Wide Variety Of Avian Species Research Articles

Molecular Identification and Phylogenetic Study Based on the Fusion Gene of Newcastle Disease Virus Isolated from Broiler Poultry Farms in Markazi Province, Iran.

Newcastle disease (ND) is an economically significant and extremely spreadable viral illness affecting a wide variety of avian species. ND can rapidly spread within poultry farms and result in considerable economic losses for the global poultry industry. This disease is endemic in Iran, and despite intensive vaccination efforts in the poultry industry, outbreaks of ND occur unexpectedly. This study aimed to isolate the Newcastle disease virus (NDV) from poultry farms with breathing problems in Markazi province, Iran, and investigate the evolutionary relationship and molecular characteristics of the isolates during 2017-2019. To this end, tissue samples (lung, brain, and trachea) were taken from 42 broiler farms exhibiting respiratory symptoms. The samples were inoculated into 9-11-day-old embryonated eggs, and the virus was isolated from 20 (47.6%) of the 42 farms. Subsequently, RT-PCR was used to amplify partial fusion gene sequences from the new isolates. The amplified products were sequenced and compared phylogenetically to the standard pilot dataset (125 selected sequences) generated by the NDV consortium. As determined by phylogenetic analysis, all nine isolates belonged to subgenotype VII.1.1 of genotype VII and were highly similar to isolates from other parts of Iran and China. Moreover, all isolates possessed a polybasic cleavage site motif (112RRQKRF117), characteristic of virulent strains. Furthermore, the present isolates shared a high nucleotide identity (96%) with viruses previously isolated from other provinces of Iran, as determined by BLAST searches and multiple alignments. In addition, they shared a high degree of sequence similarity but were distinct from the existing NDV vaccines. Therefore, the genetic dissimilarity between current vaccine strains and circulating NDVs must be considered in vaccination programs.

Immunization of broiler chickens with recombinant alpha-toxin protein for protection against necrotic enteritis

Necrotic enteritis ( NE ), caused by Clostridium perfringens ( CP ), has been reported in a wide variety of avian species including chickens. Necrotic enteritis has been reported in most parts of the world with acute outbreaks occurring sporadically and most often in broiler chickens after maternal immunity declines with bird age. This study was conducted to develop computer-aided design of a recombinant CP protein vaccine in an experimental model of necrotic enteritis. A synthetic rCPA which has been designed using predictive tools expressed and obtained through cloning, used to immunize and assess intestinal lesion scores, antibody levels, growth performance, and CP reduction in vivo. Broilers were immunized on days 7, 14, and 21 (V groups) with the purified recombinant clostridium alpha-toxin. On days 24, 25, and 26, the birds were orally challenged with CP (1.0 × 10 9 CFU/bird). Increased body weight (BW), clearance of CP, increased antibodies, and intestinal lesion scores were measured as protection parameters after oral co-infection with CP. Birds immunized and challenged with recombinant protein (CHV) experienced significantly less body weight loss and less intestinal lesions compared to the positive control group (PC). Higher anti-recombinant anti-rCPA antibody titers were observed in the challenged vaccinated (CHV) and only vaccinated (V) groups with an increased level CP clearance of intestinal contents in immunized birds. Based on these results, vaccination with computer-aided clostridial recombinant proteins, particularly rCPA toxin, enhances protective immunity against experimental necrotic enteritis in broiler chickens.

Novel adenovirus associated with common tern (Sterna hirundo) chicks.

Adenoviruses have been identified in a wide variety of avian species, and in some species, they have been shown to cause disease and increase mortality. As part of an endeavor to investigate viruses associated with common terns (Sterna hirundo), a novel adenovirus was identified in fecal samples from two common terns on Gull Island, Lake Ontario, Canada. The coding-complete genome sequence of the new adenovirus is 31,094 bp, containing 28 putative genes, and this is the first adenovirus to be associated with terns. The virus was identified in two out of 13 fecal samples from tern chicks, and it was found to be most closely related to duck adenovirus 1, with the DNA polymerase sharing 58% amino acid sequence identity. Phylogenetic analysis based on DNA polymerase protein sequences showed that the new virus forms a distinct sub-branch within the atadenovirus clade and likely represents a new species in this genus.

Genetic diversity of avian paramyxoviruses isolated from wild birds and domestic poultry in Taiwan between 2009 and 2020.

Avian paramyxoviruses (APMVs) belonging to the subfamily Avulavirinae within the family Paramyxoviridae. APMVs consist of twenty-two known species and are constantly isolated from a wide variety of avian species around the world. In this study, the APMV isolates obtained from wild birds and domestic poultry during 2009–2020 in Taiwan were genetically characterized by phylogenetic analysis of their complete fusion protein gene or full-length genome. As a result, 57 APMV isolates belonging to seven different species were obtained during this period and subsequently identified as APMV-1 (n=17), APMV-2 (n=1), APMV-4 (n=25), APMV-6 (n=8), APMV-12 (n=2), APMV-21 (n=2) and APMV-22 (n=2). Sanger sequencing was performed to provide 22 full-length genome sequences and 35 complete fusion protein gene sequences for the APMV isolates. Phylogenetic analysis showed that the recovered viruses were closely related to Eurasian strains, except five class I APMV-1 and four APMV-4 isolates were related to North America strains. Our findings provided more evidence for the intercontinental transmission of APMVs between Eurasia and North America by wild birds. In addition, according to the criteria of the classification system based on complete fusion protein gene sequences, three novel genotypes within APMV-2, APMV-12, and APMV-22 were identified. Together, this investigation provided a broader perspective on the genetic diversity, evolution, and distribution of APMVs in multiple avian host species sampled in Taiwan.

Effect of GPS tagging on behaviour and marine distribution of breeding Arctic Terns Sterna paradisaea

Tracking tags have been used to map the distributions of a wide variety of avian species, but few studies have examined whether the use of these devices has impacts on the study animals that may bias the spatial data obtained. As Global Positioning System (GPS) tags small enough for deployment on terns (family: Laridae) have only recently become available, until now tracking of this group has been conducted by following unmanipulated individuals by boat, which offers a means of comparing distributions obtained from GPS‐tracking. We compared the utilization distributions (UDs) of breeding Arctic Terns Sterna paradisaea obtained by GPS‐tracking 10 individuals over 2 weeks, with UDs derived from contemporaneous visual boat tracks from 81 individuals. The 50% and 95% UDs of both methods had high similarity scores, indicating good agreement in the density distributions derived from the two methods. The footprints of the UDs of tagged birds were ~ 75–80% larger, which may reflect an effect of tagging on foraging range or the occasional inability to follow by boat individuals which roamed further from the colony. We also compared the nest attendance and chick provisioning rates of adults that were (1) fitted with a GPS tag and leg‐flag, (2) handled and marked with a leg‐flag but not tagged and (3) fitted with a leg‐flag in a previous year but unhandled in the year of the study. There was some evidence that birds fitted with both a GPS tag and leg‐flag spent slightly less time at the nest compared with unhandled birds and those fitted with a leg‐flag only. Both treatments where birds were fitted with a leg‐flag in the year of the study had similarly lower provisioning rates to those of unhandled control birds > 48 h after handling, suggesting that negative effects on provisioning are due to capture and handling or leg‐flag attachment rather than to GPS tag attachment/loading per se. Overall brood‐provisioning rate was compensated for by the increased effort by the unhandled partner. Our study suggests that despite slight effects of GPS‐tagging on behaviour, the estimates of marine density distribution obtained were very similar to those of unmanipulated birds.

Molecular evolution and genetic variations of \u2009V and W proteins derived by RNA editing in Avian Paramyxoviruses

The newly assigned subfamily Avulavirinae in the family Paramyxoviridae includes avian paramyxoviruses (APMVs) isolated from a wide variety of avian species across the globe. Till date, 21 species of APMVs are reported and their complete genome sequences are available in GenBank. The APMV genome comprises of a single stranded, negative sense, non-segmented RNA comprising six transcriptional units (except APMV-6 with seven units) each coding for a structural protein. Additionally, by co-transcriptional RNA editing of phosphoprotein (P) gene, two mRNAs coding for accessory viral proteins, V and W, are generated along with unedited P mRNA. However, in APMV-11, the unedited mRNA codes for V protein while +2 edited mRNA translates to P protein, similar to members of subfamily Rubulavirinae in the same family. Such RNA editing in paramyxoviruses enables maximizing the coding capacity of their smaller genome. The three proteins of P gene: P, V and W, share identical N terminal but varied C terminal sequences that contribute to their unique functions. Here, we analyzed the P gene editing site, V and W sequences of all 21 APMV species known so far (55 viruses) by using bioinformatics and report their genetic variations and molecular evolution. The variations observed in the sequence and hexamer phase positions of the P gene editing sites is likely to influence the levels and relative proportions of P, V and W proteins’ expressions which could explain the differences in the pathogenicity of APMVs. The V protein sequences of APMVs had conserved motifs similar to V proteins of other paramyxoviruses including the seven cysteine residues involved in MDA5 interference, STAT1 degradation and interferon antagonism. Conversely, W protein sequences of APMVs were distinct. High sequence homology was observed in both V and W proteins between strains of the same species than between species except in APMV-3 which was the most divergent APMV species. The estimates of synonymous and non-synonymous substitution rates suggested negative selection pressure on the V and W proteins within species indicating their low evolution rate. The molecular clock analysis revealed higher conservation of V protein sequence compared to W protein indicating the important role played by V protein in viral replication, pathogenesis and immune evasion. However, we speculate the genetic diversity of W proteins could impact the degree of pathogenesis, variable interferon antagonistic activity and the wide host range exhibited by APMV species. Phylogenetically, V proteins of APMVs clustered into three groups similar to the recent classification of APMVs into three new genera while no such pattern could be deciphered in the analysis of W proteins except that strains of same species grouped together. This is the first comprehensive study describing in detail the genetic variations and the molecular evolution of P gene edited, accessory viral proteins of Avian paramyxoviruses.

Ritual Deposition of Avifauna in the Northern Burial Cluster at Pueblo Bonito, Chaco Canyon

Birds play an important role in modern and historic Puebloan ceremonialism, with feathers, wings, stuffed birds, and live birds used for ceremonies, sacrifices, and creation of ritual paraphernalia. Archaeological evidence suggests birds played an ideological and ritual role for some prehispanic Southwestern groups as well. Here, we explore the use of avian remains for ritual purposes at Pueblo Bonito by presenting new evidence from the northern burial cluster. Drawing on multiple quantitative measures, we establish the dissimilarity of this assemblage to other Chacoan sites. We demonstrate that an unusually wide variety of avian species was deposited in the northern burial cluster, particularly wing elements that may reflect wing fan deposition. These findings improve our understanding of ritual life at Pueblo Bonito and also show the utility of using both qualitative and quantitative data to create a more accurate understanding of past ceremonial activity.

Avian Paramyxovirus Type-3 as a Vaccine Vector: Identification of a Genome Location for High Level Expression of a Foreign Gene.

Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3′-to-5′ attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

West Nile Virus Temperature Sensitivity and Avian Virulence Are Modulated by NS1-2B Polymorphisms.

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.

Avian Paramyxovirus: A Brief Review

Avian paramyxoviruses (APMVs) have been reported from a wide variety of avian species around the world. Avian paramyxoviruses are economically significant because of the huge mortality and morbidity associated with it. Twelve different serotypes of APMV have been reported till date. Avian paramyxoviruses belong to the family Paramyxoviridae under genus Avulavirus. Newcastle disease virus (APMV-1) is the most characterized members among the APMV serotypes. Complete genome sequence of all twelve APMV serotypes has been published recently. In recent years, APMV-1 has attracted the virologists for its oncolytic activity and its use as a vaccine vector for both animals and humans. The recombinant APMV-based vaccine offers a pertinent choice for the construction of live attenuated vaccine due to its minimum recombination frequency, modular nature of transcription and lack of DNA phase during its replication. Although insufficient data are available regarding other APMV serotypes, our understanding about the APMV biology is expanding rapidly because of the availability of modern molecular biology tools and high-throughput complete genome sequencing.

An ex vivo avian leukocyte culture model for West Nile virus infection

West Nile virus (WNV) replicates in a wide variety of avian species, which act as amplification hosts. In particular, WNV generates high titers and elicits severe pathology in American crows (AMCRs; Corvus brachyrhynchos), a species that has been used as a sentinel for WNV transmission. Although the specific cellular targets of WNV replication in AMCRs are not well defined, preliminary evidence suggests that leukocytes may be an important target of early replication. Therefore, development of a protocol for ex vivo culture of AMCR leukocytes as a model for assessing differential avian host susceptibility is described herein. WNV growth in these cultures mirrored in vivo viremia profiles. These data indicate that ex vivo leukocyte cultures can be used for preliminary pathological assessment of novel WNV strains and potentially of other flaviviruses that use avian reservoir hosts.

The Additional Diagnostic Value of Optical Coherence Tomography (OCT) and Its Application Procedure in A Wide Variety of Avian Species

Objective: The current study introduced OCT as a novel tool in clinical veterinary ophthalmology in a much wider variety of free-living avian species than hitherto studied. Methods: OCT was tested and performed in 39 free-living birds (21 species of 12 families) and compared to direct ophthalmoscopy. Birds were examined with combinations of different restraints (manual restraint or fixation on a holding device) and different anaesthesia regimes (none, sedation, general anaesthesia). Inter- and intra-species specific variations of the general procedure, the restraint methods and the clinical findings were evaluated. Results: OCT was possible in all examined avian species (from 40 g up to 7720 g) and superseded direct ophthalmoscopy in quality and quantity of ophthalmological findings. All restraint methods enabled OCT examination, however combination of general anaesthesia and a holding device provided the most rapid and subjectively the least stressful examination technique. Stability, stress reduction, head angle and distance from the OCT device were important factors influencing volume scanning quality. Sixteen, out of 39 birds, presented ocular abnormalities detected by OCT (compared to only five birds when using direct ophthalmoscopy). OCT with included fundus images offered an objective assessment of retinal changes. Retinal abnormalities included changes of fundus pigmentation, drusenoid changes and severe retinal and choroidal degenerations. Species-specific variations of retinal layer dimensions and of foveal structures were evident. Conclusion: OCT is a promising, non-invasive method, which significantly compliments standard techniques. OCT is applicable to a wide variety of avian species; it provides high quality cross-sectional images of the retina, enabling accurate and improved diagnosis and prognosis of therapies. An assessment of the visual capabilities of traumatised birds is a major factor for their rehabilitation and survival in the wild. Finally, this method is an excellent tool in interdisciplinary retinal research, providing novel insights into the diversity of very specialized structural adaptations of avian retina.

Marking power lines to reduce avian collisions near the Audubon National Wildlife Refuge, North Dakota

Overhead power lines can pose collision risks to birds. Risks may be mitigated through marking lines with high-visibility devices, but the effectiveness of line marking remains unclear. Effectiveness is particularly poorly described for lines bisecting open water, where detection of carcasses can be difficult. We marked 3 of 9 spans (lines between adjacent structures) along a causeway crossing open water and 2 adjacent spans over lake shores between Lake Sakakawea and Lake Audubon near Audubon National Wildlife Refuge, North Dakota, USA. Over 3 years, we found 1,186 avian carcasses, including 276 attributed to power-line collision. American coots (Fulica americana; n = 83) and double-crested cormorants (Phalacrocorax auritus; n = 27) were the species most commonly associated with power-line collision, but we also found carcasses of 51 other species, including a piping plover (Charadrius melodus; n = 1). Multi-variable modeling indicated line marking over open water reduced predicted collisions per span per season (mid-April through mid-October, 2006–2008) from 10.3 to 5.8. Birds with high-aspect-ratio wings benefitted most from line marking (e.g., shorebirds and gulls). If the 9 open-water spans we studied were unmarked for 30 years, we predicted 2,775 collisions. We predicted only 1,560 collisions if all of these spans were marked. Our data demonstrate that a wide variety of avian species are at risk of collision with lines bisecting open water, marking lines can reduce collision risk, and because collisions persisted and some line markers fell off power lines, improvements to effectively mark lines are needed. © 2013 The Wildlife Society.

An Iron-Rich Organelle in the Cuticular Plate of Avian Hair Cells

Hair cells reside in specialized epithelia in the inner ear of vertebrates, mediating the detection of sound, motion, and gravity. The transduction of these stimuli into a neuronal impulse requires the deflection of stereocilia, which are stabilized by the actin-rich cuticular plate. Recent electrophysiological studies have implicated the vestibular system in pigeon magnetosensation. Here we report the discovery of a single iron-rich organelle that resides in the cuticular plate of cochlear and vestibular hair cells in the pigeon. Transmission electron microscopy, coupled with elemental analysis, has shown that this structure is composed of ferritin-like granules, is approximately 300-600 nm in diameter, is spherical, and in some instances is membrane-bound and/or organized in a paracrystalline array. This organelle is found in hair cells in a wide variety of avian species, but not in rodents or in humans. This structure may function as (1) a store of excess iron, (2) a stabilizer of stereocilia, or (3) a mediator of magnetic detection. Given the specific subcellular location, elemental composition, and evolutionary conservation, we propose that this structure is an integral component of the sensory apparatus in birds.

Genotypic and Pathotypic Characterization of Newcastle Disease Viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3′ leader and 5′ trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have “died out” after the first panzootic (1926–1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India.

Analysis of the Fusion Protein Cleavage Site of Newcastle disease virus Isolates from India Reveals Preliminary Evidence for the Existence of II, VI and VII Genotypes

Newcastle disease virus (NDV) has been a threat to poultry industry in most of the developing countries with a wide variety of avian species being susceptible, coupled with the presence of mobile wild bird reservoirs contributing not only to the vast genomic diversity of this virus but also to the diagnostic failures. NDV of multiple genotypes (I-XI) is known to be prevalent and reported worldwide. However, there is a paucity of information on the circulating genotypes of NDV in India. This study utilized the fusion protein cleavage site (FPCS) sequence to determine the different genotypes of NDV circulating in India. Our results indicate that majority of NDV isolates from southern states of India namely, Tamil Nadu, Kerala and Karnataka were found to belong to genotype II. However, some of the strains from Tamil Nadu and most from Uttar Pradesh belong to genotype groups VI and VII. Interestingly, three isolates recovered from Tamil Nadu grouped with genotype IV viruses (namely Herts/33) which had not been hitherto reported to GenBank since 1989. This preliminary information points to the existence of multiple genotypes and also the need for efficacy studies with vaccines incorporating multiple genotypes in controlling virulent NDV (vNDV) outbreaks in India.

Evaluation of the Newcastle Disease Virus F and HN Proteins in Protective Immunity by Using a Recombinant Avian Paramyxovirus Type 3 Vector in Chickens

Newcastle disease virus (NDV) belongs to serotype 1 of the avian paramyxoviruses (APMV-1) and causes severe disease in chickens. Current live attenuated NDV vaccines are not fully satisfactory. An alternative is to use a viral vector vaccine that infects chickens but does not cause disease. APMV serotype 3 infects a wide variety of avian species but does not cause any apparent disease in chickens. In this study, we constructed a reverse-genetics system for recovery of infectious APMV-3 strain Netherlands from cloned cDNAs. Two recombinant viruses, rAPMV3-F and rAPMV3-HN, were generated expressing the NDV fusion (F) and hemagglutinin-neuraminidase (HN) proteins, respectively, from added genes. These viruses were used to immunize 2-week-old chickens by the oculonasal route in order to evaluate the contribution of each protein to the induction of NDV-specific neutralizing antibodies and protective immunity. Each virus induced high titers of NDV-specific hemagglutination inhibition and serum neutralizing antibodies, but the response to F protein was greater. Protective immunity was evaluated by challenging the immunized birds 21 days later with virulent NDV via the oculonasal, intramuscular, or intravenous route. With oculonasal or intramuscular challenge, all three recombinant viruses (rAPMV3, rAPMV3-F, and rAPMV3-HN) were protective, while all unvaccinated birds succumbed to death. These results indicated that rAPMV3 alone can provide cross-protection against NDV challenge. However, with intravenous challenge, birds immunized with rAPMV3 were not protected, whereas birds immunized with rAPMV3-F alone or in combination with rAPMV3-HN were completely protected, and birds immunized with rAPMV3-HN alone were partially protected. These results indicate that the NDV F and HN proteins are independent neutralization and protective antigens, but the contribution by F is greater. rAMPV3 represents an avirulent vaccine vector that can be used against NDV and other poultry pathogens.

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.

Influenza: an emerging disease.

Because all known influenza A subtypes exist in the aquatic bird reservoir, influenza is not an eradicable disease; prevention and control are the only realistic goals. If people, pigs, and aquatic birds are the principal variables associated with interspecies transfer of influenza virus and the emergence of new human pandemic strains, influenza surveillance in these species is indicated. Live-bird markets housing a wide variety of avian species together (chickens, ducks, geese, pigeon, turkeys, pheasants, guinea fowl), occasionally with pigs, for sale directly to the public provide outstanding conditions for genetic mixing and spreading of influenza viruses; therefore, these birds should be monitored for influenza viruses. Moreover, if pigs are the mixing vessel for influenza viruses, surveillance in this population may also provide an early warning system for humans.