Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Characterization of ten paramyxovirus type 1 viruses isolated from pigeons in China during 1996-2019.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1, which mainly infects pigeons. Here, we characterized ten PPMV-1 viruses isolated from pigeons in China during 1996-2019. Phylogenetic analysis of available complete genomes, F and HN genes of PPMV-1 from China showed that multiple PPMV-1 genotypes (I, II, VI, and VII) exist in pigeons in China. Ten PPMV-1 viruses isolated in this study belonged to genotypes VI.1.2.2.2, VI.2.1.1.2.1, VI.2.1.1.2.2 and VII respectively. Genotype VI is predominant in pigeons. VI.2.1.1.2.2 contains most recently isolated PPMV-1 viruses, suggesting that VI.2.1.1.2.2 is a prevalent genotype in pigeons in China. In vitro and in vivo studies showed that four representative viruses from genotypes VI.2.1.1.2.1 (TA14), VI.2.1.1.2.2 (SD19), VI.1.2.2.2 (SD16), and VII (JN08) could replicate efficiently in chicken embryo fibroblasts, while the replication titer of JN08 (VII) virus was significantly lower than that of VI gene viruses in pigeon embryo fibroblasts. The TA14 (VI.2.1.1.2.1) and SD19 (VI.2.1.1.2.2) viruses caused 20 % and 30 % mortality in pigeons, respectively. No birds infected with SD16 (VI.1.2.2.2) died during the study period. JN08 (VII) virus did not cause obvious clinical signs in infected pigeons. All data indicated that VI.2.1.1.2.2 is the prevalent genotype circulating in China and poses a major threat to pigeons, suggesting that a matched vaccine is necessary to control the disease.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Sequence analysis and biological characterization of virulent Avian avulavirus 1 isolated from asymptomatic migratory fowl.

Continuous monitoring and surveillance of avian avulaviruses (AAvVs) in water/migratory fowl is imperative to ascertain the evolutionary dynamics of these viruses. Here, we report genomic and amino acid characteristics of two AAvVs strains isolated from asymptomatic waterfowl (Anas carolinensis). Sequence characteristics including the presence of virulent motif (112RRQKR↓F117) and biological assessment confirmed the virulent nature of study isolates. Phylogenetic analysis of complete fusion (F) and hemagglutinin-neuraminidase (HN), and hyper-variable region of F gene revealed clustering of both strains within genotype VII and sub-genotype VIIi. The inferred residue analysis of complete F and HN genes revealed a number of substitutions in functionally and structurally important motif/s compared to reference strains of each genotype (I-XI). This study concludes an evolutionary nature of avian avulavaris 1 (AAvV-1), ascertaining continuous surveillance of migratory fowl to better elucidate their infection, epidemiology and subsequent impacts on commercial and backyard poultry. Keywords: virulent AAvV-1; migratory fowl; genetic characterization; evolutionary analysis; Pakistan.

Antarctic Penguins as Reservoirs of Diversity for Avian Avulaviruses.

Wild birds harbor a huge diversity of avian avulaviruses (formerly avian paramyxoviruses). Antarctic penguin species have been screened for avian avulaviruses since the 1980s and, as such, are known hosts of these viruses. In this study, we screened three penguin species from the South Shetland Islands and the Antarctic Peninsula for avian avulaviruses. We show that Adelie penguins (Pygoscelis adeliae) are hosts for four different avian avulavirus species, the recently described avian avulaviruses 17 to 19 and avian avulavirus 10-like, never before isolated in Antarctica. A total of 24 viruses were isolated and sequenced; avian avulavirus 17 was the most common, and phylogenetic analysis demonstrated patterns of occurrence, with different genetic clusters corresponding to penguin age and location. Following infection in specific-pathogen-free (SPF) chickens, all four avian avulavirus species were shed from the oral cavity for up to 7 days postinfection. There was limited shedding from the cloaca in a proportion of infected chickens, and all but one bird seroconverted by day 21. No clinical signs were observed. Taken together, we propose that penguin species, including Antarctic penguins, may be the central reservoir for a diversity of avian avulavirus species and that these viruses have the potential to infect other avian hosts.IMPORTANCE Approximately 99% of all viruses are still to be described, and in our changing world, any one of these unknown viruses could potentially expand their host range and cause epidemic disease in wildlife, agricultural animals, or humans. Avian avulavirus 1 causes outbreaks in wild birds and poultry and is thus well described. However, for many avulavirus species, only a single specimen has been described, and their viral ecology and epidemiology are unknown. Through the detection of avian avulaviruses in penguins from Antarctica, we have been able to expand upon our understanding of three avian avulavirus species (avian avulaviruses 17 to 19) and report a potentially novel avulavirus species. Importantly, we show that penguins appear to play a key role in the epidemiology of avian avulaviruses, and we encourage additional sampling of this avian group.

Pathological and molecular findings of avian avulavirus type 1 outbreak in pigeons (Columba livia) of southern Brazil / Achados patológicos e moleculares da infecção por avulavirus aviário tipo 1 em pombos (Columba livia) do sul do Brasil

The Newcastle disease, caused by avian avulavirus type 1 strains (APMV-1) is an important avian disease involved into high rates of mortality and economic losses. Several outbreaks have been reported over the last 30 years in Columbiformes in different parts of the world, caused by a adapted variant strain of AAvV-1, called pigeon paramyxovirus type 1 (PPMV-1). A high mortality associated with an outbreak was analyzed in free-living pigeons (Columba livia) in a public square in Porto Alegre in Southern Brazil. A total of 24 pigeons moribund or freshly dead, within five weeks interval were submitted to necropsy, histopathological, immunohistochemical (anti-Newcastle), and RT-PCR followed by sequencing of the amplification products analysis. They presented neurological signs, non-suppurative encephalitis and encephalomyelitis, and mononuclear inflammatory infiltrate in different organs. Immunohistochemical analysis in nine pigeons tissue showed that anti-Newcastle was expressed in brain, kidney, liver and pancreas. The RT-PCR test for the M protein of Newcastle disease virus was positive in six pigeons. The differential diagnosis of Influenza, West Nile, Mycoplasma gallisepticum and Mycoplasma synoviae in all pigeons presented negative results. The sequence of amino acids in the cleavage site region of the F protein was 112RRQKRF117 classifying the strain as virulent. The phylogenetic analysis classified this virus strain into Class II and VI genotype.(AU)

A doença de Newcastle, causada por cepas de avulavirus aviário tipo 1 (AAvV-1), é uma doença de aves importante por causar altos índices de mortalidade e perdas econômicas. Vários surtos têm sido relatados ao longo de 30 anos em aves da ordem Columbiformes, em diferentes partes do mundo, causados por uma cepa variante específica de AAvV-1, denominada Pigeon paramyxovirus tipo 1 (PPMV-1). Foi analisado um surto de mortalidade em pombos domésticos (Columba livia), provenientes de uma praça pública em Porto Alegre, no Sul do Brasil. Vinte e quatro aves moribundas ou mortas foram submetidas, no intervalo de cinco semanas, ao exame de necropsia, exame histopatológico, imuno-histoquímico anti-Newcastle, RT-PCR e sequenciamento. Apresentaram sinais neurológicos, encefalite e encefalomielite não supurativas, além de infiltrado inflamatório mononuclear em diversos órgãos. Nove aves demonstraram exame imuno-histoquímico positivo em órgãos como cérebro, rim, fígado e pâncreas. Seis aves foram positivas no exame de RT-PCR para a proteína M do vírus da Doença de Newcastle. Nos exames de diagnósticos diferenciais de Influenza, West Nile, Mycoplasma gallisepticum e Mycoplasma synoviae, todas as aves testadas foram negativas. A sequência dos aminoácidos na região do sítio de clivagem da proteína foi 112RRQKRF117, classificando a cepa como virulenta. De acordo com a análise filogenética o vírus identificado foi classificado como pertencente à classe II e ao genótipo VI.(AU)

Evaluation of the replication and pathogenicity of a variant avian paramyxovirus serotype 6 in mice.

Avian paramyxoviruses (APMVs) have been evaluated for their potential use as vaccine vectors, sparking research efforts leading to a better understanding of APMVs' replication and pathogenicity. However, within APMV serotypes, significant genetic diversity exists, and the infectivity of variant strains in mammals has not been studied. We utilized a mouse model to evaluate the pathogenicity of a variant strain of APMV-6 (APMV-6/red-necked stint/Japan/8KS0813/2008) in comparison with the prototype APMV-6 strain (APMV-6/duck/Hong Kong/18/199/1977). Although the two viruses differ substantially, both genetically and antigenically, we found that the variant and prototype strains could similarly replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the mice. Both viruses induced a humoral immune response that could be clearly detected by ELISA but which was poorly recognized by the hemagglutination inhibition test.

Dispersal and Transmission of Avian Paramyxovirus Serotype 4 among Wild Birds and Domestic Poultry.

Avian paramyxovirus serotype 4 (APMV-4) is found sporadically in wild birds worldwide, and it is an economically important poultry pathogen. Despite the existence of several published strains, very little is known about the distribution, host species, and transmission of APMV-4 strains. To better understand the relationships among these factors, we conducted an APMV-4 surveillance of wild birds and domestic poultry in six provinces of China suspected of being intercontinental flyways and sites of interspecies transmission. APMV-4 surveillance was conducted in 9,160 wild birds representing seven species, and 1,461 domestic poultry in live bird markets (LMBs) from December 2013 to June 2016. The rate of APMV-4 isolation was 0.10% (11/10,621), and viruses were isolated from swan geese, bean geese, cormorants, mallards, and chickens. Sequencing and phylogenetic analyses of the 11 isolated viruses indicated that all the isolates belonging to genotype I were epidemiologically connected with wild bird-origin viruses from the Ukraine and Italy. Moreover, chicken-origin APMV-4 strains isolated from the LBMs were highly similar to wild bird-origin viruses from nearby lakes with free-living wild birds. In additional, a hemagglutination-negative APMV-4 virus was identified. These findings, together with recent APMV-4 studies, suggest potential virus interspecies transmission between wild birds and domestic poultry, and reveal possible epidemiological intercontinental connections between APMV-4 transmission by wild birds.

Characterization of avian paramyxovirus type 6 isolated from a Eurasian teal in the intersection of migratory flyways in Russia.

The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments.

Surveillance of avian paramyxovirus serotype-1 in migratory waterfowls in Japan between 2011 and 2013.

To further understand the epidemiology of avian paramyxovirus serotype-1 (APMV-1) in migratory waterfowls in Japan, we conducted the surveillance of this virus from feces derived from the migratory waterfowls collected in 41 Japanese prefectures between October 2011 and March 2013. Six APMV-1 viruses were isolated from total 661 samples. All isolates were identified as the avirulent (lentogenic) type on the basis of intracerebral pathogenicity tests. Genetic analysis showed that these viruses possessed the deduced amino acid sequence of 112GKQGR-L117 or 112ERQER-L117 at the cleavage site of the F0 protein, which was identical to the motif in the avirulent type. Phylogenetic analysis based on the partial fusion protein gene classified these APMV-1 isolates into 2 major genetic groups. Four isolates were classified as class II genotype I, and they were genetically closely related to strains isolated in Asian countries, including Japan. In contrast, two isolates were classified as class I, and they were genetically closely related to strains mainly isolated in the U.S.A.

Surveillance of avian paramyxovirus in migratory waterfowls in the San-in region of western Japan from 2006 to 2012.

Relatively little is known about the distribution of avian paramyxoviruses (APMVs) among wild birds in Japan. Surveillance of APMV in migratory waterfowl was conducted in the San-in region of western Japan during winters of 2006 to 2012. A total of 16 avian paramyxoviruses consisting of 3 lentogenic Newcastle disease viruses (NDVs), 12 APMV-4 and 1 APMV-8 were isolated from 1,967 wild-bird fecal samples. The results show that NDV and APMV-4 are relatively widely distributed among wild waterfowl that migrate to Japan from northern regions. Phylogenetic analysis revealed that there was no genetic relationship between the isolates from wild birds and domestic poultry in Japan. However, surveillance of APMVs in wild waterfowl needs to be conducted due to the pathogenic potential of these isolates in domestic poultry.

Evaluation of the replication, pathogenicity, and immunogenicity of avian paramyxovirus (APMV) serotypes 2, 3, 4, 5, 7, and 9 in rhesus macaques.

Avian paramyxoviruses (APMV) serotypes 1-9 are frequently isolated from domestic and wild birds worldwide. APMV-1 (also called Newcastle disease virus, NDV) is attenuated in non-human primates and is being developed as a candidate human vaccine vector. The vector potential of the other serotypes was unknown. In the present study, we evaluated nine different biologically- or recombinantly-derived APMV strains for the ability to replicate and cause disease in rhesus macaque model. Five of the viruses were: biologically-derived wild type (wt) APMV-2, -3, -5, -7 and -9. Another virus was a recombinant (r) version of wt APMV-4. The remaining three viruses were versions of wt rAPMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for clinical disease, virus shedding from the upper and lower respiratory tract, and seroconversion. Virus shedding was not detected for wt APMV-5. Very limited shedding was detected for wt rAPMV-4 and modified rAPMV-4, and only in a subset of animals. Shedding by the other viruses was detected in every infected animal, and usually from both the upper and lower respiratory tract. In particular, shedding over a number of days in every animal was observed for modified rAPMV-2, wt APMV-7, and modified rAPMV-7. Modification of the F protein cleavage site appeared to increase shedding by wt rAPMV-2 and marginally by wt rAPMV-4. All APMVs except wt APMV-5 induced a virus-specific serum antibody response in all infected animals. None of the animals exhibited any clinical disease signs. These results indicate that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype. This suggests that they are not likely to significantly infect primates in nature, and represent promising attenuated candidates for vector development.

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.

Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 4 confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks.

To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks.

Phylogenetic analysis and comparison of eight strains of pigeon paramyxovirus type 1 (PPMV-1) isolated in China between 2010 and 2012.

Eight strains of pigeon paramyxovirus type 1 (PPMV-1) were isolated and identified in this study, from diseased pigeon flocks suspected to be infected with PPMV-1 in China between 2010 and 2012. These PPMV-1 isolates were purified using specific-pathogen-free (SPF) chicken embryo cells before full-length genomic sequencing. The complete genome of these isolates contained 15,192 nucleotides, similar to those of Newcastle disease virus (NDV) strains in genotypes V-XI, with the gene order 3'-NP-P-M-F-HN-L-5'. A six-nucleotide insertion was found to be located in the 5' non-coding region of the nucleoprotein gene in our eight PPMV-1 strains when compared with those of genotypes I, II, III, IV and V. The cleavage site of the fusion protein was (112)RRQKRF(117), a feature generally associated with virulent NDV strains. The structural proteins were in accordance with those of other PPMV-1 strains, with the exception of the W protein of pigeon/CHINA/LJL/100605. The length of the W protein was 227 amino acids, in common with PPMV-1 strains, whereas that of pigeon/CHINA/LJL/100605 was only 181 amino acids. Phylogenetic analysis, based on the genomic sequences and sequences of the fusion gene, revealed that our eight isolates should be classified as class II genotype VIb NDVs. To our knowledge, this is the first report to show that the strain pigeon/CHINA/LLN/110713 is similar to strains isolated abroad, but it was isolated in China, which implies that it may have been introduced to China from overseas. Differences between the Chinese and foreign strains were identified in three regions (nucleotide positions 1632-2229, 3023-3310 and 6103-6439). In addition, the values of ICPI and MDT demonstrated that PPMV-1 isolates were mesogenic or lentogenic, and virulence studies showed that these PPMV-1 strains were non-pathogenic in chickens, but they induced the generation of antibodies in vivo.

Full genomic sequence of an African avian paramyxovirus type 4 strain isolated from a wild duck.

A random amplification/deep sequencing approach was applied to determine the complete genomic sequence of an Avian Paramyxovirus Type 4 (APMV-4) strain isolated from a wild duck in South Africa in 2010. This sequence represents the fourth full genome of APMV-4 in public sequence databases and the first for the African continent. A total of 87,402,081 Illumina paired-end reads were obtained of which 47,338,867 (54.16 %) mapped to the reference genome EU877976. The entire genomic sequence of 15,054 nt, including the intact termini, was recovered at a high redundancy (coverage per base: average = 198,861.06, minimum = 52 and maximum = 1,790,889). Pairwise comparison of full genomic nucleotide sequences indicated that APMV-4/Egyptian goose/South Africa/N1468/10 shared 97.3 % sequence identity with APMV-4/KR/YJ/06, 96.4 % sequence identity with APMV-4/mallard/Belgium/15129/07 and 90.8 % nucleotide sequence identity with APMV-4/duck/HK/D3/75. Genomic features were consistent with previously sequenced viruses including predicted open reading frames for the NP, P, F and L genes, but variations in coding regions for the M and HN genes were identified. The sequencing approach adopted in this study could successfully indicate quasispecies in the viral stock.

Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1-9 in chickens and ducks.

Avian paramyxovirus (APMV) serotypes 1-9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2-9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2-9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2-9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2-9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1-9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.

Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 2 increase cleavability and syncytium formation but do not increase viral virulence in chickens.

Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety of avian species around the world. In this study, we constructed a reverse genetics system for recovery of infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The reverse genetics system was used to analyze the role of the cleavage site of the fusion (F) protein in viral replication and pathogenesis. The cleavage site of APMV-2/Yuc (KPASR↓F) contains only a single basic residue (position -1) that matches the preferred furin cleavage site [RX(K/R)R↓]. (Underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Contrary to what would be expected for this cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is avirulent in chickens. A total of 12 APMV-2 mutants with F protein cleavage site sequences derived from APMV serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these cleavage sites are associated with protease dependence and lack of syncytium formation in their respective native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence, syncytium formation, and increased replication in cell culture. Examination of selected mutants during a pulse-chase experiment demonstrated an increase in F protein cleavage compared to that for wild-type APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F protein cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that structural features in addition to the cleavage site play a major role in the cleavability of the F protein and the activity of the cleaved protein. Furthermore, cleavage of the F protein is not a determinant of APMV-2 pathogenicity in chickens.

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9.

Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.