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Introduction and Objectives: A new interaction between avian influenza virus (AIV) and infectious bronchitis virus (IBV) replication interference was identified in this work using chicks by ELISA antibody, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cytokine analyses and histochemistry. Materiel and Methods: In this study chicks were infected with IBV vaccine strain (H120) and a H9N2 LPAIV (A/CK/TUN/145/2012) simultaneously or sequentially three days apart. Our research studied first the eventual replication interference of AIV and IBV in vivo using qualitative and quantitative real-time reverse transcription-polymerase chain reaction (real-time qRT-PCR) to accurately detect either AIV and IBV genomes or the number of viral copies during mixed infections. Second, we determined the amount of IL-1 beta by ELISA and antibody of the infected chick's sera with AIV alone, IBV alone, Mixed AIV + IBV, IBV 3 days AIV and AIV 3 days IBV. Our data revealed that the second virus inoculated inhibits the growth of the first one independently of its nature. Results and Conclusion: In conclusion, co-infection of chicks with AIV and IBV affected the replication dynamics of these viruses and affect clinical signs. These results propose that infection with heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated. In conclusion, the second virus inoculated inhibits the first one by different mechanisms. [ABSTRACT FROM AUTHOR]

A one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR as the "gold standard", the specificity, the sensitivity and the reproducibility of the new multiplex assay were evaluated and validated using 70 clinical samples. The performances of the singleplex and the multiplex assays were assessed using the confusion matrix. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the one-step multiplex rRT-PCR assay presents higher sensitivity and specificity. Correlation coefficients (R2) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95 % LoDs of multiplex assay were in the range [3-19] copies genomic/µl, and its corresponding cutoff cycles were in the range [34.16-36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex rRT-PCR assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease (RHD) causes high mortality and morbidity in European rabbits ( Oryctolagus cuniculus ). In Africa, the presence of the causative agent, the rabbit hemorrhagic disease virus (RHDV), was first confirmed in 1992 (genotype Lagovirus europaeus /GI.1). In 2015, the new genotype Lagovirus europaeus /GI.2 (RHDV2/b) was detected in Tunisia. Currently, GI.2 strains are present in several North and Sub-Saharan African countries. Considerable economic losses have been observed in industrial and traditional African rabbitries due to RHDV. Like other RNA viruses, this virus presents high recombination rates, with the emergence of GI.2 being associated with a recombinant strain. Recombination events have been detected with both pathogenic (GI.1b and GII.1) and benign (GI.3 and GI.4) strains. We obtained complete genome sequences of Tunisian GI.2 strains collected between 2018 and 2020 and carried out phylogenetic analyses. The results revealed that Tunisian strains are GI.3P-GI.2 strains that were most likely introduced from Europe. In addition, the results support the occurrence of multiple introductions of GI.2 into Africa, stressing the need for characterizing complete genome sequences of the circulating lagoviruses to uncover their origin. Continued monitoring and control of rabbit trade will grant a better containment of the disease and reduce the disease-associated economic losses.

Background: Low pathogenic avian influenza (LPAI) H9N2 and infectious bronchitis virus (IBV) are important pathogens of poultry, causing important economic losses for the sector. Replication interference between these two viruses was described using cell cultures (CC) and embryonated chicken eggs (ECE). Chicken embryo lung (CEL) and ECE were simultaneously or sequentially infected with IBV vaccine strain (H120) and LPAIV-H9N2 (A/Ck/TUN/145/2012) to evaluate viral interactionsin vitro and in ovo, respectively. Real-time RT-PCR was developed to specifically quantify both AIV and IBV genomes as well as viral gene copy numbers during mixed infections. The amount of IL-1 beta, in supernatants of co-infected cell cultures, was determined using an ELISA assay., Results: Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus., Conclusion: It appears that either AIV or IBV has a negative impact on the other virus growth when they are inoculated simultaneously or sequentially. The ELISA results showed that higher level of secreted IL-1beta varies, depending on the viral interference conditions between both viruses, during mixed infections., (Copyright © 2018. Published by Elsevier B.V.)

A one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR, the specificity, the sensitivity and the reproducibility of the new assay were evaluated and validated using 70 clinical samples. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the multiplex assay presents higher sensitivity and specificity. Correlation coefficients (R 2 ) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LoDs of multiplex assay were in the range [3-19] copies genomic/ µl, and its corresponding cutoff cycles were in the range [34.16-36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples., (Copyright © 2017 Elsevier Inc. All rights reserved.)

H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus. First, a panel of specific ssDNA aptamers was selected via a one step high stringency protocol. Next, the panel of selected aptamers was characterized for their affinities and their specificity to H9N2 virus. The aptamer showing the highest binding affinity to the virus was used as ligand to develop a highly sensitive sandwich Aptamer I-PCR. A 3-log increase in analytical sensitivity was achieved as compared to a routinely used ELISA antigen test, highlighting the potential of this approach to detect very low levels of virus particles. The test was validated using clinical samples and constitutes a rapid and a label-free platform, opening a new venue for the development of aptamer -based viability sensing for a variety of microorganisms of economic importance in Tunisia and surrounding regions., (Copyright © 2017 Elsevier B.V. All rights reserved.)