Complicating disease association studies are the extent of viral genetic diversity shown by some recently characterized viral groups [66
]. As an example, a high number of serotype/genotype can be found within each human enterovirus species and each associated with no or a variety of symptoms (http://www.picornaviridae.com/enterovirus/prototypes/hev-b_prototypes.htm
]. Other groups of similarly genetically divergent viruses may therefore be as equally diverse phenotypically. When determining the pathogenicity of such new viral groups each serotype/genotype should therefore be considered separately increasing the number of samples required to detect sufficient numbers of infections to measure possible disease association.
Many viruses are common but only cause symptoms in a very small subset of infections. To detect a disease association for a virus frequently detected in healthy controls, it must be found in a still higher fraction of the unexplained matched disease cases. If a virus is responsible for only a small fraction of the unexplained cases it may be difficult for that increase in prevalence to rise above the high background of asymptomatic infections. Two groups of very common human viruses found in plasma, the anellovirus genus and GBV-C in the Flaviviridae
family, were initially detected in hepatitis patients and at first thought to be associated with that condition [71
]. Further studies showed anelloviruses to be extremely diverse and nearly universal in human plasma and likely transmitted through close family contact very early after birth [74
]. GBV-C was also shown to be a very common infection, especially in individuals exposed to blood products [76
]. Association of either group of virus with hepatitis was not confirmed [75
]. Given that anelloviruses exhibit an extreme degree of genetic variability it remains possible that a subset are associated with some disease in a situation analogous to those of papillomaviruses, but their ubiquitous detection makes association studies challenging [79
Co-infections may also aggravate symptoms. If disease induction is greatly increased in the context of other infections then only the total number of infections or particular combinations of viruses may be associated with symptoms. Deciphering such complex interactions will require metagenomics or microarray technologies that query all virus families rather then single virus assays in order to identify all participating viruses.
Ultimately, convincingly demonstrating disease association requires confirmation by different research groups using different sets of patient and control samples. The reduction of symptoms and disease prevalence seen after using specific anti-virals and vaccinations are direct ways to demonstrate human virus pathogenicity. Since such measures are only developed for highly pathogenic and prevalent viral infections, the ultimate confirmation of their pathogenicity first depends on disease association tests such as described above. For animal viruses their direct inoculation into their host species can greatly facilitate the determination of their pathogenicity.