Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe, Asia, and Africa

Abstract Background The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. Results Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014–2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. Conclusions We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions

Presence of vaccine-derived newcastle disease viruses in wild birds

Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife119sem informaçã

Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds

<div><p>Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order <i>Columbiformes</i> (n = 23), followed in frequency by the order <i>Anseriformes</i> (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.</p></div

Phylogenetic tree of isolates and their relationship to class II NDV viruses.

<p>Phylogenetic analysis based on the complete nucleotide sequence of the fusion gene of isolates representing NDV class II. The evolutionary history was inferred by using the Maximum Likelihood method based on Tamura 3-parameter model with 500 bootstrap replicates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162484#pone.0162484.ref070" target="_blank">70</a>]. The tree with the highest log likelihood (-108983.3717) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.0936). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.7777% sites). The tree is drawn to scale with branch lengths measured in the number of substitutions per site and the percentage of trees in which the associated taxa clustered together are shown below the branches. The analysis involved 81 nucleotide sequences with a total of 1662 positions in the final dataset. Isolates studied in this work are designated in front of the taxa name as follows: USA—●; Ukraine—○; Brazil—□, Bulgaria—■. Evolutionary analyses were conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162484#pone.0162484.ref067" target="_blank">67</a>]. The Roman numerals presented in the taxa names in the phylogenetic trees represent the respective genotype for each isolate, followed by the GenBank identification number, host name (if available), country of isolation, strain designation and country of isolation.</p

Collated Isolates from GenBank and SEPRL samples.

<p>A total of 54 isolates from the following taxonomic orders are tabulated below: <i>Accipitriformes</i> (n = 1); <i>Anseriformes</i> (n = 13); <i>Charadriiformes</i> (n = 3); <i>Columbiformes</i> (n = 23); <i>Falconiformes</i> (n = 1); <i>Galliformes</i> (n = 4); <i>Passeriformes</i> (n = 2); <i>Pelecaniformes</i> (n = 1); <i>Phoenicopteriformes</i> (n = 1); <i>Psittaciformes</i> (n = 4); Unknown (n = 1). GenBank accession numbers bolded are strains sequenced from this study.</p

Rock Pigeon virus isolation, HI antibody titer, and intracerebral pathogenicity indices (ICPIs).

<p>Field swabs collected from the oral and cloacal cavities positive for virus are listed with a + symbol, otherwise an N/A is listed. ICPI experimental infection swabs collected in the lab from the oral and cloacal cavities positive for virus are listed below with their corresponding ICPI values, otherwise an N/A is listed.</p