Natural Norovirus Infections in Rhesus Macaques

Using a recently developed real-time reverse transcription PCR, I retested 500 fecal samples from rhesus macaques collected in 2008. Previous conventional reverse transcription PCR testing identified 1 isolate of GII norovirus; retesting found GI, GII, and possible GIV noroviruses in the samples, indicating the natural circulation of noroviruses in nonhuman primate colonies.

N oroviruses are a leading cause of acute gastroenteritis in humans and have also been described in several animal species. Although the pathologic role of most animal noroviruses is not clearly established, evidence of the close genetic relatedness of animal and human noroviruses and the detection of animal norovirus-specific antibodies in humans and of human norovirus specific-antibodies in different animal species strongly suggest zoonotic or interspecies transmission of noroviruses (1)(2)(3)(4). However, zoonotic norovirus infections have not yet been reported, perhaps because zoonotic transmission of noroviruses is rare or the routine norovirus detection techniques are not designed to detect animal strains. On the other hand, experimental infection of several animal species has clearly shown that human norovirus strains are able to replicate in animals, including gnotobiotic pigs and calves and nonhuman primates (5)(6)(7)(8). In addition, molecular detection of GII.12 and GII.4 noroviruses, which are usually human strains, has been reported in swine, cattle, and pet dogs (9,10).

The Study
A previous study evaluated samples that were collected during 2008 from the rhesus macaque (Macaca mulatta) colony housed at the Tulane National Primate Research Center (Covington, LA, USA) to determine the presence of caliciviruses (11). Using a broadly reactive primer pair (P289/P290), this study detected diverse recovirus strains and 1 GII norovirus (FT244, GenBank accession no. HM035148) in 1 macaque (11). Recently, a quantitative real-time reverse transcription PCR (RT-PCR) for the detection of GI, GII, and GIV noroviruses was developed (12). In 2015, I used this highly specific and sensitive assay to retest the 500 rhesus macaque fecal samples from the previous study, including the sample that contained FT244, to determine the presence of noroviruses.

Conclusions
A previous study reported the molecular detection of a GII norovirus in 1 of 500 rhesus macaques tested (11). Although this detection rate was extremely low (0.2%), the finding indicated the occurrence of natural norovirus infections in colony macaques. In this study, retesting the 500 samples by a more sensitive and specific real-time RT-PCR confirmed the previously detected presence of the GII norovirus in 1 sample but also identified additional samples positive for GI, GII, and GIV noroviruses. Furthermore, additional amplification of the viral genome and sequencing confirmed the presence of GI.1 and GII.7 noroviruses but not GIV noroviruses.
The detection of a GI.1 norovirus in samples collected in 2008, with 100% homology to the prototype Norwalk virus, is somewhat surprising. The prototype virus was originally described in an outbreak occurring during 1968 (15). According to outbreak surveillance data, GI.1 norovirus infections are extremely rare; consequently, establishing whether the Norwalk strain is still in circulation or has completely disappeared is difficult. Data on human norovirus strains circulating in the community or present in environmental samples at the time of the rhesus macaque fecal sample collection were not available from the local health department. The fecal samples were collected by rectal loops from individual macaques, so environmental contamination of samples, which might be an issue in other studies where manure is collected (9), can be ruled out. Samples positive for Norwalk virus were not present in the laboratories involved in the study, and rhesus macaque samples were stored in a separate freezer under Biosafety Level 2+ protocol. This finding is supported by a report of recent circulation of noroviruses with 100% nt homology to the Norwalk virus (GenBank accession nos. JX455860, JX455863, JX455870, and JX455872) that were found in sewage samples in Tunisia during [2007][2008][2009].
The observations from this study and the previous study (11) indicate a strong plausibility for nonhuman primate reservoirs of human norovirus infections and the genetic mixing of animal and human caliciviruses under natural conditions from which new strains or emerging pathogens may arise. Additional studies are needed to establish the frequency, identity, and relevance of norovirus infections in nonhuman primates.