Brundaban Panigrahy

Recombination Resulting in Virulence Shift in Avian Influenza Outbreak, Chile

Influenza A viruses occur worldwide in wild birds and are occasionally associated with outbreaks in commercial chickens and turkeys. However, avian influenza viruses have not been isolated from wild birds or poultry in South America. A recent outbreak in chickens of H7N3 low pathogenic avian influenza (LPAI) occurred in Chile. One month later, after a sudden increase in deaths, H7N3 highly pathogenic avian influenza (HPAI) virus was isolated. Sequence analysis of all eight genes of the LPAI virus and the HPAI viruses showed minor differences between the viruses except at the hemagglutinin (HA) cleavage site. The LPAI virus had a cleavage site similar to other low pathogenic H7 viruses, but the HPAI isolates had a 30 nucleotide insert. The insertion likely occurred by recombination between the HA and nucleoprotein genes of the LPAI virus, resulting in a virulence shift. Sequence comparison of all eight gene segments showed the Chilean viruses were also distinct from all other avian influenza viruses and represent a distinct South American clade.

Phylogenetic Relationships among Virulent Newcastle Disease Virus Isolates from the 2002-2003 Outbreak in California and Other Recent Outbreaks in North America

ABSTRACT Isolates from the 2002-2003 virulent Newcastle disease virus (v-NDV) outbreak in southern California, Nevada, Arizona, and Texas in the United States were compared to each other along with recent v-NDV isolates from Mexico and Central America and reference avian paramyxovirus type 1 strains. Nucleotide sequencing and phylogenetic analyses were conducted on a 1,195-base genomic segment composing the 3′ region of the matrix (M) protein gene and a 5′ portion of the fusion (F) protein gene including the M-F intergenic region. This encompasses coding sequences for the nuclear localization signal of the M protein and the F protein cleavage activation site. A dibasic amino acid motif was present at the predicted F protein cleavage activation site in all v-NDVs, including the California 2002-2003, Arizona, Nevada, Texas, Mexico, and Central America isolates. Phylogenetic analyses demonstrated that the California 2002-2003, Arizona, Nevada, and Texas viruses were most closely related to isolates from Mexico and Central America. An isolate from Texas obtained during 2003 appeared to represent a separate introduction of v-NDV into the United States, as this virus was even more closely related to the Mexico 2000 isolates than the California, Arizona, and Nevada viruses. The close phylogenetic relationship between the recent 2002-2003 U.S. v-NDV isolates and those viruses from countries geographically close to the United States warrants continued surveillance of commercial and noncommercial poultry for early detection of highly virulent NDV.

Avian Influenza Virus Subtypes Inside and Outside the Live Bird Markets, 1993–2000: A Spatial and Temporal Relationship

SUMMARY. Between 1993 and 2000, gallinaceous birds, waterfowl, and environmental specimens from the live bird markets (LBMs) of the northeastern United States and non-LBM premises were tested for the presence of avian influenza virus (AIV), pathogenic properties of AIV subtypes, especially of hemagglutinin (H) subtypes H5 and H7, and a possible association between LBM and non-LBM infections. Ten H subtypes of AIV were isolated from the LBM specimens: H1, H2, H3, H4, H5, H6, H7, H9, H10, and H11. During this period, the 10 subtypes also were isolated from birds in non-LBM premises. In the LBMs, subtypes H2, H3, H4, H6, H7, and H11 were present for 5–8 yr despite efforts to clean and disinfect the premises. The H5 or H7 subtypes present during the same year in both LBMs and non-LBMs within a state or in contiguous states were (subtype/year): H5N2/1993, 1999, and H7N2/1994–99. The AIV subtypes including the H5 and H7 that were evaluated for pathogenicity in chickens were low pathogenic. The deduced amino acid sequence at the H cleavage site of H5 and H7 subtypes was consistent with those of low pathogenic AIV. Although the H5N2 and H7N2 subtypes remained low pathogenic, they did undergo mutations and acquired an additional basic amino acid at the H cleavage site; however, the minimum number of basic amino acids in correct sequence (B-X-B-R, where B = basic amino acid, X = need not be basic amino acid, and R = arginine) required for high pathogenicity was lacking. A low pathogenic H5 or H7 subtype may become highly pathogenic by acquiring additional basic amino acids at the H cleavage site. The LBMs have been and will likely continue to be a source of AIV for commercial poultry.

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

Effect of composting poultry carcasses on survival of exotic avian viruses: highly pathogenic avian influenza (HPAI) virus and adenovirus of egg drop syndrome-76.

Eight-week-old chickens were inoculated with one of two exotic viruses to determine the effect of composting on virus survival. Group 1 chickens were inoculated with highly pathogenic avian influenza (HPAI) virus via the caudal thoracic air sac. Group 2 chickens were inoculated with the adenovirus that causes egg drop syndrome-76 (EDS-76) by the oral route. Five days after inoculation, lung, trachea, and air sacs for HPAI and spleen, cecal tonsils, and bursa of Fabricius for EDS-76 were collected and composted with poultry carcasses. At the end of the first 10 days of composting, virus-isolation efforts showed that the HPAI virus had been inactivated, and only 1 of 20 tissue samples yielded the adenovirus of EDS-76. The viruses of HPAI and EDS-76 were completely inactivated at the end of the second 10-day period of the two-stage composting process. Control tissues collected at necropsy and frozen at -70 C for virus isolation were all positive for virus.

Molecular and biological characteristics of H5 and H7 avian influenza viruses in live-bird markets of the northeastern United States, 1994-2001.

Abstract Surveillance for H5 and H7 subtypes of avian influenza virus (AIV) in the live-bird markets (LBMs) of the northeastern United States has been in effect since 1986 when the markets were first recognized as a potential reservoir for AIV. Long-term maintenance of AIV in the LBM system has been documented. However, little is known about the influence of successive cycles of replication in unnatural avian hosts (gallinaceous birds) on the genetics of the virus, especially in the region of the hemagglutinin (HA) gene that can influence pathogenicity. Isolation of low-pathogenicity H5 AIVs from the LBMs has been sporadic; however, in 1994 a low-pathogenicity H7N2 virus was isolated that has persisted in the LBMs for more than 7 yr. Efforts to eliminate the H7 virus from the markets have been unsuccessful. During the 7-yr period, several molecular changes have occurred at the hemagglutinin cleavage site of the H7 virus. These changes include substitutions of proline for threonine and lysine for asparagine, respectively, at the −2 and −5 positions of the HA1 protein. In addition, there has been a 24 nucleotide base-pair deletion in the receptor binding region of the HA1. The accumulation of an additional basic amino acid at the cleavage site is a cause for concern to regulatory authorities, and, therefore, efforts to eliminate the virus from the LBM system have been intensified.

Susceptibility of pigeons to avian influenza.

Susceptibility to infection with avian influenza virus (AIV) was studied in pigeons inoculated via oculonasal (Experiment 1) or intravenous (Experiment 2) route. Chickens were included as susceptible hosts in both experiments. Two subtypes each of the highly pathogenic AIV (HPAIV; HP CK/PA H5N2 and HP CK/Australia H7N7) and non-pathogenic AIV (NPAIV; NP CK/PA H5N2 and NP emu/TX H7N1) at a dose of 10(5) embryo infective dose per bird were used as inoculum. The pigeons inoculated with HP CK/PA H5N2 or HP CK/Australia H7N7 remained apparently healthy throughout the 21-day observation period, did not shed viruses on 3, 7, 14, and 21 days postinoculation (DPI), and had no demonstrable levels of antibodies on 21 DPI. On the other hand, 9 of 12 chickens inoculated with the HPAIV died of highly pathogenic avian influenza; the viruses were recovered from their respiratory and intestinal tissues, and the surviving chickens had antibodies to AIV. Regarding responses of pigeons to inoculation with NP CK/PA H5N2 or NP emu/TX H7N1, the pigeons remained clinically healthy throughout the 21-day observation period and did not have detectable levels of antibodies on 21 DPI; only one pigeon yielded the NP emu/TX H7N1 on 3 DPI. The virus was isolated from a tracheal swab and was believed to be the residual inoculum virus. Based on the responses of pigeons to NPAIV and HPAIV, it was concluded that the pigeons were resistant or minimally susceptible to infection with HPAIV or NPAIV.

Experimental and Serologic Observations on Avian Pneumovirus (APV/turkey/Colorado/97) Infection in Turkeys

An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States.

An outbreak of duck viral enteritis (duck plague) in domestic Muscovy ducks (Cairina moschata domesticus) in Illinois.

Duck viral enteritis (DVE) was diagnosed in an outbreak of the disease in a resident population of Muscovy ducks (Cairina moschata domesticus) on a privately owned multispecies game bird production facility in Illinois, where it claimed 625 ducks. This disease condition had not been reported previously in domestic ducks in Illinois. Although other varieties and age groups of domestic waterfowl (i.e., black ducks, rhumen ducks, Pekin ducks, ducklings, and geese) were present on the game bird farm, the morbidity and mortality (100%) in this epornitic was solely limited to adult ducks of the Muscovy lineage. The clinical signs in the affected ducks were lethargy, diarrhea, dehydration, and death within 2-3 hr of onset of symptoms. Gross pathologic changes were nonspecific and included ecchymotic hemorrhage, effusion of fluid and blood within body cavities reflective of an acute systemic infectious disease. Light microscopic findings were necrosis of primarily digestive lining epithelium and variable lymphohistiocytic infiltration within mucosal and serosal connective tissues. Intranuclear inclusions resembling characteristic herpetic (i.e., Cowdry type A) inclusions were observed primarily in the digestive, respiratory, and reproductive tracts; liver; and spleen. Esophageal candidiasis, bacteriosis, and systemic Pasteurella anatipestifer infections, thought to be concurrent or opportunistic infections, were present in several ducks. DVE virus was demonstrated in infected Muscovy duck embryo fibroblast cells by direct DVE virus-specific fluorescent antibody staining.

Identification and Characterization of H2N3 Avian Influenza Virus from Backyard Poultry and Comparison with Novel H2N3 Swine Influenza Virus

SUMMARY. In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.