism for lymphoid tissues, confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA. Viral N protein was detected early in the cytoplasm of cells in the center of the splenic ellipsoids. The apoptosis assays (TUNEL and IHC/caspase-3) showed increased apoptosis in the splenic ellipsoids as well. Apparently, apoptosis is an important mechanism in lymphoid depletion during NDV infection.

Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.

Highly pathogenic avian influenza (HPAI) in poultry causes high morbidity and mortality, and it is a List A disease of the Office International des Epizooties. An outbreak of HPAI in commercial poultry not only causes direct disease losses but often results in trade restrictions for the affected country. Because HPAI viruses can mutate from H5 and H7 low pathogenic avian influenza viruses, it is necessary to monitor and control even the low pathogenic form of the virus. We report a practical approach for screening large numbers of isolates that uses amplification by reverse transcriptase-polymerase chain reaction of a segment of the hemagglutinin (HA) gene (536-560 bp) of H7 avian influenza viruses followed by the heteroduplex mobility assay (HMA). The HMA test compares the amplified polymerase chain reaction product from unknown samples with reference isolates, which allows the identification of new variants. The HMA test results were compared with sequence analysis of the isolates used in the study. On the basis of the HMA, we could identify several new variant viruses present in the live bird markets in the northeastern United States. New strains gave a distinct pattern of bands in the gels in accordance with the different heteroduplexes formed when their HA region amplification products were incubated together with the same amplification product of a reference strain. These differences correlate with phylogenetic analysis from sequence data.

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.

Formalin-fixed, paraffin-embedded spleen and intestinal tissues were harvested at 2 days postinfection from 4-wk-old white rock chickens infected with five different strains of Newcastle disease virus (NDV). These tissues were examined for the presence of macrophage antigen expression, virus replication, and interferon gamma (IFN gamma) production. The five strains represented all three NDV pathotypes. Viral replication and IFN gamma, as determined by riboprobe in situ hybridization, were detected only in those chickens infected with velogenic viscerotropic NDV (VVNDV) strains. Macrophage antigen expression, an indicator of macrophage activation, was determined by immunohistochemistry with a macrophage-specific antibody, CVI-ChNL-68.1. Presence of macrophage antigen was most prominent in VVNDV-infected chickens. The distribution of this antigen within tissues was far more diffuse than the staining for viral mRNA. The presence of IFN gamma mRNA was detected in the spleen and intestinal lymphoid tissue of VVNDV-infected chickens. There was also increased macrophage antigen expression in the mesogen-infected birds, but it was less dramatic than in tissues from VVNDV-infected chickens. One of two lentogen-infected birds had evidence of increased macrophage antigen expression only in the spleen.

SUMMARY. Newcastle disease virus (NDV) remains a major pathogen of poultry where highly virulent strains require reporting to the Office of International Epizootes. NDV is a paramyxovirus existing as different strains classified on the basis of severity of the disease they cause. The present study was conducted in Argentina to determine the prevalence of highly virulent velogenic NDV strains in commercial poultry farms. Tracheal and cloacal swabs from 693 flocks, representing 14% of the broiler production, were collected and pooled. A pool amplified twice in embryonated eggs presented a limited hemagglutination titer. We performed reverse transcription coupled to polymerase chain reaction to amplify fusion and matrix protein gene sequences of the isolate and the strain Trenque Lauquen, isolated in Argentina during an outbreak in 1970-71 and previously characterized as velogenic viscerotropic by biological methods. The amino acid sequences were deduced from nucleotide sequences of the amplification products and the pathotype predicted according to the sequences obtained. From the samples analyzed, we found only one type of NDV, being the isolate identified as lentogenic NDV. This strain is probably the one used in vaccination of flocks where that sample was obtained. These data have allowed us to consider a velogenic NDV-free status in Argentina's commercial poultry.

Three infectious bursal disease field viruses, identified as U28, 3212, and MISS and isolated in the early 1980s from the southeastern United States, were characterized both antigenically and genotypically. A panel of monoclonal antibodies (MAbs) was utilized in an antigen-capture enzyme-linked immunosorbent assay (ELISA) for antigenic characterization. The ELISA data indicated that U28 and the Delaware variants are antigenically related. The 3212 and the GLS variants were more closely related antigenically to each other than to other viruses analyzed. However, the MISS isolate reacted with MAbs that were specific for both classic and variant strains of infectious bursal disease virus (IBDV). Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify nucleotide sequences from the genome coding for the variable region of VP2 from IBDV field isolates U28, 3212, and MISS. Phylogenetic analysis of the deduced amino acid sequences revealed that U28 was most similar (98.3%) to the IBDV Delaware variants and that 3212 was most similar (97.1%) to the GLS variant. The MISS isolate was most similar (97.4%) to the classic 52/70 strain. Positive correlation occurred with the use of different methods to characterize IBDV isolates.

SUMMARY. Fifty-seven Newcastle disease virus (NDV) isolates from chickens, turkeys, a rhea, a parrot, and an anhinga were pathotyped and characterized by monoclonal antibody (mAb) inhibition profile, elution rate, and hemagglutinin thermostability. Nucleotide sequence analysis of portions of the fusion protein and matrix protein genes of the parrot isolate was done for comparisorL with prior sequence analysis of the anhinga isolate and NDV reference strains. Seven of the 43 chicken isolates were recovered from flocks in Canada. The remaining isolates, including 1 1L from turkeys, were isolated in the United States. All isolates except that of the anhinga were of low virulence by mean death time in embryos, intracerebral pathogenicity index, and/or intravenous pathogenicity index procedures and were classified as lentogens. The anhinga isolate was more virulent than the other strains and was pathotyped as a mesogen. However, nucleotide sequence analysis of the anhinga isolate had revealed a homology with the virulent cormorant isolates of 1992 rather than the classical U.S. mesogens characterized by the Roakin strain. Variability was evident among the lentogenic isolates. Two isolates from turkeys had inAb profiles that differed from B 1 and La Sota reference and vaccine strains, and 38% (21/56) of the isolates had more thermostable hemagglutinins than those reference strains. There was no evidence that any of the isolates from poultry were more virulent than the lentogenic pathotype.

SUMMARY. Nine Newcastle disease virus (NDV) isolates obtained from Puerto Rico, Georgia, Alabama, Mississippi, and Texas were analyzed for in vivo pathogenicity, biological properties (hemagglutination of mammalian erythrocytes), and for sequence variation at the amino acid and sense RNA level of the fusion protein cleavage site. Intracerebral pathogenicity index values ranged from 0 to 0.3 and the intravenous pathogenicity index obtained for all isolates was 0. Four isolates hemagglutinated bovine erythrocytes, whereas no hemagglutination was observed using equine erythrocytes. By direct sequencing of reverse transcription polymerase chain reaction products, all the isolates had a predicted fusion cleavage sequence comparable to lentogenic NDV strains. Based on nucleotide sequence, the viruses could be grouped phylogenetically with the B1 vaccine-type virus. However, nucleotide sequences were not 100% similar to the B1 or La Sota NDV strains, indicating that minor genetic heterogeneity occurs among lentogenic field isolates of NDV.