Report of a new meq gene size: The first study on genetic characterisation of Marek's disease viruses circulating in Iranian commercial layer and backyard chicken.

1. In recent months, several outbreaks with clinical signs of MDV-1 were reported in Iranian parent and laying hen farms, in addition to backyard chickens. Several meq gene sequences from these outbreaks were amplified and molecularly characterised.2. The meq protein sequences revealed three different sizes, namely the standard 339 aa, a shorter form of 338 aa lacking a proline residue at position 191, and a very short (vs) size of 265 aa. Based on sequence and size, the 265 aa meq has never been reported from international research groups before. The protein has only one PPPP repeat motif suggesting it belongs to a highly virulent strain.3. The standard meq sequences showed 100% BLAST identity to the vv+ isolate Polen5. However, the 338 aa form clustered to the clade usually reported from North America.4. This is the first report on genetic analysis of MDV-1 from Iran, but further study is required to obtain a better picture of the diversity and prevalence of different MDV-1 strains circulating in the country's farms, backyard poultry and other bird species.

Characterization and full genome sequencing of a velogenic Newcastle disease virus (NDV) strain Ck/IR/Beh/2011 belonging to subgenotype VII(L).

Avian avulavirus 1, better known as Newcastle disease virus (NDV), causes substantial loss to the poultry industry in many developing countries. In this study we have characterized and fully sequenced the genome of a velogenic NDV strain named Beh (Ck/IR/Beh/2011) that has been used in our lab for a number of challenge and immunological studies over the last few years. This strain was isolated from poultry in the city of Behshahr, Mazandaran Province, Iran after an outbreak reported in the region in 2011. The intracerebral pathogenicity index (ICPI) was 1.8 in one-day-old chicks, characteristic of a velogenic NDV strain. Later, the virus was purified using a sucrose gradient centrifugation and used for next-generation sequencing (NGS). The results showed that the genome length was 15192 bp, similar to those of class II velogenic strains. In addition, the phylogenetic analysis based on the complete F gene showed that the NDV strain Beh has an F protein cleavage site 112RRQKR↓ F117 and belongs to the newly identified subgenotype VII(L). Based on the biological and genetic characterization, NDV strain Beh is now the best documented reference isolate representing the novel subgenotype VII(L) in Iran. Keywords: NDV; NGS; velogenic strain, subgenotype VII(L); phylogenetic analysis.

Authorizing of Immunogenicity of Concentrated and Purified Newcastle Disease Virus V4 Strain using Downstream Processing.

Newcastle disease virus (NVD) from the Paramyxoviridae family is a single-stranded negative-sense RNA virus. This infection can affect both domestic poultry and almost all other bird species. It has been considered a very severe difficulty for the poultry industry all over the world. Even though it remains a potential threat to poultry industries, this virus is a powerful oncolytic virus as well. In this study, a process was accomplished to achieve concentrated and highly purified NDV V4 strain particles. Downstream processing of Newcastle virus strain V4 was characterized by amplifying virus in embryonated chicken eggs. Through a sequence of steps, harvesting allantoic fluid, clarification by centrifuge, concentration by ultrafiltration, and size exclusion separation, the reduced volume and pure virus particles were considered for the amount of ovalbumin, hemagglutinin activity, transmission electron microscopy (TEM), electrophoresis, and additionally immunogenicity of prepared antigens. The results presented a high recovery of HA activity in concentrated and purified virus with the removal of ovalbumin and the typical morphology based on TEM. Sepharose CL-4B was determined as the best media among all used resins to purify the virus. Prepared formulations as vaccines demonstrated positive hemagglutinin inhibition for 6 months and stability for 2 years. Strong evidence from organized studies supports the effectiveness of this method in concentrating and purifying intact NDV, which could be valuable in vaccine research, antiserum preparation, or even as an alternative oncotic agent to traditional methods. Despite further studies being conducted, this method can be utilized particularly on a semi-industrial scale to produce various vaccine components.

Effectiveness of Thermostable Vaccine for Newcastle Disease Produced by the Razi Institute on Backyard Poultry in Iran during 2015.

Newcastle disease causes many economic losses to the poultry industry in most countries. This disease is endemic in Iran. Backyard poultry is considered the reservoir of Newcastle virus; however, there is either no vaccination program against Newcastle, or it is performed in a restricted manner. Commercial live vaccines are inactive and sensitive to temperature; moreover, vaccine delivery to villages and remote areas requires special equipment and high cost to maintain the cold chain. This study evaluated the effectiveness of a thermostable Newcastle vaccine produced by the Razi Institute (ND.TR.IR) on the backyard poultry. In four provinces, at least 4 villages were selected as the treatment group, and the same number was selected as the control group. At least, 30 birds were sampled in each village. In each group, blood samples were collected before vaccination and 2 weeks later, and the serum titer of the samples was examined with the haemagglutination inhibition test. The arithmetic mean and standard deviation of the sample titers at the rural level were compared using paired t-test before and after vaccination in each group. Moreover, Repeated Measures ANOVA was utilized to compare the vaccinated and control groups in terms of the titer changes before and after vaccination. In this study, 584 and 389 samples were taken from the treatment (53 households in 20 villages) and control groups (33 households in 14 villages). The mean serum titer values of Newcastle were 4.51±3.03 and 6.64±2.48 in the treatment group before and after vaccination, respectively (P<0.001). The increase in mean titer of the treatment group (2.31 log) was statistically higher than that in the control group (0.66 log) (P<0.001). Out of 584 birds, 517 (88.5%) ones had titer above 3 in the second turn in the treatment group. The thermostable vaccine (ND.TR.IR) produced by the Razi institute is suitable for backyard poultry, which immunizes them against Newcastle disease. Appropriate vaccination programs for backyard poultry should be made; moreover, vaccination of backyard poultry can be effective in preventing the circulation of the field viruses.

Molecular characterization of haemagglutinin-neuraminidase gene among virulent Newcastle disease viruses isolated in Iran.

BACKGROUND: Virulent Newcastle disease virus (vNDV) causes great economic losses to the poultry industry throughout the world. Despite the endemicity of Newcastle disease (ND) and occurrence of recurrent outbreaks, the nature and genetic features of circulating NDV strains in Iran are largely unknown. Aims: This study was conducted to characterize 13 NDV isolates obtained from different outbreaks in various regions of Iran during 1999-2000 by sequencing and phylogenetic analysis of complete coding sequences of haemagglutinin-neuraminidase (HN) gene. METHODS: All isolates were analyzed based on the previously determined in vivo pathogenicity indices and amino acid (aa) sequences of fusion (F) protein cleavage site (FPCS). RESULTS: Phylogenetic analysis based on the HN gene coding region revealed a very close relationship of these viruses with the recently defined genotype XIII, and more specifically, subgenotype XIIIa viruses. Analysis of HN gene nucleotide (nt) sequences revealed that all studied isolates encode for a protein length of 571 aa and there is no C-terminal extension on HN aa sequences. Sequence analysis revealed multiple aa residue substitutions at antigenic sites or neutralizing epitopes on the HN glycoprotein of studied viruses compared with commonly used vaccinal strains. CONCLUSION: In this study, molecular characterization of vNDV isolates, obtained from commercial poultry farms in Iran, were conducted through complete sequencing and analysis of HN gene. Isolation and molecular characterization of further NDV isolates from other parts of Iran and from neighboring countries in the region will be helpful to identify the nature and origin of indigenous viruses.