Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

Epidemiological and Phylogeographic Study of Equid Herpesviruses in Tunisia.

Equid herpesvirus (EHV) is a contagious viral disease affecting horses, causing illness characterized by respiratory symptoms, abortion and neurological disorders. It is common worldwide and causes severe economic losses to the equine industry. The present study was aimed at investigating the incidence of EHVs, the genetic characterization of Tunisian isolates and a spatiotemporal study, using 298 collected samples from diseased and clinically healthy horses. The global incidence of EHV infection was found to be about 71.81%. EHV2 and EHV5 were detected in 146 (48.99%) and 159 (53.35%) sampled horses, respectively. EHV1 was detected in 11 samples (3.69%); EHV4 was not detected. Co-infections with EHV1-EHV2, EHV1-EHV5 and EHV2-EHV5 were observed in 0.33%, 1.34% and 31.54% of tested horses, respectively. Phylogenetic analyses showed that gB of EHV2 and EHV5 displays high genetic diversity with a nucleotide sequence identity ranging from 88 to 100% for EHV2 and 97.5 to 100% for EHV5. Phylogeography suggested Iceland and USA as the most likely countries of origin of the Tunisian EHV2 and EHV5 isolates. These viruses detected in Tunisia seemed to be introduced in the 2000s. This first epidemiological and phylogeographic study is important for better knowledge of the evolution of equid herpesvirus infections in Tunisia.

Phylogenetic analysis and assessment of the pathogenic potential of the first H9N2 avian influenza viruses isolated from wild birds and Lagoon water in Tunisia.

H9N2 avian influenza virus (AIV) has been isolated from various species of wild birds and domestic poultry worldwide. It has been reported since the late 1990s, that H9N2 AIV has infected humans as reported in some Asian and North African countries. This subtype has already been circulating and constituting a serious threat to the poultry industry in Tunisia back in 2009. To investigate zoonotic potential and pathogenicity of H9N2 AIV in chickens and mice in Tunisia, five strains have been isolated during the period from 2014 to 2018. Samples were withdrawn from several wild bird species and environment (Lagoon water) of Maamoura and Korba Lagoons as well as Kuriat Island. Phylogenetic analyzes demonstrated that the isolated H9N2 strains belonged to the G1-like sublineage and were close to AIV H9N2 poultry viruses from North Africa, West Africa and the Middle East. All strains carried in their hemagglutinin the residue 226 L, which is an important marker for avian-to-human viral transmission. The hemagglutinin cleavage site has several motifs: PSKSSR/G, PARSSR/G and HARSSR/G. The neuraminidase showed S372A and R403W substitutions that have been previously detected in H3N2 and H2N2 viruses that were reported in human pandemics. Many mutations associated with mammalian infections have been detected in internal proteins. Pathogenicity evaluation in chickens showed that GF/14 replicates effectively in the lungs, tracheas, spleens, kidneys and brains and that it was transmitted among contact chickens. However, GHG/18 replicates poorly in chickens and has not an efficient transmission in contact chickens. GF/14 and GHG/18 could not kill mice though they replicated in their respiratory tract and caused a significant body weight loss (p < 0.05). This study highlights the importance of H9N2 AIV monitoring in both migratory birds and the environment to prevent virus transmission to humans.

Inhibition of avian influenza virus H9N2 infection by antiviral hexapeptides that target viral attachment to epithelial cells.

The H9N2 subtype of influenza A virus circulates frequently among poultry in Asian and North African countries causing economic loss in the poultry sector. The antigenic variations of the H9N2 virus were at the origin of its genetic evolution through the emergence of viral strains transmissible to humans and resistant to chemical antivirals, which require a strengthening of the fight means against this virus. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select new antiviral peptides that inhibit the infectivity of H9N2 virus. After three rounds of stringent selection and amplification, polyclonal phage-peptides directed against H9N2 virus were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to H9N2 virus by monoclonal phage ELISA. The DNA of 27 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences were deduced. Sixteen different phage-peptides were able to bind specifically the H9N2 virus, among them, 13 phage-peptides interacted with the hemagglutinin H9. Two selected peptides, P1 (LSRMPK) and P2 (FAPRWR) have shown antiviral activity in ovo and P1 was more protective in vivo then P2 when co-administered with the H9N2 virus. Mechanistically, these peptides prevent infection by inhibiting the attachment of the H9N2 virus to the cellular receptor. Molecular docking revealed that the peptides LSRMPK and FAPRWR bind to hemagglutinin protein H9, but interact differently with the receptor binding site (RBS). The present study demonstrated that the peptide P1 (LSRMPK) could be used as a new inhibitory molecule directed against the H9N2 virus.

Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia.

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green-based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Molecular characterization of a unique variant of avian infectious bronchitis virus in Tunisia.

Avian infectious bronchitis virus (IBV) is responsible of significant economic losses for poultry industry around the world, through evolution of its pathogenicity, inadequacy of vaccines, and virus evasion. Such evasion is related to the unstable nature of its RNA, in particular the S glycoprotein encoding gene, which raises great challenges with regard to the control of the disease, along with the lack of proof reading mechanisms of the RNA polymerase. The emergence of new variants might be a reason for the endemic outbreaks that are being reported in Tunisia, in addition to poor vaccination techniques and ineffective prophylactic programs. In the present study, partial nucleotide sequences of the S1 glycoprotein gene and the 3'-untranslated region (UTR) of 2 Tunisian isolates, TN1011/16 and TN1012/16, identified in 2016, were determined. Specific mutations were found in S1 gene as well as in 3'UTR region. Phylogenetic analysis of the S1 nucleotide sequences showed that both isolates are closely related to the Algerian strains, and formed a common cluster within the genotype I. In addition, these isolates were non-recombinant ones, confirming that they are unique variants. Based on their S1 gene sequences, TN1011/16 and TN1012/16 strains were distant from the H120 vaccine strain, commercially used in Tunisia along with the variant vaccine 793B type (4/91). A comparison between nucleotide sequences of their 3'UTR region and S1 gene showed a difference in IBV classification. The obtained results have confirmed that the IBVsequence continues to drift and brings valuable information in relation with its evolution, vaccine development and better control of the disease.

Thermoprotective properties of Opuntia ficus-indica f. inermis cladodes and mesocarps on sheep lymphocytes.

This study aims to investigate the thermoprotective properties of Opuntia ficus-indica f. inermis. Extracts were prepared from cladodes (CE) and mesocarps (ME), then subjected to a spectrophotometric and LC-MS analyses. Lymphocytes were isolated from peripheral blood of non-stressed sheep, supplemented with CE, ME, betanin or α-tocopherol, and subjected to two thermal treatments: 40 and 41 °C, for 6 h. Viable lymphocytes and H2O2 production were evaluated. The antioxidant activity of ME was 3.43 folds higher than CE. The LC-MS analysis of CE and ME allowed identifying 11 phenolic acids, 2 flavanones, 6 flavones, 3 flavonols and 1 betanin type betacyanin. Lymphocytes mortality increased linearly as function of the severity and the duration of heat stress. This mortality was correlated with H2O2 production. At 41 °C, only ME allowed maintaining lymphocytes viability. Moreover, ME was more efficient than CE in reducing H2O2 production. This thermoprotection was ensured by betaxanthin and betacyanin pigments. Interestingly, betanin was more efficient than α-tocopherol in preventing hyperthermia-induced lymphocytes' mortality. We report here for the first time the thermoprotective properties of cladodes and mesocarps of Opuntia ficus-indica f. inermis. Betanin was able to maintain lymphocyte viability through reducing H2O2 production, and therefore the oxidative-induced heat stress.

Evaluation of protection conferred by a vaccination program based on the H120 and CR88 commercial vaccines against a field variant of avian infectious bronchitis virus.

Due to serotype variations among different avian infectious bronchitis viruses isolated in Tunisia since 2000, protection of chicks, especially broiler flocks, with Mass H120 vaccine often fails. Therefore, association of CR88 (793B type) with H120 vaccines was used for better response. Challenge experiments were then conducted to evaluate tracheal and renal cross-protection in chickens immunized via nasal and eye drops. Conferred protection was measured by clinical signs and macroscopic lesions observed, based on scores attributed according to their severities. The results showed a low protection conferred by H120 alone, as vaccination did not reduce tracheal and kidney lesions (70% scored as 3) after TN20/00 virus challenge, which also led to 10% mortality. Conversely, the challenge results indicated that the combination of the 2 strains (H120/CR88) allow high protection. Based on the results of the challenge experiments, a vaccination protocol coupling CR88 to H120 was applied for industrial broiler flocks. Clinical observations and serological results confirmed that association of heterologous serotypes (H120 and CR88 vaccines) increased the levels of protection against infectious bronchitis viruses compared with the H120 vaccine given alone.

Characterization of new variants of avian infectious bronchitis virus in Tunisia.

Three infectious bronchitis virus (IBV) strains, isolated from suspected Tunisian broiler flocks, were characterized as variant viruses using genotyping and serotyping techniques. They were compared with commonly used vaccine strains, including 793/B, D274, and Massachusetts types. Reverse transcription-PCR-restriction fragment length polymorphism, nucleotide sequencing, and GenBank BLAST database analyses of the hypervariable region of the S1 subunit of the virus spike gene showed that the three isolates, designated TN20/00, TN200/01, and TN335/01, share from 64% to 82% homologies between each other but are very different from the H120 strain, the only infectious bronchitis vaccine used in Tunisia. In addition, they showed from 57% to 78% similarities with the European genotypes, including D274 and 793/B. Phylogenetic data allowed classification of the three Tunisian isolates as new genotypes placed inside the same genetic group as the CR88121 and D274 genotypes but very distant from the Massachusetts genotype. Cross-virus neutralization tests confirmed the genotyping results and showed that both TN200/01 and TN335/01 isolates are serologically related, whereas the TN20/00 is closer to TN335/01 than to TN200/01. Moreover, all three Tunisian isolates are closely related to the European variant serotypes, including the CR88121 and the D274 strains, but none is serologically related to the H120 vaccine strain. These data demonstrated, for the first time in Tunisia, the cocirculation of IBV variant serotypes along with the Massachusetts type, causing severe clinical diseases and high economic losses to the poultry industry.