First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.

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.

Retrospective Phylodynamic and Phylogeographic Analysis of the Bluetongue Virus in Tunisia.

Bluetongue virus (BTV) is an arbovirus considered as a major threat for the global livestock economy. Since 1999, Tunisia has experienced several incursions of BTV, during which numerous cases of infection and mortality have been reported. However, the geographical origin and epidemiological characteristics of these incursions remained unclear. To understand the evolutionary history of BTV emergence in Tunisia, we extracted from Genbank the segment 6 sequences of 7 BTV strains isolated in Tunisia during the period 2000 to 2017 and blasted them to obtain a final dataset of 67 sequences. We subjected the dataset to a Bayesian phylogeography framework inferring geographical origin and serotype as phylodynamic models. Our results suggest that BTV-2 was first introduced in Tunisia in the 1960s and that since 1990s, the country has witnessed the emergence of other typical and atypical BTV serotypes notably BTV-1, BTV-3 and BTV-Y. The reported serotypes have a diverse geographical origin and have been transmitted to Tunisia from countries in the Mediterranean Basin. Interserotype reassortments have been identified among BTV-1, BTV-2 and BTV-Y. This study has provided new insights on the temporal and geographical origin of BTV in Tunisia, suggesting the contribution of animal trade and environment conditions in virus spread.

Phylogenetic and phylodynamic analyses of subtype-B metapneumovirus from chickens in Tunisia.

Swollen Head Syndrome (SHS) is an economically important viral disease of chickens caused by avian metapneumovirus (aMPV). The virus comprises 6 different subtypes (A,B,C,D, New-1 and New-2). To date, no information was available on the presence of the virus in Tunisian poultry. The present work aims to detect the presence of (aMPV) in broiler chicken in Tunisia, then to characterise the isolates in order to determine their subtype and to estimate their geographic origin of introduction. A total of 289 samples were collected, aMPV detection was detected by real time RT-PCR and molecular characterization was warried out by Sanger sequencing on the glycoprotein (G) gene. Phylogenetic analysis was carried out using Beast 2 software. Out of the 289 samples, 21 were revealed positive to aMPV. Only 2 isolates have been confirmed by sequencing analysis ; one isolate sampled in 2015 and another in 2019. Based on the partial G gene sequence, analysis of these 2 Tunisian isolates showed that they belong to subtype B. The isolate sampled in 2015, appeared to be phylogenetically related to derived vaccine strain. However, the one sampled in 2019 appeared to be a field strain. Phylodynamic analysis provided evidence that this field strain derived from a Spanish strain and probably the virus has been introduced from Spain to North Africa back in 2016. This study is the first that highlighted the circulation of (aMPV) in Tunisia. It is possible that aMPV has been circulating in Tunisia and neighboring countries without being detected. Also, multiple strains could be present and therefore multiple introductions have happened. Through this study, we shed the light on the importance of reinforcing farms biosecurity as well as virological surveillance.

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.

Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization.

During 2009-2012, several outbreaks of avian influenza virus H9N2 were reported in Tunisian poultry. The circulating strains carried in their hemagglutinins the human-like marker 226L, which is known to be important for avian-to-human viral transmission. To investigate the origins and zoonotic potential of the Tunisian H9N2 viruses, five new isolates were identified during 2012-2016 and their whole genomes were sequenced. Bayesian-based phylogeny showed that the HA, NA, M and NP segments belong to the G1-like lineage. The PB1, PB2, PA and NS segments appeared to have undergone multiple intersubtype reassortments and to be only distantly related to all of the Eurasian lineages (G1-like, Y280-like and Korean-like). The spatiotemporal dynamic of virus spread revealed that the H9N2 virus was transferred to Tunisia from the UAE through Asian and European pathways. As indicated by Bayesian analysis of host traits, ducks and terrestrial birds played an important role in virus transmission to Tunisia. The subtype phylodynamics showed that the history of the PB1 and PB2 segments was marked by intersubtype reassortments with H4N6, H10N4 and H2N2 subtypes. Most of these transitions between locations, hosts and subtypes were statistically supported (BF > 3) and not influenced by sampling bias. Evidence of genetic evolution was observed in the predicted amino acid sequences of the viral proteins of recent Tunisian H9N2 viruses, which were characterized by the acquisition of new mutations involved in virus adaptation to avian and mammalian hosts and amantadine resistance. This study is the first comprehensive analysis of the evolutionary history of Tunisian H9N2 viruses and highlights the zoonotic risk associated with their circulation in poultry, indicating the need for continuous surveillance of their molecular evolution.

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.

Non-cytotoxic Thymus capitata extracts prevent Bovine herpesvirus-1 infection in cell cultures.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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.

Risk factors and characteristics of low pathogenic avian influenza virus isolated from commercial poultry in Tunisia.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Genetic evolution of low pathogenecity H9N2 avian influenza viruses in Tunisia: acquisition of new mutations.

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*GLF341 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.

First serological investigation of peste-des-petits-ruminants and Rift Valley fever in Tunisia.

This study, carried out between September 2006 and January 2007, is the first cross-sectional serological investigation of peste-des-petits-ruminants (PPR) and Rift Valley fever (RVF) in Tunisia. The objective was to assess the potential need to develop a dual, recombinant PPR-RVF vaccine and how such a vaccine might be utilised in Tunisia. An overall PPR seroprevalence of 7.45% was determined, a finding supported by the high specificity (99.4%) and sensitivity (94.5%) of the ELISA used. On assessment of the diversity and density of mosquitoes in the sampling area, four species of RVF-vectors of the genus Aedes and Culex were identified. However, no serological evidence of RVF was found despite the use of a highly sensitive ELISA (99-100%). Larger scale investigations are underway to confirm these findings and the continuation of the emergency vaccination program against these two diseases remains valid.