Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing

Lauck, Michael; Hyeroba, David; Tumukunde, Alex; Weny, Geoffrey; Lank, Simon M.; Chapman, Colin A.; O'Connor, David H.; Friedrich, Thomas C.; Goldberg, Tony L.

Our results highlight the utility of direct pyrosequencing for detecting novel viruses and for characterizing viral genomic architecture during natural infection. The combination of random hexamer-primed reverse transcription and double-stranded cDNA synthesis used in this study obviated the need for PCR amplification to generate suitable amounts of DNA for pyrosequencing libraries. This method also prevented the potential introduction of amplification-induced error and template bias associated with whole genome amplification [26] . Despite marked divergence from each other and from type strain LVR 42-0/M694, SHFV-krc1 and SHFV-krc2 share the same 39 genomic architecture as the type strain, consisting of additional ORFs 2a, 2b, and 3, which are not present in the other arteriviruses ( Figure 1 ). Because our methods did not involve tissue culture, which can introduce genetic anomalies as viruses replicate in, and potentially adapt to, cells or cell lines derived from non-natural host species, we conclude that the unique 39 genomic architecture of SHFV in comparison to the other arteriviruses is authentic and characteristic of SHFV. Future studies of isolated viruses should help clarify the stability of SHFV in cell culture and should facilitate investigations of biological differences among SHFV variants, including their ability to be detected by available serologic diagnostics tests [27] . Finally, our results have implications for primate health and conservation. The animal sampled was apparently healthy at the time of capture and, as of the time of this writing, continues to behave normally within its social group. Preliminary data (unpublished) indicate that this particular animal is also infected with the recently discovered simian immunodeficiency virus SIVkrc [20] , and that other apparently healthy red colobus in the same location are infected with SHFV-krc. This evidence supports the idea that wild primates can harbor SHFV subclinically, although long-term field observations of infected and uninfected animals will be required to detect any negative effects of infection and co-infection with other viruses on host fitness. Nevertheless, our findings underscore that wild primates can be reservoirs for unknown pathogens of potential concern for global health, and that extreme care should therefore be taken when introducing primates into new environments. Indeed, we suggest that direct pyrosequencing or other similar technologies for broad viral screening might prove useful in settings such as zoos, primate colonies, sanctuaries, and primate reintroduction programs where asymptomatic carriers of novel viral pathogens might come into contact with clinically susceptible hosts.