Chapter 3 Novel avian metaavulavirus isolated from birds of the family
3.4 Discussion
This study demonstrated that a unique APMV strain has been long-term circulating in resident doves and pigeons in Taiwan. In addition to the novel AHRI33 virus, 13 AHRI33-like APMVs were isolated from three different species of the family Columbidae in 2009-2017. Although the samples taken in the avian influenza
surveillance program covered many species of domestic poultry, migratory birds, resident birds, and imported birds, the novel APMV strain was isolated from members of the family Columbidae, implying that pigeons and doves may be one of the natural hosts of AHRI33-like viruses. Future surveillance of wild birds in Taiwan may help to better elucidate the host distribution of AHRI33-like viruses.
Sequencing of the viral genome of the AHRI33 isolate revealed characteristic APMV coding regions and the non-coding terminal sequences (e.g., the 55 nucleotide non-coding leader sequence at the 3' end that is present in all APMVs) (Fig. 3.2). The genome length of 16,914 nt is compatible with the “rule of six”, which plays an
important role in the replication of APMVs (Kolakofsky et al., 1998), and it is the third longest among the twenty species of APMVs reported to date, shorter than only those of APMV-11 and APMV-5. The schematic diagram of AHRI33 and the protovirus
APMV-7 genome made clear the significant disparities in length of the complete genome, all six of genes, the intergenic regions, and the 5’-trailer region (Fig. 3.2). The length difference of 1,434 nt between AHRI33 and APMV-7 (15,480 bp) is much greater than the largest difference, 120 nt, between intra-species of APMV-2/Yucaipa and APMV-2/Bangor. The phylogenetic relationship with APMV-7 is consistent throughout the genome, forming a monophyletic group, suggesting that these viruses share a more recent common ancestor than do other lineages (Fig. 3.3). The deduced amino acid sequence of the F-gene cleavage site was STQQER/LFG, which was significantly different from that of APMV-7/Tennessee (LPSSR/FAG) and all other
APMVs. This motif lacks multiple basic residues and phenylalanine at the N-terminus of the F1 protein, a characteristic that typically corresponds with non-pathogenic variants, which is concordant with the results of the ICPI test.
Traditionally, APMVs were classified based on their antigenic differences, and nine serotypes were defined by HI assay in the 1970s (Alexander, 1987). In the present study, HI assay revealed weak cross-reactivity between APMV-7 and AHRI33 (R=0.125), and there were extremely low relationships between AHRI33 and representatives of the other species. HI cross-reactivity is not rare between different species of APMV (e.g., between APMV-1 and APMV-12 (Terregino et al., 2013), and APMV-9 and APMV-16 (Lee et al., 2017)), and lack of HA activity observed in APMV-5 (Samuel et al., 2010) and one novel APMV-6 (Chen et al., 2018), and all this makes classification into serotypes problematic.
In contrast, Terregino et al. (2013) proposed a classification based on nucleotide sequence identities of the whole genome as one simple method. According to this classification method based on genome identities, AHRI33 is closest to APMV-7, at 62.4%, which less than those between APMV-1 and -16 (68.1%), APMV-A and -B (67.4%), APMV-12 and -13 (64.5%), and APMV-B and -C (62.7%) (Table 3.2). These results of genetic analyses indicate that the AHRI33 isolate evolved from a common ancestor of APMV-7 and -11 and is now a distinct branch of the APMV groups.
In the last proposal for taxonomy changes of the family Paramyxoviridae, the ICTV Study Group has decided that the classification should be based on a sequence comparison of the RdRps of the viruses (ICTV, 2019). Based on the phylogenetic tree topology (clustered into monophyletic branch within the clade of the genus
Metaavulavirus) and the branch length measured in the number of substitutions per site above 0.03, the AHRI33-like isolates met the criteria for designation as distinct species.
To sum up, we identified new APMVs from the birds of family Columbidae in Taiwan from 2009 to 2017. The new APMV isolates are more closely related to
APMV-7 based on estimates of nucleotide identities of the full-length genome; however, these heterogeneous levels are comparable to, or even greater than, those of several inter-species distances separating other accepted species. This, together with the analysis according to new RdRp phylogeny-based classification system, suggests that the newly-isolated APMV should be considered as a novel species and the prototype strain of a new APMV-22 group, with the full name
APMV-22/dove/Taiwan/AHRI33/2009.
Table 3.1 Antigenicity between newly isolated AHRI33 virus and representative avian paramyxoviruses (APMVs), measured by
cross-hemagglutination inhibition tests. The following representative viruses were used as antigens and to prepare homologous chicken antisera:
APMV-1/Ulster/2C/70, APMV-2/chicken/California/Yucaipa/56, APMV-3/parakeet/Netherlands/449/75, APMV-4/duck/Hong Kong/D3/75, APMV-6/duck/Hong Kong/199/77, APMV-7/dove/Tennessee/4/75, APMV-8/goose/Delaware/1053/75, and APMV-9/duck/New York/22/78.
Virus APMV-1
a HI titres are expressed as the reciprocal of the highest dilution causing inhibition of 4 HA units of virus.
Table 3.2 Percentage identity of nucleotide sequences of genome (lower left) and amino acid sequences of RdRp gene (upper right). Shaded cells represent the inter-species heterogeneous levels that are lower than those between APMV-7 and AHRI33.
APMV -1 -2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 -17 -18 -19 -20 AHRI33
Table 3.3 Percentage nucleotide (nt) and deduced amino acid (aa) sequence identities between the AHRI33 isolate and avian paramyxoviruses (APMVs) representing the species in the subfamily Avulavirinae.
Virus N P M F HN L
Figure 3.1 Paramyxovirus virion found in allantoic fluid of embryonated chicken egg inoculated with swab sample extract from a red collared dove in Taiwan. The virion consists of a fringed envelope, and the nucleoprotein helices protruded through the envelope. (×150,000.)
Figure 3.2 Schematic diagram of avian paramyxovirus (APMV) AHRI33 isolate and APMV-7 genome. Each rectangle indicates a gene and the letters within each rectangle represents the genes: N (nucleoprotein gene), P (phosphoprotein gene), M (matrix protein gene), F (fusion protein gene), HN (hemagglutinin-neuraminidase gene), and L (large polymerase gene). The length of the genes and predicted proteins are shown above and under the rectangle, respectively. The lengths of the non-translated upstream and downstream regions are underlined. Intergenic regions are located between each box.
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Figure 3.3 Phylogenetic tree of the avian paramyxoviruses (APMVs), based on comparison of their full-length genomes. The solid triangles mark the isolates of APMV isolated from doves and pigeons in Taiwan in 2009-2017. The evolutionary history was inferred by using the Maximum Likelihood method based on the general time reversible model with discrete gamma distribution and invariant sites. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. The final dataset had a total of 12,555 positions. Numbers at the nodes indicate bootstrap confidence value (1000 replicates) for the group composed of the viruses right to the node.
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Figure 3.4 Phylogenetic tree of the fusion gene. The solid triangles mark the isolates of APMV isolated from doves and pigeons in Taiwan in 2009-2017. The evolutionary history was inferred by using the Maximum Likelihood method based on the general time reversible model with discrete gamma distribution and invariant sites. There were a total of 1,455 positions in the final dataset.
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Figure 3.5 Phylogenetic tree of the hemagglutinin-neuraminidase gene. The solid triangles mark four isolates of APMV isolated from doves and pigeons in Taiwan in 2009, 2010, 2016, and 2017. The evolutionary history was inferred by using the
Maximum Likelihood method based on the general time reversible model with discrete gamma distribution and invariant sites (4 categories +G, parameter = 1.8716; [+I], 5.94% sites). The final dataset had a total of 1,514 positions.
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Figure 3.6 Maximum Likelihood phylogenetic tree of the amino acid sequences of RdRp gene. The solid triangles mark the isolates of APMV isolated from doves and pigeons in Taiwan in 2009-2017. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood (-92897.20) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The final dataset had a total of 2,344 positions.
Chapter 4
Phylogenetic analysis of avian paramyxoviruses 1 isolated in Taiwan from 2010 to 2018 and evidence for their intercontinental dispersal by migratory birds
4.1 Introduction
Newcastle disease (ND) is one of the most important diseases of poultry and caused by virulent strains of avian paramyxovirus 1 (APMV-1), also known as
Newcastle disease virus (NDV). Newcastle disease is classified as a notifiable disease by the World Organization for Animal Health (OIE) resulting in implementing control measures and trading restrictions to prevent the spread of the disease (OIE, 2012).
Based on its genetic characteristics, the APMV-1 was re-assigned into the new genus Orthoavulavirus within a new subfamily Avulavirinae of the family Paramyxoviridae
by the International Committee on Taxonomy of Viruses in 2019 (Amarasinghe et al., 2019). The virus has high genetic diversity and the infection of APMV-1 has been reported in a wide variety of avian species around the world (Dimitrov et al., 2016).
Based on phylogenetic analyses of nucleotide sequences of viral fusion protein gene, APMV-1 has been divided into two distinct clades, class I and class II (Czeglédi et al., 2006). An unified and objective classification system of APMV-1 was proposed in 2012 based on the coding sequences of the complete fusion protein gene (Diel et al., 2012), and this system (hereafter “former” for the purposes of this chapter) and
nomenclature criteria for APMV-1 were revised and updated by a global consortium in 2019 (Dimitrov et al., 2019). The viruses within class I APMV-1 were assigned to a single genotype (genotype 1) and those within class II APMV-1 were further identified
as 21 genotypes (genotype I-XXI) per criteria put forth by Dimitrov et al. APMV-1 are categorized as lentogenic, mesogenic, and velogenic depending on clinical signs in chickens and the cleavage site amino acid sequence of fusion protein (Miller and Koch, 2013). Almost all of the class I viruses are lentogenic strains and have been isolated primarily from waterfowl of the family Anatidae worldwide and occasionally from poultry in live bird markets (Kim et al., 2007). APMV-1 isolates of class II, genotype I consist of lentogenic strains and have been widely recovered from a diversity of wild and domestic waterfowl. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1 (PPMV-1), is commonly isolated from columbids and can cause ND-like infectious disease in wild and domestic birds. Strains of genotype VII are regarded as the major pathogen responsible for the recent ND outbreaks in Europe, the Middle East, Africa, and Asia, including Taiwan (Dimitrov et al., 2016).
In Taiwan, despite that intensive vaccination programs against ND have been implemented for decades, NDV still has caused sporadic outbreaks among poultry flocks up to now. The antigenic and genetic characterization of velogenic NDV in Taiwan’s poultry population has been studied previously (Tsai et al., 2004; Lien et al., 2007), and all of the 20 isolates (Lien et al., 2007) and 30 isolates (Ke et al., 2010) collected from ND outbreaks in Taiwan from 2003 to 2006 and from 2002 to 2008, respectively, were assigned to former class II genotype VIIe, with an exception of a VIIc isolate by phylogenetic analysis of partial fusion protein gene sequences. However, the full extent of the distribution, evolution, and host species of APMV-1 circulating in domestic and wild birds has remained unexplored. Moreover, the emerging virulent NDV isolates from sub-genotypes VIIi and VIIh were rapidly spreading throughout Asia in recent years and had potential to cause a new ND panzootic (Miller et al., 2015).
Based on the global phylodynamic analysis, a study provides evidence for East
Asia representing a critically important node for the global dispersion of APMV-1 (Hicks et al., 2019). In a study of fusion protein gene sequences of two sub-genotypes of class II APMV-1 strains isolated from wild birds in Eurasia and North America,
evidence of intercontinental dispersal by wild birds has been found (Ramey et al., 2013).
The phylogentic study for global APMV-4 isolates presented limited evidence for historical viral movement between continents (Reeves et al., 2016). Phylogenetic network analysis also supported the introduction of Asia-origin clade 2.3.4.4 H5N8 avian influenza viruses into North America via intercontinental associations of waterfowl (Lee et al., 2015). Collectively, these findings suggest that migratory birds may play a potential role in the global spread of kinds of avian infectious agents.
In the present study, the APMV-1 isolates obtained from migratory birds and poultry in Taiwan were characterized by sequencing of complete fusion protein gene sequences and were compared to those available in GenBank. Based on the results of the phylogenetic analyses, we aim to illustrate the genetic diversity of APMV-1 in various avian hosts, present new epidemiological information on ND in Taiwan, and provide evidence for the potential intercontinental transmission of APMV-1 by migratory birds.
4.2 Materials and methods
4.2.1 Sample collection and virus isolation
The samples of this study were collected from migratory, resident, and domestic birds in Taiwan as part of an avian influenza surveillance program and clinical cases submitted to Animal Health Research Institute from 2010 to 2018. The cloacal swab samples, fecal samples from healthy birds and tissue samples of the brain, trachea, lung, liver, spleen, heart, and kidney from clinical cases were inoculated into the allantoic
cavities of 9- to 11-day-old specific-pathogen-free embryonated chicken eggs (Animal Drugs Inspection Branch, Animal Health Research Institute, Miaoli, Taiwan) and then incubated at 37℃ for 72 hr. The allantoic fluid from each inoculated embryo was examined for hemagglutination (HA) activity. If no HA activity was detected, a second passage was then performed. When HA activity was positive, then the allantoic fluid was tested by a commercial rapid test strip, NDV Ag Test Kit (BioNote Inc.,
Hwaseong-si, South Korea). Samples that were tested positive by the kit were subjected to further analyses.
4.2.2 RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Viral RNA was extracted from infective allantoid fluid using the MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The detection of APMV-1 RNA was performed using specific fusion protein gene-targeting RT-PCRs with SuperScript III One-Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA) to generate amplicons of either 328 bp (class I) or 292 bp (class II). The primers to amplify the specific region and the complete coding region of the fusion protein genes of class I and class II APMV-1 isolates are listed in Table 4.1. The full-length genome sequences of the representatives of each genotype and virulent class II genotype VII NDVs were determined with five different sets of primers according to the genotypes of isolates, and the sequences of theses primers are available upon request. The cycling parameters were reverse transcription at 50°C for 40 min, followed by heating at 95°C for 2 min, 35 cycles of denaturing at 95℃ for 40 sec, annealing at 50℃ for 50 sec, and extension at 72℃ for
1 min, and completed with a final extension step at 72℃ for 7 min. The RT-PCR products were separated by electrophoresis using 2% agarose gel and were visualized with ethidium bromide stain and ultraviolet transillumination.
4.2.3 Nucleotide sequencing of fusion protein gene and full-length genome The RT-PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). These products were then cloned with TOPO TA Cloning kit (Invitrogen) using the standard protocol, and the inserted cDNA segments were amplified using M13 forward and reverse primers provided by the kit. Amplified products with expected size were sequenced using the 3700XL DNA analyzer (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) by a commercial sequencing service (Mission Biotech, Taipei, Taiwan). Sequences were assembled and edited with the Lasergene 6.0 software package (DNASTAR Inc., Madison, WI, USA).
4.2.4 Phylogenetic analysis
The curated complete fusion protein gene of class I and class II datasets, provided by Dimitrov et al. (2019) and four referenced sequences of class I novel genotype isolates retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank) were
analyzed with those obtained in this study. For the construction of the phylogenetic trees, the evolutionary history was inferred using the maximum-likelihood (ML) method based on the general time reversible model with discrete gamma distribution and invariant sites by using RaxML version 8.2.12 (Stamatakis, 2014) with 1,000 bootstrap replicates through the CIPRES Science Gateway (Miller et al., 2010). The parameters for building Maximum likelihood trees using the CIPRES Science Gateway were set according to the step-by-step guidelines (Dimitrov et al., 2019). Trees were visualized
using Molecular Evolutionary Genetics Analysis version 7, or MEGA 7 (Kumar et al., 2016).
The estimates of average evolutionary distance between class I genotype 1 and genotype 2 were inferred using MEGA 7 (Kumar et al., 2016). Analyses were conducted using the Maximum Composite Likelihood model (Tamura et al., 2004). The rate
variation among sites was modeled with a gamma distribution (shape parameter=1). The analysis involved 292 nucleotide sequences. Codon positions included were
1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated.
4.3 Results
4.3.1 Sample collection and virus isolation
Forty APMV-1 isolates from different avian species were collected in this study (Table 4.2). The isolates were confirmed to be APMV-1 by isolation in
specific-pathogen-free embryonated eggs with hemagglutination activity, NDV rapid test strip, specific F gene RT-PCR and sequencing. Vaccine-like isolates of NDV obtained from samples of chickens, turkeys and pet parrots were excluded from the dataset in order to analyze only APMV-1 representing natural circulation and evolution in Taiwan during 2010-2018.
4.3.2 GenBank accession numbers
GenBank accession numbers of the APMV-1 strains described in this study are listed in Tables 4.2. The accession numbers for the full-length genome sequences of 18 isolates are MN632509-MN632526. The accession numbers for the complete coding region of the fusion protein genes of the other 22 isolates are MN632527-MN632548.
4.3.3 Genetic analysis of class I APMV-1
All of the 10 class I isolates were obtained from the samples of waterfowl of the order Anseriformes and domestic ducks. The fusion protein gene sequences of the 10 class I isolates were most closely related to those previously identified as former sub-genotype 1c (n = 2), sub-genotype 1d (n = 7) or phylogenetically divergent (n = 1) from class I genotypes 1, illustrated in the ML phylogenetic tree (Fig. 4.1). The
sequences of two former sub-genotype 1c isolates were grouped with those derived from wild bird samples collected in Japan, China, Russia, Kazakhstan, Germany, and Finland. The sequences of seven former sub-genotype 1d isolates were grouped with those originated from samples collected in Alaska, Connecticut, Delaware, Florida, Idaho, Louisiana, Massachusetts, Michigan, Minnesota, New Jersey, Ohio, Oregon, Pennsylvania, and Texas of the United States. The fusion protein gene sequence of the isolate, Anseriformes/Taiwan/AHRI67/2011, and other four sequences of APMV-1 collected from China, France, and Finland formed a strongly supported monophyletic clade. In estimated evolutionary mean distance analyses, this undesignated clade
(genotype 2) had average distances of 0.246 (standard error 0.018) base pairs per site as compared to the sequences in genotype 1.
The deduced amino acid motif at the fusion protein cleavage site sequence for nine class I genotype 1 isolates were 112E(R/Q)QER↓L117, and that of one genotype 2 isolate was 112ERQGR↓L117.
4.3.4 Genetic analysis of class II APMV-1
The fusion protein gene sequences of class II isolates in this study were most
closely related to those previously identified as genotype I (n = 9), genotype VI (n = 16), or genotype VII (n = 5). The nine genotype I isolates were obtained from waterfowl (Anseriformes), and shorebirds (Charadriiformes), except one isolate, AHRI137, from sparrow (Passeriformes). The fusion protein gene sequences of the nine isolates were assigned to genotype Ib with scattered distribution in the clade and were grouped with those derived from samples collected in China, Japan, Russia, and South Korea in ML phylogenetic analyses (Fig. 4.2). The deduced amino acid motif at the fusion protein cleavage site of the nine sub-genotype I.2 (former Ib) isolates was 112GKQGR↓L117, except that of one isolate from domestic ducks was 112GEQGR↓L117.
Of the 16 isolates of pigeon paramyxovirus 1 (PPMV-1), a genetic variant of NDV that belongs to genotype VI, 15 were obtained from birds in the family Columbidae (pigeon, red collared dove, spotted dove, and rufous turtle dove) and 1 from that of the family Corvidae (magpie). Two of the 16 PPMV-1 isolates were placed in sub-genotype VI.2.1.1.2.1 (former VIj) and the remaining 14 isolates in sub-genotype VI.2.1.1.2.2 (former VIk). Both sub-genotypes of the PPMV-1 isolates from Taiwan were related to the viruses previously circulating in pigeons and doves in China, and seem to originate from the ancestral pigeon/Belgium/248/1998 (JX901110) and
pigeon/Belgium/3936-8/2005 (JX901120) strains, respectively (Fig. 4.3). The deduced amino acid motif at the fusion protein cleavage site sequence of all PPMV-1 isolates was 112RRQKR↓F117.
pigeon/Belgium/3936-8/2005 (JX901120) strains, respectively (Fig. 4.3). The deduced amino acid motif at the fusion protein cleavage site sequence of all PPMV-1 isolates was 112RRQKR↓F117.