It is clear from the studies in tissue culture that the NSDV isolate has adapted in some way to allow it to grow well in most of the cell lines tested. At the same time, this isolate has essentially lost virulence in sheep. These findings are in accord with those of Terpstra , who found that NSDV of the 55th to 71st tissue culture passage had greatly reduced virulence, while generating a protective immune response in some animals. The nature of the attenuation remains to be determined. The attenuated virus clearly still grows in animals, though less than the pathogenic virus. This is not due to a defect in the replication machinery or assembly of the attenuated virus, as it is clear from the tissue culture studies that this virus replicates well; direct comparisons in which the two isolates are used to infect a compatible cell line (Vero cells) have shown that the NSDV isolate appeared to produce new viral protein and progeny virions slightly faster than the pathogenic isolate. Other studies in our laboratory have shown that both isolates block the actions of type 1 and type 2 interferons and the induction of type 1 interferon  equally well, suggesting that the decreased pathogenicity of the NSDV isolate is not associated with any loss of function in this area. One possible difference between the two isolates is a change in one or both surface glycoproteins of the virus to allow the adapted isolate to enter the cell lines tested more easily, but which has reduced the effectiveness of the virus at growing in the natural target cells in the animal. Further studies to identify the native receptor NSDV/GV are required before we can examine the receptor preference of these two isolates.
There have been no detailed studies on the nature of the pathogenesis in GV/NSDV infections; GV has only recently been identified as a widespread infection in India [3, 9], and it is likely that the virus has been, in the past, frequently ignored or confused with diseases having similar signs in sheep/goats (e.g. peste des petits ruminants, Rift Valley fever), on either continent. The pyrexia seen here with the pathogenic isolate is similar to that reported previously [1, 6]; the profound leucopoenia has not previously been reported for NSDV infections, although it is a common clinical sign of viral hemorraghic fever, and may be caused by the same large scale apoptosis of leukocytes seen in CCHFV-infected mice  or Ebola virus haemorrhagic fever . Loss of white cells has been reported in CCHFV-infected humans .
The cytokine responses observed in this study suggest a similar pattern to that seen in CCHFV infections in humans (reviewed in ) and in some other haemorrhagic fevers. The pathogenesis of CCHFV is poorly understood, not least because most cases occur in areas with limited clinical pathology facilities, and work on the disease requires specialized buildings and equipment (BSL4 containment). Nevertheless, serology on CCHF patients has shown increases in IL-6 and IL-10 and increased TNFα in clinically severe (hospitalised) cases [26, 27], and monocyte-derived dendritic cells infected with CCHFV release IL-6, IL-10 and TNFα , while we showed that pathogenic NSDV/GV was associated with increases in these cytokines as well as of IL-12, and a decrease in IL-4, all concordant with a Th1, proinflammatory response, which has been proposed for CCHFV [26, 29]. One study found reduced levels of IL-12 in CCHF patients , but this may be a matter of timing, since the levels of IL-12 in NSDV/GV infection declined rapidly after 7 days. The observed cytokine responses would be expected to give rise to lymphohistocytosis (often associated with CCHF ), while both IL-6 and TNFα are associated with the increase in endothelial permeability that is common in viral hemorrhagic fevers [31, 32]. Elevated TNFα is found in a number of other hemorrhagic fevers, including infection with Hantaan virus , Ebola virus  or Puumala virus . It does need to be pointed out that most of those studies have measured serum cytokine proteins, while in this instance we have looked only at the levels of specific mRNAs, since specific assays for ovine cytokines have not yet been developed. This means that we will have missed some changes due to cytokines secreted by other organs (e.g. IL-6 produced by the liver); on the other hand, the real-time PCRs are very sensitive, and the serial samples allow us to pick up quite small changes in transcription patterns.
The real-time PCR detection of viral genome was much more sensitive than virus isolation, as has been seen with other viruses. Interestingly, white cell RNA was almost as sensitive as whole blood RNA for detecting virus, especially the more wild-type, pathogenic isolate, despite the fact that low yields of RNA from the white cell preparation meant that it was necessary to use less of this RNA in the RT-PCR than whole blood RNA, suggesting that the viral RNA in the blood is mostly associated with white cells, and that EDTA blood or other anticoagulated blood will be a suitable sample for laboratory testing/diagnosis.