Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.     

Genetic variation of Italian avian paramyxoviruses serotype 9

The haemagglutinin-neuraminidase (HN) and fusion protein (F) gene of four avian paramyoviruses serotype 9 (APMV9) recently isolated from wild birds in Italy have been sequenced. A comparison between the sequences of these four isolates and the prototype virus PMV-9/domestic Duck/New York/22/78 revealed significant sequence variation that suggests that different lineages exist among APMV-9 viruses similar to that seen for APMV-1 (Newcastle disease).     

Characterization of avian paramyxovirus type 1 strains isolated in Germany during 1992 to 1996.

In Germany all avian paramyxoviruses (APMV) isolated in regional laboratories are collected and characterized by the National Reference Laboratory. From 1992 until 1996, 635 APMV-1 virus isolates were submitted from almost all regions. Of these viruses, 371 were isolated from chickens, 39 from other poultry, 171 from pigeons and 54 from exotic birds. All isolates were examined for virulence in intracerebral pathogenicity index (ICPI) tests, for their ability to react with a panel of monoclonal antibodies (mAb) and their thermostability. In addition, the nucleotide sequences of the cleavage site of the fusion protein of a few virus isolates were determined. Most isolates from chickens and other poultry were of the velogenic pathotype. This virus was responsible for the epizootic in 1993 to 1995 in many small flocks. The same virus was obtained from some pigeons and some exotic birds. The pathogenicity of the velogenic/epizootic virus was high with most viruses giving ICPI values of 1.8 to 1.9, and the sequences of the cleavage site of all velogenic isolates tested were closely related. However, viruses isolated at the beginning of the epizootic period differed from viruses isolated towards the end in their reaction with some mAbs. 149 virus isolates were identified as pigeon variant PMV-1 (PPMV-1). Most of these were obtained from pigeons but a few were isolated from chickens and other birds. Most lentogenic isolates proved to be vaccine virus strains.     

Genetic diversity of avian paramyxovirus type 4 isolates from wild ducks in Korea from 2006 to 2011

Thirteen isolates of avian paramyxovirus type 4 (APMV-4) isolated from wild ducks in Korea from 2006 to 2011 were genetically characterized by sequence analysis of the N-terminal region of the APMV-4 fusion (F) protein gene. The results revealed that the amino acid sequence homology within Korean isolates was 97.5 % or greater. The homologies of the Korean isolates with the APMV-4/duck/HK/D3/75 and APMV-4/duck/BE/15129/07 strains were 86.9–88.0 and 95.5–96.1 %, respectively. All Korean isolates had sequence motifs of ¹¹⁶DIQPR↓F¹²¹ at the F0 cleavage site. Phylogenetic analysis based on the N-terminal region of the F protein gene of APMV-4 isolates revealed that all 2006–2011 Korean isolates formed a single genotypic cluster that was phylogenetically different from APMV-4/duck/HK/D3/75 or APMV-4/duck/BE/15129/07 strains. Korean APMV-4 isolates were more closely related to APMV-4/goose/ZA/N1468/10 (isolated in South Africa) than to the Belgium APMV-4 virus. Korean APMV-4 isolates were further divided into at least two subgroups (A and B) based on phylogenetic analysis. Subgroup A viruses were isolated throughout Korea, whereas subgroup B viruses were detected only in isolates from Cheju island in 2011, suggesting that Korean APMV-4 exhibits marked genetic diversity and differs from viruses currently circulating in Europe and other locations.     

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand

Summary.  A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to ≥1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00–0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France. Received October 29, 2001; accepted February 26, 2002 Published online May 24, 2002     

Isolation and characterization of avian paramyxovirus type 1 (Newcastle disease) viruses from a flock of ostriches (Struthio camelus) and emus (Dromaius novaehollandiae) in Europe with inconsistent serology.

During a 95-day study period in 1995 in Denmark, 18 ostriches in a flock of 77 ostriches and four emus held in quarantine died. Clinical and pathological observations did not indicate the presence of transmissible infectious disease in the flock. Management failures and indoor housing were believed to have contributed significantly to the number of deaths. Samples from 17 of the dead ostriches were examined virologically. Three isolates of avian paramyxovirus serotype 1 (APMV-1) were obtained from intestines and intestinal contents of dead ostriches submitted for laboratory investigations. In ICPI tests in day-old chicks values for the three APMV-1 isolates were in the range 1.63-1.69. Characterization by means of mouse monoclonal antibodies and by restriction site analysis revealed that the three isolates were indistinguishable and similar to APMV-1 viruses present in a simultaneous epizootic of Newcastle disease in back yard poultry in Denmark. Blood samples were taken from all live birds in the flock after 25 and 95 days of quarantine and all were negative for antibodies to APMV-1 in haemagglutination inhibition tests. All samples taken after 95 days of quarantine were also negative for antibodies to APMV-1 in serum neutralization tests performed in chicken embryo cells. Blood samples taken after 95 days of quarantine were tested in a commercial ELISA for antibodies to APMV-1. In this test 35% of the samples were positive, 35% were border line and 30% were negative.     

Wood ducks (Aix sponsa) as potential reservoirs for avian influenza and avian paramyxoviruses

Influenza A viruses (IAVs) and avian paramyxoviruses (APMVs) are important pathogens of poultry worldwide, and both commonly occur in wild waterfowl, especially ducks in the family Anatidae. Although wood ducks (Aix sponsa) are members of the Anatidae, their behaviour differs from most other species in this family, which could affect the transmission of IAVs and APMVs. We collected cloacal and oropharyngeal swab and blood samples from more than 700 wood ducks across nine states in the eastern United States of America. No IAVs were isolated, and based on blocking enzyme-linked immunoassay ELISA results, antibodies to IAVs were only detected in 0.2% of samples. In contrast, 23 (3%) APMVs were isolated (22 Newcastle disease virus and 1 APMV-6), and antibodies to multiple serotypes of APMVs were detected in more than 60% of the samples. After-hatch-year birds were more likely to be antibody positive for APMV-4 and APMV-6 compared to hatch-year birds. Female birds were more likely to be antibody positive for APMV-4 than were male birds. Our results indicate that wood ducks are probably not an important host for IAV but are frequently infected with APMVs.     

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12)

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.     

The panorama of the diversity of H5 subtype influenza viruses

To elucidate the global diversity of H5 influenza viruses from a dynamic view, haemagglutinin (HA) sequences of 170 isolates were selected and analyzed in this study. Our results showed that H5 influenza isolates could be divided into two distinct lineages that circulated in the Eastern Hemisphere and the Western Hemisphere, respectively. This may be due to the separate migration routes and habitats of birds in the two hemispheres. The two distinct lineages, having existed at least for decades, possibly began divergence in 1850s. Each of the two distinct HA lineages could be further divided into some sublineages, but there was little correlation between the minor lineages and their isolation places, isolation time, neuraminidase subtypes, host species or virulence. The panorama of the diversity of H5 influenza viruses presented here integrated all known H5 epidemics including the current severe H5N1 avian epidemics in the Eastern Hemisphere and suggested that H5 virulent viruses could originate from multiple sublineages and associate with multiple NA subtypes. Our study provided a framework for the studies on the evolution and epidemiology of H5 influenza viruses.     

Virulent Newcastle disease virus in Nigeria: identification of a new clade of sub-lineage 5f from livebird markets

Newcastle disease (ND), caused by Avian Paramyxovirus Type 1, is a highly contagious and devastating viral disease of poultry of worldwide distribution with an enormous economic impact. Although ND is reported to be endemic in Nigeria, little information exists on the molecular epidemiology and the lineage distribution of the Newcastle disease viruses (NDVs) in the country, especially in the live bird markets (LBMs). Recent studies reported the identification of three unique sub-lineages. namely; 5f, 5g and 5h in West Africa, and sub-lineages 5f and 5g in particular in non-commercial farms in Nigeria. In this study, 33 NDV isolates, which included NDVs recovered from LBMs in Nigeria, during active surveillance from 2007 to 2008 and viruses recovered from outbreaks in backyard and commercial chicken farms within the same period were analysed. Based on determination of the F₀ cleavage site amino acid sequence and phylogenetic analysis, the isolates were classified as virulent; 16 strains were identified as sub-lineage 5g and 17 as sub-lineage 5f. Interestingly, 13 strains from the 5f group formed a distinct cluster that was not identified by other groups in similar studies. The close genetic similarities identified, provided evidence for the first time of the epidemiological link between the viruses circulating in the LBMs and those recovered from outbreaks in backyard and commercial chicken farms in Nigeria between 2007 and 2008. The emergence and identification of new sub-lineages provide an insight into the high rate of genetic drift occurring in NDV strains in Nigeria, and raises a lot of concerns about the efficacy of current ND control measures in the country.     

Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype

A 380 nucleotide region (bases 613 to 992) of the HA1 part of the haemagglutinin (H) gene was obtained for 35 influenza viruses of H9 subtype isolated from around the world over the past 33 years. These were analyzed phylogenetically and compared with sequences from 19 H9 subtype viruses available in the Genbank database. These viruses do not show such clear geographical lineages as other subtypes (i.e. H5 or H7) and there is a high degree of variation at the cleavage site of the haemagglutinin. Genetically distinct lineages of H9 viruses have circulated contemporaneously in different locations. Thus, it is likely that the numerous infections of poultry and other birds with H9 subtype influenza viruses during the 1990s originate from separate introductions from feral birds. The observed heterogeneity of these viruses may reflect the gene pool for H9 viruses, which is maintained in shorebirds and gulls (Charadriiformes).     

The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012

To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade 2.3.2.1 and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.     

Genetic characterization of the NS gene indicates co-circulation of two sub-lineages of highly pathogenic avian influenza virus of H5N1 subtype in Northern Europe in 2006

The non-structural (NS) gene of highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI-H5N1) isolated in Baltic Sea area of Sweden in 2006 was studied. The phylogenetic analysis data demonstrated that two distinct sub-lineages of HPAI-H5N1 were circulating during the outbreak in Northern Europe in Spring 2006. Sub-lineage I viruses fell into the same clade as viruses found in Denmark and Germany and formed a sub-clade which also included viruses isolated in the Russian Federation in late 2005. Sub-lineage II viruses formed a sub-clade closely related to European, Middle Eastern and African isolates reported in 2006. Analysis of the inferred amino acid sequences of the NS1 protein showed a deletion of five amino acids at positions 80–84. No viruses represented in this study contained Glu92 in the NS1 and all isolates contained the avian-like ESKV amino acid sequences at the NS1 C-terminal end. Sub-lineage I isolates contained unique substitutions V194I in NS1 and G63E in Nuclear export protein (NEP).     

Matrix Protein Gene Sequence Analysis of Avian Paramyxovirus 1 Isolates Obtained from Pigeons

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.     

Pathobiological characterization of low-pathogenicity H5 avian influenza viruses of diverse origins in chickens, ducks and turkeys

We undertook one of the most comprehensive studies on the replication and intraspecies transmission characteristics of low-pathogenicity avian influenza viruses in ducks, chickens and turkeys. Our results indicated that most of these isolates could replicate and be transmitted in poultry without inducing clinical disease. However, differences in transmission to contact control birds were noted, emphasizing the importance of having contact control cage mates in biological characterization experiments. Ducks supported the replication of viruses of wild aquatic bird origin in their respiratory and digestive tracts equally well. The viruses from wild aquatic birds were not effectively transmitted among chickens. In contrast, the wild-bird isolates and viruses of domestic bird origin from live-bird markets and commercial poultry operations replicated and were transmitted more efficiently in turkeys than in chickens or ducks. We also found a lower minimal infectious dose requirement for infection of turkeys compared to chickens and ducks. Our data support an important role of turkeys as being more susceptible hosts for avian influenza viruses than domestic ducks and chickens. These results highlight the role of turkeys as intermediate or bridging hosts in the transmission of influenza viruses from wild birds to land-based domestic poultry or among different land-based bird species.     

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D¹⁴⁴ mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ408506-DQ408529.     

Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions

We report a molecular epidemiological study of rabies in Arctic countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from domestic animals (dogs/cats) and from two human cases were investigated. The resulting 400 bp N-gene sequences were compared with isolates representing neighbouring Arctic or Baltic countries from North America, the former Soviet Union and Europe. Phylogenetic analysis demonstrated similarities between sequences from the Arctic and Arctic-like viruses, which were distinct from rabies isolates originating in the Baltic region of Europe, the Steppes in Russia and from North America. The Arctic-like group consist of isolates from India, Pakistan, southeast Siberia and Japan. The Arctic group was differentiated into two lineages, Arctic 1 and Arctic 2, with good bootstrap support. Arctic 1 is mainly comprised of Canadian isolates with a single fox isolate from Maine in the USA. Arctic 2 was further divided into sub-lineages: 2a/2b. Arctic 2a comprises isolates from the Arctic regions of Yakutia in northeast Siberia and Alaska. Arctic 2b isolates represent a biotype, which is dispersed throughout the Arctic region. The broad distribution of rabies in the Arctic regions including Greenland, Canada and Alaska provides evidence for the movement of rabies across borders.     

Evolutionary pattern of influenza B viruses based on the HA and NS genes during 1940 to 1999: origin of the NS genes after 1997

Summary.  Phylogenetic analysis was carried out for genes encoding hemagglutinin (HA) (24 new and 25 previously reported sequences) and nonstructural proteins (NS) (22 new and 14 previously reported sequences) of influenza B virus isolates obtained from 1940 to 1999. Two antigenically and genetically distinct HA lineages are presently known to exist. Divergence into these two lineages was estimated to have occurred around 1969. Phylogenetic analysis of NS genes revealed that their phylogenetic relationships were not linked to the two HA lineages but suggested that reassortment of viral genes between the viruses of two HA lineages had occurred. In addition two distinct NS lineages which were not linked to the two HA lineages were observed. Viruses isolated after 1997 formed their own lineage in combination with B/Houston/84 while other virus isolates obtained from 1973 to 1995 comprised the other NS lineage. Accepted May 26, 1999 Received March 26, 1999