Background: Since the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. To monitor variations of Influenza viruses during the outbreaks, Tunisian H9N2 virus isolates were identified and genetically characterized. Methods: The genomic RNA segments of Tunisian H9N2 strains were subjected to RT-PCR amplifications followed by sequencing analysis. Results: Phylogenetic analysis demonstrated that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 viruses represent multiple reassortant lineages, with genes coming from Middle East strains, and share the common ancestor Qa/HK/G1/97 isolate which has contributed internal genes of H5N1 virus circulating in Asia. Some of the internal genes seemed to have undergone broad reassortments with other influenza subtypes. Deduced amino acid sequences of the hemagglutinin (HA) gene showed the presence of additional glycosylation site and Leu at position 234 indicating to binding preference to α (2, 6) sialic acid receptors, indicating their potential to directly infect humans. The Hemagglutinin cleavage site motif sequence is ³³³ PARSSR*GLF ³⁴¹ which indicates the low pathogenicity nature of the Tunisian H9N2 strains and the potential to acquire the basic amino acids required for the highly pathogenic strains. Their neuraminidase protein (NA) carried substitutions in the hemadsorption (HB) site, similar to those of other avian H9N2 viruses from Asia, Middle Eastern and human pandemic H2N2 and H3N2 that bind to α -2, 6 -linked receptors. Two avian virus-like aa at positions 661 (A) and 702 (K), similar to H5N1 strains, were identified in the polymerase (PB2) protein. Likewise, matrix (M) protein carried some substitutions which are linked with increasing replication in mammals. In addition, H9N2 strain recently circulating carried new polymorphism, "GSEV" PDZ ligand (PL) C-terminal motif in its non structural (NS) protein. Two new aa substitutions (I) and (V), that haven't been previously reported, were identified in the polymerase and matrix proteins, respectively. Nucleoprotein and non-structural protein carried some substitutions similar to H5N1 strains. Conclusion: Considering these new mutations, the molecular basis of tropism, host responses and enhanced virulence will be defined and studied. Otherwise, Continuous monitoring of viral genetic changes throughout the year is warranted to monitor variations of Influenza viruses in the field.

Estimate the seroprevalence of influenza A virus in various commercial poultry farms and evaluate specific risk factors as well as analyze their genetic nature using molecular assays. This report summarizes the findings of a national survey realized from October 2010 to May 2011 on 800 flocks in 20 governorates. Serum samples were screened for the presence of specific influenza virus antibodies using cELISA test. Additionally, swab samples were tested by real time and conventional RT-PCR and compared with results obtained by others assays. Phylogenetic and genetic analyses of the glycoproteins were established for some strains. Out of the 800 chicken and turkey flocks tested by cELISA, 223 showed positive anti-NP antibodies (28.7%, 95% CI: 25.6–32.1). Significantly higher seroprevalence was found among the coastal areas compared to inland and during the autumn and winter. Broiler flocks showed significantly lower seroprevalence than layers and broiler breeders. The influenza virus infection prevalence increased after the laying phase among layer flocks. In addition, AIV seropositivity was significantly associated with low biosecurity measures. The Ag EIA and rRT-PCR tests revealed significantly higher numbers of AI positive samples as compared to cell cultures or egg inoculation. All new strains were subtyped as H9N2 by real time and conventional RT-PCR. Drift mutations, addition or deletion of glycosylation sites were likely to have occurred in the HA and NA glycoproteins of Tunisian strains resulting in multiple new amino acid substitutions. This fact may reflect different evolutionary pressures affecting these glycoproteins. The role of these newly detected substitutions should be tested. Our findings highlight the potential risk of AIV to avian health. Strict enforcement of biosecurity measures and possible vaccination of all poultry flocks with continuous monitoring of poultry stations may ensure reduction of AIV prevalence and avoid emergence of more pathogenic strains.

Background: Genotype analyses of avian reoviruses isolated from organ samples collected from chickens with suspicious clinical symptoms, between 1997–2008, was based on sequences for both σC and σB genes and aligned with those published in the Genbank, making it possible to carry out studies of molecular classification and relationships.Methods: The full length of the known variable protein σC and part of the σB encoding genes, were amplified with RT-PCR, using conserved primers. PCR products were sequenced and the sequences were analyzed and aligned with avian reovirus sequences from the Genbank database.Results: The sequences of σC-encoding genes of all the isolated strains indicated their close relationship with the American, Chinese and Indian strains. Taking the American strain S1133 as a reference, the two Tunisian isolates 97.1 and 97.2 showed some nucleotide substitutions. For isolate 97.1, the substitution was silent whereas for strain 97.2 the mutation was at the first position of the corresponding codon and induced the substitution of the amino acid encoded. For the σB-encoding gene, the sequences of the Tunisian strains showed mutations at positions two or three of the corresponding codons, inducing substitutions of amino acids at these positions. The phylogenic trees based on σC and σB encoding genes indicated closer relationship between Tunisian, American and Taiwanese isolates of genotype I.Conclusion: Our study describes the genotype of avian reoviruses that are not yet well characterized genetically. The characterization and classification of these viruses might be significant for understanding the epidemiology of malabsorption syndrome and viral arthritis, and improving our knowledge of the genotype of strains circulating in Tunisian flocks. Furthermore, the study of their variable pathogenicity could be extremely important in the choice of the appropriate vaccine strain to control disease.

An efficient and simple approach has been developed for the synthesis of eight dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonates through the Pudovik-type reaction of dialkyl/arylphosphite with imines, obtained from 5-phenyl-1,3,4-oxadiazol-2-amine and aromatic aldehydes, under microwave irradiation. Five of them were hydrolyzed to lead to the corresponding phosphonic acids. Selected synthesized compounds were screened for their in vitro antiviral activity against the avian bronchitis virus (IBV). In the MTT cytotoxicity assay, the dose-response curve showed that all test compounds were safe in the range concentration of 540–1599 µM. The direct contact of novel synthesized compounds with IBV showed that the diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethoxyphenyl)methyl]phosphonate (5f) (at 33 µM) and the [(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl] phosphonic acid (6a) (at 1.23 µM) strongly inhibited the IBV infectivity, indicating their high virucidal activity. However, virus titers from IBV-infected Vero cells remained unchanged in response to treatment with the lowest non-cytotoxic concentrations of synthesized compounds suggesting their incapacity to inhibit the virus replication inside the host cell. Lack of antiviral activity might presumably be ascribed to their polarity that hampers their diffusion across the lipophilic cytoplasmic membrane. Therefore, the interactions of 5f and 6a were analyzed against the main coronavirus protease, papain-like protease, and nucleocapsid protein by molecular docking methods. Nevertheless, the novel 1,3,4-oxadiazole-based α-aminophosphonic acids and α-amino-phosphonates hold potential for developing new hygienic virucidal products for domestic, chemical, and medical uses.

Bovine herpes virus type 1 (BHV-1) still causes great economic loss to the livestock industry and trade because there aren’t any available drugs that proved to be fully effective against it. In this study, the cytotoxicity and the antiviral activities of the Thymus capitata extracts were evaluated for the development of new, non toxic and specific anti-herpesvirus drug. Aqueous extracts (AE), ethanolic extracts (EE) and essential oil (EO) of the aerial parts of Thymus capitata were analyzed to determine their chemical compositions by gas chromatography, and high performance liquid chromatography combined with mass spectrometry. Their cytotoxicity and antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by quantifying the reduction of the viral cytopathic effect using Madin-Darby Bovine Kidney cell line with colorimetric assay. T. capitata extracts were added at different stages of the viral infection to investigate and better quantify their potential inhibitory effects. Polyphenols and flavonoids were the major compounds found in T. capitata EO, EE and AE. The cytotoxic concentrations at 50% were 48.70, 189 and 289 μg ml−1 for EO, EE and AE, respectively. The inhibitor concentrations at 50% for the EO, EE and AE, were 3.36, 47.80 and 164 μg ml−1, respectively. The selectivity index anti-BHV-1 values were 14.49, 3.95 and 1.81 for EO, EE and AE, respectively. Thus, the EO extracts were the most efficient antiviral compounds. T. capitata extracts affect mainly the adsorption of BHV-1 virus to host cells. T. capitata extracts inhibit the viral replication by interfering with the early stages of viral adsorption and replication. Thus, T. capitata is a potential candidate for anti-herpesvirus treatment.

Newcastle disease virus (NDV) is one of the most serious contagions affecting domestic poultry and other avian species. It causes high morbidity and mortality, resulting in huge economic losses to the poultry industry worldwide. Despite vaccination, NDV outbreaks increase the need for alternative prevention and control means. In this study, we have screened fractions of Buthus occitanus tunetanus (Bot) scorpion venom and isolated the first scorpion peptide inhibiting the NDV multiplication. It showed a dose dependent effect on NDV growth in vitro, with an IC₅₀ of 0.69 µM, and a low cytotoxicity on cultured Vero cells (CC50 > 55 µM). Furthermore, tests carried out in specific pathogen-free embryonated chicken eggs demonstrated that the isolated peptide has a protective effect on chicken embryos against NDV, and reduced by 73% the virus titer in allantoic fluid. The N-terminal sequence, as well as the number of cysteine residues of the isolated peptide, showed that it belongs to the scorpion venom Chlorotoxin-like peptides family, which led us to designate it “BotCl”. Interestingly, at 10 µg/mL, BotCl showed an inhibiting effect three times higher than its analogue AaCtx, from Androctonus australis (Aa) scorpion venom, on NDV development. Altogether, our results highlight the chlorotoxin-like peptides as a new scorpion venom AMPs family.