Dennis A. Senne

Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

ABSTRACT A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103 to 104 gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

Effect of Vaccine Use in the Evolution of Mexican Lineage H5N2 Avian Influenza Virus

ABSTRACT An outbreak of avian influenza (AI) caused by a low-pathogenic H5N2 type A influenza virus began in Mexico in 1993 and several highly pathogenic strains of the virus emerged in 1994-1995. The highly pathogenic virus has not been reported since 1996, but the low-pathogenic virus remains endemic in Mexico and has spread to two adjacent countries, Guatemala and El Salvador. Measures implemented to control the outbreak and eradicate the virus in Mexico have included a widespread vaccination program in effect since 1995. Because this is the first case of long-term use of AI vaccines in poultry, the Mexican lineage virus presented us with a unique opportunity to examine the evolution of type A influenza virus circulating in poultry populations where there was elevated herd immunity due to maternal and active immunity. We analyzed the coding sequence of the HA1 subunit and the NS gene of 52 Mexican lineage viruses that were isolated between 1993 and 2002. Phylogenetic analysis indicated the presence of multiple sublineages of Mexican lineage isolates at the time vaccine was introduced. Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccines sublineage. Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages. Vaccine protection studies further confirmed the in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates. These findings indicate that multilineage antigenic drift, which has not been observed in AI virus, is occurring in the Mexican lineage AI viruses and the persistence of the virus in the field is likely aided by its large antigenic difference from the vaccine strain.

The Evolutionary Genetics and Emergence of Avian Influenza Viruses in Wild Birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient “genome constellations,” continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.

Survey of the Hemagglutinin (HA) Cleavage Site Sequence of H5 and H7 Avian Influenza Viruses: Amino Acid Sequence at the HA Cleavage Site as a Marker of Pathogenicity Potential

The deduced amino acid sequence at the hemagglutinin (HA) cleavage site of 76 avian influenza (AI) viruses, subtypes H5 and H7, was determined by reverse transcription-polymerase chain reaction and cycle sequencing techniques to assess pathogenicity. Eighteen of the 76 viruses were isolated in 1993 and 1994 from various sources in the United States. In addition, 34 H5 (4 highly pathogenic [HP] and 30 non-highly pathogenic [non-HP]) and 24 H7 (3 HP and 21 non-HP) repository viruses, isolated between 1927 and 1992, were sequenced and the sequences compared to those in recent isolates. All repository HP H5 and H7 viruses studied had multiple basic amino acids adjacent to the HA cleavage site and most had basic amino acids in excess of the proposed minimum motif B-X-B-R (B = basic amino acids arginine or lysine, X = nonbasic amino acid, R = arginine) that has been associated with high pathogenicity. Of the non-HP viruses studied, 35 of 38 for H5 and 30 of 31 for H7 conformed to the motif B-X-X-R and B-X-R, respectively. Two non-HP H5 viruses had the motif X-X-X-R at the cleavage site and a third had the motif B-X-X-K (K = basic amino acid lysine). One non-HP H7 (A/Pekin robin/CA/30412-5/94) had four basic amino acids (K-R-R-R) adjacent to the cleavage site. Although the Pekin robin isolate did not produce disease in chickens under the conditions of the study it did have the amino acid sequence compatible with that in HP AI viruses and, therefore, is considered potentially HP. This is the first account of an H7 virus that is non-HP in chickens but meets the molecular criterion to be classified as HP.

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.

Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples

ABSTRACT A real-time reverse-transcription PCR (RRT-PCR) was developed to detect avian paramyxovirus 1 (APMV-1) RNA, also referred to as Newcastle disease virus (NDV), in clinical samples from birds. The assay uses a single-tube protocol with fluorogenic hydrolysis probes. Oligonucleotide primers and probes were designed to detect sequences from a conserved region of the matrix protein (M) gene that recognized a diverse set (n = 44) of APMV-1 isolates. A second primer-probe set was targeted to sequences in the fusion protein (F) gene that code for the cleavage site and detect potentially virulent NDV isolates. A third set, also directed against the M gene, was specific for the North American (N.A.) pre-1960 genotype that includes the common vaccine strains used in commercial poultry in the United States. The APMV-1 M gene, N.A. pre-1960 M gene, and F gene probe sets were capable of detecting approximately 103, 102, and 104 genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 101 50% egg infective doses (EID50), and the F gene assay could detect approximately 103 EID50. The RRT-PCR test was used to examine clinical samples from chickens experimentally infected with the NDV strain responsible for a recent epizootic in the southwestern United States. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.

Phylogenetic Diversity among Low-Virulence Newcastle Disease Viruses from Waterfowl and Shorebirds and Comparison of Genotype Distributions to Those of Poultry-Origin Isolates

ABSTRACT Low-virulence Newcastle disease viruses (loNDV) are frequently recovered from wild bird species, but little is known about their distribution, genetic diversity, or potential to cause disease in poultry. NDV isolates recovered from cloacal samples of apparently healthy waterfowl and shorebirds (WS) in the United States during 1986 to 2005 were examined for genomic diversity and their potential for virulence (n = 249). In addition 19 loNDV isolates from U.S. live bird markets (LBMs) were analyzed and found to be genetically distinct from NDV used in live vaccines but related to WS-origin NDV. Phylogenetic analysis of the fusion protein identified nine novel genotypes among the class I NDV, and new genomic subgroups were identified among genotypes I and II of the class II viruses. The WS-origin viruses exhibited broad genetic and antigenic diversity, and some WS genotypes displayed a closer phylogenetic relationship to LBM-origin NDV. All NDV were predicted to be lentogenic based upon sequencing of the fusion cleavage site, intracerebral pathogenicity index, or mean death time in embryo assays. The USDA real-time reverse transcription-PCR assay, which targets the matrix gene, identified nearly all of the class II NDV tested but failed to detect class I viruses from both LBM and WS. The close phylogenetic proximity of some WS and LBM loNDV suggests that viral transmission may occur among wild birds and poultry; however, these events may occur unnoticed due to the broad genetic diversity of loNDV, the lentogenic presentation in birds, and the limitations of current rapid diagnostic tools.

Development of Real-Time RT-PCR for the Detection of Avian Influenza Virus

Abstract A real-time reverse transcriptase/polymerase chain reaction (RRT-PCR) assay was developed using hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene that are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions that are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live-bird markets (LBMs) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBMs that were the sources of VI positive samples. Of the 58 markets that were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and LBMs.

Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat

ABSTRACT Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.

Transmission of Eurasian avian H2 influenza virus to shorebirds in North America.

Influenza A virus of the H2 subtype caused a serious pandemic in 1957 and may cause similar outbreaks in the future. To assess the evolution and the antigenic relationships of avian influenza H2 viruses, we sequenced the haemagglutinin (HA) genes of H2 isolates from shorebirds, ducks and poultry in North America and derived a phylogenetic tree to establish their interrelationships. This analysis confirmed the divergence of H2 HA into two geographical lineages, American and Eurasian. One group of viruses isolated from shorebirds in North America had HA belonging to the Eurasian lineage, indicating an interregional transmission of the H2 gene. Characterization of HA with a monoclonal antibody panel revealed that the antigenicity of the Delaware strains differed from the other avian strains analysed. The data emphasizes the importance of avian influenza surveillance.