Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR).

Cutaneous basophilia in the resistance of goats to Amblyomma cajennense nymphs after repeated infestations.

The acquisition of resistance in goats against Amblyomma cajennense after repeated infestations and the role of inflammatory cells in this mechanism were investigated. Ten naive goats aged 6 months were distributed into two groups: test (n= 5), infested thrice at 30-day intervals; and control (n= 5), infested once. Nymphs (n= 115 per animal per infestation) were released inside alimentary chambers glued to the animal's dorsum and the following biological parameters were evaluated: yielding rate, nymphal engorgement weight, engorgement period, ecdise rate, and ecdise period. Skin fragments of tick bite sites were collected at 24, 48, 72, and 120 h post attachment for histopathology and inflammatory cells counts. The engorgement weight decreased significantly (P < 0.05) from the first infestation onward; nymphs weighed 41.7% and 37.1% less after the second and third infestations, respectively, as compared to those collected after the first infestation (12.55 mg +/- 3.21). Furthermore, the ecdise period increased significantly (P < 0.05) in the third infestation (18 days +/- 2.83) in comparison with the first infestation (15 days +/- 0.82) and the ecdise rate was significantly lower after the second infestation (71.91%+/- 17.38) in comparison with the first infestation (96.89%+/- 3.38). There were no significant differences with regard to both the engorgement period and yielding rate. A cutaneous basophilia was evidenced between 48 and 72 h (P < 0.05) after both the second and third infestations. We conclude that goats develop resistance against nymphs of A. cajennense and that basophils may play an important role in such a mechanism.