Multidrug-resistant tuberculosis was diagnosed in 21 HIV-negative, nonhospitalized male patients residing in northern Tunisia. A detailed investigation showed accelerated transmission of a Mycobacterium tuberculosis clone of the Haarlem type in 90% of all patients. This finding highlights the epidemic potential of this prevalent genotype.

Typing analyses of 378 Mycobacterium tuberculosis isolates collected between the years 2001 and 2005 from three northern representative regions of Tunisia revealed a highly homogeneous population. Indeed, 84.9 % of all tuberculosis (TB) cases were attributed to the Haarlem, LAM or T families. Strikingly, within each family, more than 60 % of TB cases were due to a single genotype. ST50 (Haarlem3) and ST42 (LAM9) genotypes were exceptionally predominant, representing 46.3 % of all typed isolates. ST50 showed an increased tendency for clustering and was more predominant in the extreme north of the country. By contrast, the more widespread ST42, which was apparently prevalent 17 years ago, displayed weak cluster individualization and a low transmission rate, consistent with its stable association with the Tunisian population. It is believed that both mass BCG vaccination, strictly applied for four decades, and the high endogamy rate that characterizes the Tunisian population could have profoundly shaped the population structure of M. tuberculosis by concurrently favouring the selection and accommodation of particular genotypes.

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Outbreaks of infectious bursal disease (IBD) still continue to afflict the Tunisian poultry industry even in those flocks where the vaccination program is strictly applied. To characterize the viruses that circumvent protection provided by vaccination, field isolates of infectious bursal disease virus (IBDV) obtained from vaccinated flocks that have repeatedly experienced IBDV outbreak episodes were analyzed from bursal samples by reverse transcription coupled with polymerase chain reaction and dideoxynucleotide sequencing of the VP2 hypervariable region. Although sequence data were obtained from samples collected from three distinct flocks over a period of 3 years, only limited sequence variation has been observed. The few nucleotide changes were silent and the deduced amino acid sequences were identical. Thus, the virus population that predominates in the field seems to represent a homogeneous antigenic pool. Compared with the VP2 sequences of several IBDV strains, this predominant pool was found to be closely related to the very virulent (vv) IBDV viruses described in Europe and Asia. Sequence and phylogenetic analysis of the precursor polyprotein coding sequence of a representative Tunisian isolate further confirmed its assignment to the vv genotype. The deduced amino acid sequence of the whole polyprotein of the Tunisian isolate was found to be identical to a South Korean IBDV strain. Alignment of the polyprotein amino acid sequence of 35 IBDV strains identified additional mutations outside the VP2 variable domain and which occur frequently in vv strains. Based on this comparative analysis, the set of amino acid residues that should represent a typical vv profile involves Ala222, Ile242, Ile256, Ile294, Leu451, Tyr680, N685, Ser715, Asp751, Val990, and Ala1005. Such a combination of amino acid changes was observed for the majority of vvIBDV strains that define a distinct phylogroup.

We describe a simplified RNA polymerase II-based reverse genetics approach that allows for the efficient rescue of high-titer infectious bursal disease virus (IBDV) from cloned cDNAs of genomic segments A and B. Unlike the previously reported RNA polymerase II-based methods, the developed strategy does not necessitate the introduction of a ribozyme sequence at both ends of the genomic cDNA sequences. This was achieved by fusing the 5' terminal sequence of the cDNA of each segment to the transcription start site of the immediate early cytomegalovirus promoter, while a ribozyme sequence was only introduced at the 3' end. Using this strategy, and without complementing with IBDV structural proteins, titers as high as 10(11) tissue culture infectious dose 50 were reproducibly obtained in chicken embryo fibroblast cells immediately upon co-transfection with cDNAs of both segments. We anticipate that this modification could improve reverse genetics for any other RNA virus and may be beneficial for vaccine development and dissection of the viral life cycle.