Pathogenicity In Chickens Research Articles

Prostate-Specific Antigen-Retargeted Recombinant Newcastle Disease Virus for Prostate Cancer Virotherapy

Oncolytic virus (OV) therapies of cancer are based on the use of replication-competent, tumor-selective viruses with limited toxicity. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising OV and is inherently tumor selective and cytotoxic only to tumor cells. Replication is restricted in normal cells. Despite encouraging phase I/II clinical trials with NDV, further refinements for tumor-specific targeting are needed to enhance its therapeutic index. Systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity, and the extracellular matrix. Overcoming these hurdles is paramount to realizing the exceptional oncolytic efficacy of NDV. We engineered the F protein of NDV and generated a recombinant NDV (rNDV) whose F protein is cleavable exclusively by prostate-specific antigen (PSA). The rNDV replicated efficiently and specifically in prostate cancer (CaP) cells and 3-dimensional prostaspheres but failed to replicate in the absence of PSA. Induction of intracellular PSA production by a synthetic androgen analog (R1881) enhanced fusogenicity in androgen-responsive CaP cells. Further, PSA-cleavable rNDV caused specific lysis of androgen-independent and androgen-responsive/nonresponsive CaP cells and prostaspheres, with a half-maximal effective concentration (EC50) ranging from a multiplicity of infection of 0.01 to 0.1. PSA-retargeted NDV efficiently lysed prostasphere tumor mimics, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating no pathogenicity for chickens. Prostate-specific antigen targeting is likely to enhance the therapeutic index of rNDV owing to tumor-restricted replication and enhanced fusogenicity.

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.

Prevalence and characterization of influenza viruses in diverse species in Los Llanos, Colombia

While much is known about the prevalence of influenza viruses in North America and Eurasia, their prevalence in birds and mammals in South America is largely unknown. To fill this knowledge gap and provide a baseline for future ecology and epidemiology studies, we conducted 2 years of influenza surveillance in the eastern plains (Los Llanos) region of Colombia. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) identified influenza viruses in wild birds, domestic poultry, swine and horses. Prevalence ranged from 2.6% to 13.4% across species. Swine showed the highest prevalence and were infected primarily with 2009 pandemic H1N1 (pH1N1) viruses genetically related to those in humans. In addition, we isolated H5N2 viruses from two resident species of whistling ducks (genus Dendrocygna) that differed completely from previous South American isolates, instead genetically resembling North American wild bird viruses. Both strains caused low pathogenicity in chickens and mammals. The prevalence and subtype diversity of influenza viruses isolated from diverse species within a small area of Colombia highlights the need for enhanced surveillance throughout South America, including monitoring of the potential transmissibility of low-pathogenic H5N2 viruses from wild birds to domestic poultry and the emergence of reassortant viruses in domestic swine.Emerging Microbes & Infections (2013) 2, e20; doi:10.1038/emi.2013.20

鳥インフルエンザの正体とその克服法

Highly pathogenic avian influenza virus (HPAIV) causes lethal infection in gallinaceous poultry such as chickens. HPAIV is generated when a nonpathogenic virus brought in by migratory birds from nesting lakes in the north is transmitted to chickens via domestic ducks, geese, quails, etc. and acquires pathogenicity for chickens with repeated multiple infections in the chicken population. Now H5N1 HPAIV has spread to 62 countries in Eurasia and Africa. H5N1 HPAIVs were isolated from dead water birds in Mongolia and Hokkaido, Japan on the way back to their nesting lakes in Siberia in April to May 2005, 2006, 2008, 2009 and 2010. It is concerned that these HPAI viruses may perpetuate in the lakes where migratory water birds nest in summer. On 14th October in 2010, 2 H5N1 HPAIVs were isolated from fecal samples of ducks who flew from Siberia to Ohnuma Lake in Wakkanai, Hokkaido, Japan. Since then, 24 outbreaks of avian influenza due to infection with the H5N1 viruses closely related to the viruses isolated from fecal samples of ducks in Hokkaido in October 2010 have occurred in 9 different prefectures in Japan until the end of March 2011. The other serious concern is that 620 people have been infected with the H5N1 virus, 60% of whom died in 15 countries since 2004 (as of 15 February 2013). It is noted that most of the human cases (86%) are in China, Viet Nam, Indonesia, and Egypt where bird flu vaccines are used. It is strongly proposed to eradicate the H5N1 HPAIVs from Asia by stamping-out without misuse of vaccine through international collaboration under the umbrella of ”One World, One Health” concept.

What's in a strain? Viral metagenomics identifies genetic variation and contaminating circoviruses in laboratory isolates of pigeon paramyxovirus type 1

We used next generation sequencing on random amplified viral nucleic acids to determine the genome sequence of 11 pigeon paramyxovirus type 1 (PPMV-1) isolates from Belgium (period 1998–2011). The PPMV-1 deep sequence data allowed identification of sequence variability in multiple PPMV-1 isolates, including one STOP codon in the Matrix gene which was present in 15% of the viral population of one isolate. Notably, mutations that were previously associated with pathogenicity in chickens were identified as minor sequence variants in one parent laboratory strain. A phylogenetic analysis of the consensus PPMV-1 genome sequences was performed. In addition to providing nearly complete paramyxovirus genome sequences, our sequence-independent approach identified the presence of pigeon circovirus (PiCV) sequences in four of these viral stocks. Real-time quantitative RT-PCR analysis specific for PMV-1 and PiCV showed that these contaminations were present in seven viral stocks consisting of allantoic fluids and was occasionally also detected in stocks passaged in embryonated chicken eggs. Phylogenetic analysis of the PiCV consensus genome sequences showed a circulation of PiCV covering the full genetic diversity of known PiCV.This study shows the value of novel sequence independent technologies for access to sequence information for the control of reference virus stocks and other biological materials, as co-infecting viruses or sequence variants from the original sample may persist in the stocks without being identified by the routine virus-specific diagnostic tools. The exact role of PiCV in pigeon disease – in particular Newcastle disease – and its potential interference with PPMV-1 diagnostics remains to be investigated.

Virulent Avian Infectious Bronchitis Virus, People’s Republic of China

A virulent avian infectious bronchitis virus (IBV) was isolated from 30-day-old broiler chickens that exhibited respiratory symptoms, nephropathologic lesions, and a high proportion of deaths in the People's Republic of China during 2005. The strain, designated YN, was genetically and pathologically characterized. Phylogenetic analysis showed that YN and most of the previously characterized IBV isolates found in China were phylogenetically classified into 2 main genetic clusters. The YN isolate caused severe lesions and resulted in deaths of 65% in experimental infections of 30-day-old specific-pathogen-free chickens. Tracheal and severe kidney lesions developed in all infected birds, confirming the ability of YN strain to induce both respiratory and renal disease. IBV antigens were detected by immunohistochemical analysis in the trachea, lung, kidney, and bursa, consistent with histopathologic observations, virus isolation, and reverse transcription PCR detection. We showed that YN IBV exhibits severe pathogenicity in chickens, and that similar viruses are prevalent in China.

Pathogenicity and transmissibility of reassortant H9 influenza viruses with genes from pandemic H1N1 virus

Both H9N2 avian influenza and 2009 pandemic H1N1 viruses (pH1N1) are able to infect humans and swine, which has raised concerns that novel reassortant H9 viruses with pH1N1 genes might be generated in these hosts by reassortment. Although previous studies have demonstrated that reassortant H9 viruses with pH1N1 genes show increased virulence in mice and transmissibility in ferrets, the virulence and transmissibility of reassortant H9 viruses in natural hosts such as chickens and swine remain unknown. This study generated two reassortant H9 viruses (H9N2/CA09 and H9N1/CA09) in the background of the pH1N1 A/California/04/2009 (CA09) virus by replacing either both the haemagglutinin (HA) and neuraminidase (NA) genes or only the HA gene with the respective genes from the A/quail/Hong Kong/G1/1997 (H9N2) virus and evaluated their replication, pathogenicity and transmission in chickens and pigs compared with the parental viruses. Chickens that were infected with the parental H9N2 and reassortant H9 viruses seroconverted. The parental H9N2 and reassortant H9N2/CA09 viruses were transmitted to sentinel chickens, but H9N1/CA09 virus was not. The parental H9N2 replicated poorly and was not transmitted in pigs, whereas both H9N2/CA09 and H9N1/CA09 viruses replicated and were transmitted efficiently in pigs, similar to the pH1N1 virus. These results demonstrated that reassortant H9 viruses with pH1N1 genes show enhanced replication and transmissibility in pigs compared with the parental H9N2 virus, indicating that they may pose a threat for humans if such reassortants arise in swine.

Listeria monocytogenes in retailed raw chicken meat in Malaysia

This study aimed to determine the prevalence Listeria monocytogenes in raw chicken meat samples at hypermarkets and wet markets. Chicken drumsticks, breasts, and thighs were randomly selected. The most probable number (MPN) PCR method was used to quantify the L. monocytogenes in the samples. Listeria monocytogenes was detected in 20% of the samples. Occurrence of L. monocytogenes was highest in breast (42.03%) followed by drumstick (11.27%) and thigh (7.14%). Samples from hypermarkets showed higher occurrence (25.71%) of L. monocytogenes compared with wet markets (14.29%). The density of L. monocytogenes found in samples ranged from <3.0 to 16 MPN•g(-1). The presence of L. monocytogenes in raw chicken meat is unwanted but unpreventable. Thus, further research on the processing method to reduce and eliminate this kind of bacteria in chicken meat before consumption is necessary. The presence of L. monocytogenes in chicken samples suggests the importance of this pathogen in chicken. Thus, more study is needed to find ways to eliminate this pathogen from poultry.

Age-dependent response to CpG oligodeoxynucleotides against organ invasion of Salmonella enteritidis in broiler chickens

Bacterial infections cause significant economic losses in the broiler industry throughout the production cycle. It is known that CpG-ODN stimulated innate immune activities and enhanced the resistance to infectious pathogens in chickens. In this study, the effectiveness of CpG-ODN was evaluated in organ colonization by Salmonella enteritidisat different ages of chickens. Pre-treatment of 2-day old chickens with CpG-ODN by subcutaneous injection and subsequent challenge with S. enteritidis 3 h after treatment resulted in significant reduction of organ invasion by S. enteritidis. In a second experiment, these chickens were repeatedly treated with CpG-ODN at day 20, and 24 h later were orally infected with S. enteritidis. There was no significant reduction in organ invasion byS. enteritidis compared to PBS treated controls. In a third experiment, five-day old chickens were treated with CpG-ODN and 24 h later orally infected with S. enteritidis. There was no reduction in organ invasion compared to PBS treated control group. Our study shows that CpG-ODN increase resistance against S. enteritidis invasion in broiler chicken in early age. Key words: CpG-ODN, Salmonella enteritidis, immunoprotection, poultry.

Rescue of virulent class I Newcastle disease virus variant 9a5b-D5C1

BackgroundThe virulent class I Newcastle disease virus (NDV) variant 9a5b was generated from a nonvirulent NDV isolate Goose/Alaska/415/91 via nine consecutive passages in the chicken air sac, followed by five passages in the chick brain. The evolutionary mechanism of virulence in the class I NDV isolate is not fully understood. To elucidate this evolutionary mechanism, a reverse genetics manipulation specific for class I NDV is indispensable.ResultsA full-length cDNA clone of 9a5b and the helper plasmids pCI-NP, pCI-P, and pCI-L were constructed from segments of cDNA. After these plasmids were co-transfected into BSR T7/5 cells, infectious viral particles were obtained. The rescued viruses were genetically and biologically identical to the parental strain and showed similar pathogenicity in chickens.ConclusionA stable recovery method for class I NDV was established. Reverse genetics of the class I NDV variant 9a5b allowed for the generation of genetically altered and virulent NDV, and can be used as a foundation for research on the evolution of virulence in class I NDV isolates.

Campylobacter jejuni is highly susceptible to killing by chicken host defense peptide cathelicidin-2 and suppresses intestinal cathelicidin-2 expression in young broilers

Little is known about the interactions of chicken host defense peptides (HDPs) with Campylobacter jejuni in young chicks. To examine the role of the chicken HDP, cathelicidin-2 (CATH-2) in host–pathogen interactions we challenged 4-day-old Ross 308 broilers with a chicken-derived C. jejuni isolate (WS356) and used the chicken pathogen Salmonella enterica Enteritidis phage type 4 (FGT1) as a reference. Immunohistochemical staining was used to localize CATH-2, C. jejuni and Salmonella enteritidis. Intestinal CATH-2 mRNA expression levels were determined by quantitative PCR. Antibacterial activities of CATH-2 peptide against C. jejuni and S. enteritidis isolates were assessed in colony count assays. In contrast to S. enteritidis, C. jejuni was not seen to attach to intestinal epithelium and C. jejuni challenge did not result in recruitment of CATH-2 containing heterophils to the small intestinal lamina propria. Minimal inhibitory concentrations found for CATH-2 peptide against human- and chicken-derived C. jejuni isolates were similar (0.6–2.5μM) and much lower than for S. enteritidis (20μM). Compared to wild-type C. jejuni 81116, the lipooligosaccharide (LOS)-deficient 81116ΔwaaF mutant was much more susceptible to CATH-2. Interestingly, CATH-2 mRNA expression levels in the small intestine were significantly lower 48h p.i. in C. jejuni-challenged chicks. These findings indicate that human clinical and chicken-derived C. jejuni are equally highly susceptible to chicken CATH-2 peptide and that C. jejuni uses LOS to protect itself to some extent against HDPs. Moreover, suppression of intestinal CATH-2 expression levels may be part of the C. jejuni immune evasion strategy.

Fowl Adenovirus (FAdV) in India: Evidence for Emerging Role as Primary Respiratory Pathogen in Chickens

Adenoviruses have been isolated from both clinically healthy and diseased birds worldwide. The pathogenic role of most of the FAdVs is still questionable. They can quickly take on the role of opportunistic pathogens when additional factors, particularly concurrent infections, adversely affect the health of the avian host. Immnosuppressing agents especially chicken infectious anemia and infectious bursal disease viruses are known to enhance the pathogenicity of FAdVs upon coinfection. The aim of the present study was to screen for the involvement of FAdV in poultry flocks affected with respiratory disease complex by RT-PCR. The samples were also screened by RT-PCR/PCR for other respiratory pathogens. Thirty two commercial poultry flocks with the history of respiratory disease complex from various parts of India. FAdV nucleic acid could be detected in tissue samples of 13 out of 34 farms investigated. Out of 13 FAdV positive farms, FAdV and CIAV were alone detected in 4/13 (31%) whereas, in other farms more than two respiratory pathogens were detected together. CIAV was detected in all the farms (34/34) investigated. Eosinophilic intranuclear inclusion bodies were noticed in FAdV infected laryngeal and tracheal epithelium under light microscopy. The findings of the study assert that FAdV can play the role of primary respiratory pathogen in immunocompromised birds and also in the presence of other respiratory pathogens.

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 101.9 TCID50/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.

Prophylactic treatment with Toll-like receptor ligands enhances host immunity to avian influenza virus in chickens

Avian influenza viruses (AIV) pose a threat towards the health of both poultry and humans. To interrupt the transmission of the virus, novel prophylactic strategies must be considered which may reduce the shedding of AIV. One potential is the prophylactic use of Toll-like receptor (TLR) ligands. Many cells of the immune system express TLRs, and cellular responses to TLR stimulation include activation and the production of cytokines. TLR ligands have been employed as prophylactic treatments to enhance host resistance to pathogens both in mammals and chickens. Therefore, the present study was conducted to determine whether TLR ligands may be used prophylactically in chickens to enhance host immunity to AIV. Chickens received intramuscular injections of either low or high doses of the TLR ligands poly I:C, lipopolysaccharide (LPS) and CpG ODN. Twenty-four hours post-treatment, chickens were infected with the low pathogenic avian influenza virus H4N6, and both oropharyngeal and cloacal virus shedding were assessed on days 4 and 7 post-infection. To identify potential correlates of immunity, spleen and lungs were collected on days 2, 4 and 7 post-infection for RNA extraction. The results suggested that all of the TLR ligand treatments induced a significant reduction in virus shedding, with the TLR3 ligand poly I:C conferring the greatest AIV immunity compared to control birds, followed by CpG ODN and LPS. Furthermore, transcriptional analysis of gene expression in the spleen and lungs suggest IFN-α and IL-8 as correlates of immunity conferred by poly I:C, and IFN-γ for CpG ODN and LPS. In conclusion, TLR ligands, have the ability to enhance host immunity against AIV, and future studies should consider exploring the combinatory effects of poly I:C and CpG ODN prophylaxis in conjunction with AIV vaccination.

Two Highly Pathogenic Avian Influenza H5N1 Viruses of Clade 2.3.2.1 with Similar Genetic Background but with Different Pathogenicity in Mice and Ducks

A number of genetic markers for virulence of avian influenza viruses (AIVs) in different hosts have been identified. However, we isolated two H5N1 AIVs, A/Chicken/Jiangsu/k0402/2010(CK/10) and A/Goose/Jiangsu/k0403/2010(GS/10) with similar genetic background, but most well-defined molecular markers for virulence in mammals and avian species were not found in both viral genomes. In addition, pathogenicity of this pair of viruses in different hosts remains unclear. Therefore, we evaluated their pathogenicity in chickens, mice, ducks and guinea pigs. Infection of CK/10 and GS/10 in chickens caused 100% mortality within 24 h. Mouse experiment showed that CK/10 was highly pathogenic (MLD50 = 0.33 log10 EID50), whereas GS/10 was avirulent (MLD50 > 6.32 log10 EID50). Interestingly, the virulence of CK/10 in ducks (DLD50 = 3.83 log10 EID50) was higher than that of GS/10 (DLD50 = 7.7 log10 EID50), which correlated with viral pathogenicity in mice. Although CK/10 and GS/10 showed distinct pathogenicity in mice, they both were lethal to guinea pigs, with CK/10 replicating to higher titres in airways than GS/10. Collectively, these findings suggest that AIVs with similar genetic backgrounds may exhibit distinct pathogenicity in specific hosts and that some unknown molecular markers for virulence may exist and need to be identified.

Epidemiological survey and genetic evolution of H9 subtype influenza viruses in Shanghai, China, from 2006 to 2010

The H9N2 influenza virus is endemic in poultry. We report its occurrence in live-poultry markets, fair-trade markets and poultry farms in the Shanghai region between September 2006 and December 2010. An analysis of partial sequences of the HA, NA, PB1, PB2 and NP genes of eleven distinct H9N2 isolates revealed that all carried an RSSR motif at the cleavage site of HA, diagnostic of low pathogenicity in chickens. A phylogenetic analysis indicated that these isolates are derived from the lineage represented by Duck/HK/Y280/97, but they have evolved a range of reassortments. Their PB1 and NP sequences resembled those of H5N1 strains, indicating a hybrid origin involving both H9 and H5 strains. The HA and NA sequences present in all eleven isolates resembled those of the Duck/HK/Y280/97-like lineage. Infection by H9N2 is commonplace in Shanghai live-poultry markets, allowing the viruses to have evolved rapidly.

An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers

The colonization of broiler chickens with Salmonella can pose serious health and economic risks for both consumers and the poultry industry. Because colonization with Salmonella can lead to subsequent contamination of chicken carcasses during processing, preemptive control measures should include the reduction of this pathogen in chickens before slaughter. In this study, we evaluated the effect of sodium bisulfate, a potential antimicrobial feed additive, on Salmonella colonization of experimentally infected broiler chickens. Two hundred and forty 1-d-old chickens were infected orally with Salmonella enterica serotype Enteritidis and divided into 4 groups (each comprised of 60 chickens). Three groups received different concentrations of sodium bisulfate integrated into their feed, while the feed of the fourth group (positive control) was not treated. At time points before the broilers' slaughter age, different organs/tissues (liver, spleen, cecum, and bone marrow) and feces were aseptically collected and tested for the occurrence and density of Salmonella Enteritidis. Our results show that at 3 d postinfection, high colonization with Salmonella Enteritidis was detected and affected all tested tissues and fecal samples. Although colonization decreased across time, Salmonella Enteritidis persisted in the cecum, feces, spleen, and bone marrow, but not in the liver, until slaughter age. Furthermore, the addition of sodium bisulfate to the feed did not significantly reduce Salmonella Enteritidis numbers in infected chickens or affect the shedding of the pathogen.

Diagnostic Strategy for Identifying Avian Pathogenic Escherichia coli Based on Four Patterns of Virulence Genes

In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories.

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.

English

&nbsp; Mycoplasma synoviae&nbsp;(MS)&nbsp;is one of the important pathogens in chicken and turkey which cause great economic losses in poultry industry.&nbsp;M. synoviae&nbsp;has one serotype but there is heterogeneity among MS strains. The aim of this study was to analyze the DNA sequence of Mycoplasma&nbsp;synoviae&nbsp;isolates from Mazandran province poultry flocks of Iran and compare them with MS from other countries. For this purpose, 32 samples were collected from choanal cleft, trachea and air sac of flocks with respiratory symptom and cultured in specific PPLO medium. Ten out of the 32 samples were positive in culture&nbsp;and reacted with specific antisera. The 10 positive samples were subsequently used for molecular study. The PCR products containing 16s rRNA genome were generated with specific primers and then sequenced. Multiple alignment and phylogenic analysis of DNA sequences was performed with DNASTAR program. Alignment and comparison of the sequences in DANSTAR program revealed that 7 of 10 isolate are quite similar and 3 other isolate are different from group one. Phylogenic analysis of&nbsp;M. synoviae&nbsp;isolated from Mazandran province and 21&nbsp;M. synoviae&nbsp;from the entire world revealed low homology among most of them. However, some of the Mazandran isolates showed high homology with&nbsp;M. synoviaeisolated from Brasilia. Overall, these results revealed the different molecular structure and heterogeneity among&nbsp;M. synoviae&nbsp;isolated from Mazandran province in this study. &nbsp; Key words:&nbsp;Mycoplasma sinoviae, sequence, phylogenetic, poultry, Mazandran.