Despite a high prevalence of hepatitis B virus (HBV) infection in endangered apes, no HBV infection has been reported in small, old-world monkeys. In search for a small, nonhuman primate model, we investigated the prevalence of HBV infection in 260 macaque (Cercopithecidae) sera of various geographical origins (i.e., Morocco, Mauritius Island, and Asia). HBV-positive markers were detected in cynomolgus macaques (Macaca fascicularis) from Mauritius Island only, and, remarkably, HBV DNA was positive in 25.8% (31 of 120) and 42% (21 of 50) of serum and liver samples, respectively. Strong liver expression of hepatitis B surface antigen and hepatitis B core antigen was detected in approximately 20%-30% of hepatocytes. Furthermore, chronic infection with persisting HBV DNA was documented in all 6 infected macaques during an 8-month follow-up period. Whole HBV genome-sequencing data revealed that it was genotype D subtype ayw3 carrying substitution in position 67 of preS1. To confirm infectivity of this isolate, 3 Macaca sylvanus were inoculated with a pool of M. fascicularis serum and developed an acute HBV infection with 100% sequence homology, compared with HBV inoculum. We demonstrated the presence of a chronic HBV infection in M. fascicularis from Mauritius Island. This closely human-related HBV might have been transmitted from humans, because the initial breeding colony originated from very few ancestors 300 years ago when it was implemented by Portuguese who imported a handful of macaques from Java to Mauritius Island. Conclusion: This report on natural, persisting HBV infection among cynomolgus macaques provides the first evidence for the existence of a novel, small simian model of chronic HBV infection, immunologically close to humans, that should be most valuable for the study of immunotherapeutic approaches against chronic hepatitis B. (Hepatology 2013;58:1610–1620)
Despite the existence of an effective vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem, responsible for 55% of hepatocellular carcinomas worldwide. Current chronic hepatitis B (CHB) treatments (e.g., interferon and nucleos(t)ide analogs) remain long lasting, expensive, partially efficient (25%), and frequently lead to the emergence of resistant variants. Because chronic HBV carriers are crippled by weak, functionally impaired immune responses, immunotherapeutic approaches that are able to stimulate or restore humoral and cellular virus-specific immune responses are currently considered as a priority goal for CHB therapy.[2, 3] However, major hurdles are the lack of suitable in vivo models of HBV infection close to humans, with the exception of chimpanzees, which are now a protected and unaffordable species. Therefore, there is an urgent need for the development of a novel primate model for CHB infection studies that should be immunologically very close to humans, regarding innate and cellular responses, and that will permit accurate evaluation of new immunotherapeutic anti-HBV approaches.
In the last 20 years, HBV transmission to old-world primates maintained in captivity has been reported. An “HBV-like” virus was also found in nonhuman primates (NHPs), including Hominidae (chimpanzee, gorilla, and orangutan),[4-11] Hylobatidae (gibbon),[4, 12-15] and Atelidae (woolly monkey). Those species are distributed over Africa (chimpanzee and gorilla), Southeast Asia (orangutan and gibbon), and South America (woolly monkey). HBV-like viruses infecting various NHP species or subspecies are genetically distinct from one another as well as from human HBV genotypes. Their clustering in specific groups suggests that they could represent indigenous virus populations. However, the origin of these hepatitis B–like viruses remains controversial. The hypothesis of cross-species transmission from humans cannot be ruled out, because the various HBV genotypes from chimpanzees from different geographical regions cluster with human HBV genotype D, although the refined phylogenic analysis data indicate the presence of distinct viral isolates, supporting the concept that these viruses may have evolved with their hosts. Previous epidemiological studies have reported a high prevalence of HBV infections in great apes that was comparable to human populations in Gabon and Congo. However, the presence of natural HBV infection among small monkeys has hitherto never been demonstrated.
Our previous studies already opened the possibility of using macaques for HBV studies, which are the NHPs most commonly used in biomedical research. We have demonstrated both successful in vivo HBV transfection and in vitro HBV transduction with baculovirus vector in macaques, although only transient viral infection could be generated by this method in these animals.[20, 21]
In the current study, we therefore searched for the presence of a natural HBV infection among macaques of various geographical origins by analyzing sera and liver samples from macaques (Cercopithecidae) originating from Asia (China, Indonesia, and the Philippines), Morocco, and Mauritius Island.
To develop a novel small simian model for the study of novel therapeutic approaches of CHB, we extensively searched for the presence of natural HBV infection in NHPs currently used for biomedical research, especially among macaques (Cercopithecidae) of various geographical origin. After investigation in M. fascicularis from China, Indonesia, the Philippines, and Mauritius Island, as well as M. sylvanus from Morocco, we report here the detection of HBV infection in macaques from Mauritius Island only. To date, occurrence of HBV infections was reported in approximately 16% of great-ape populations, based on PCR positivity and HBsAg detection,[4, 17, 29] and it was shown that human HBV isolates could also infect gibbons or chimpanzees.[30, 31] Here, we provide the first description of chronic HBV infection in small monkeys that can be used under laboratory conditions.
HBV DNA was found positive in 25.8% and 42% of Mauritius M. fascicularis sera and livers, respectively. Interestingly, 6 macaques were repeatedly positive for serum HBV DNA over an 8-month follow-up period, indicating the presence of chronic infection, and the majority of them exhibited only modest viremia variations. By contrast, the viremia of 1 animal (OGD6) varied greatly from relatively high (month 1) to undetectable values (month 8). Similarly, we and others have also observed and reported on important variations in viremia overtime in some cases of occult hepatitis B patients.
Importantly, phylogenetic analysis of a complete viral genome showed that it was HBV genotype D and, more specifically, subgenotype D3, serotype ayw3. The detailed analysis of the pre-S1 sequence revealed proline-to-serine substitution at position 67, which seems to be more specific to NHP HBVs. This substitution is located within the pre-S region that is known to play a crucial role in viral entry, suggesting a possible effect on viral pre-S1 domain conformation and subsequently on the species specificity of this isolate.
Recently, the article by Yan et al. described a receptor for HBV in humans. It does also report on the few differences in amino acid between the human Na+/taurocholate cotransporting polypeptide (NCTP) and the macaque cynomolgus NCTP, therefore underlying that the species sensitivity to HBV infection may rely on those amino acids. Moreover, Yan et al. demonstrated a specific interaction between NCTP and the pre-S1 domain, which mediated the binding of the virions. Because we were not able to infect primary Mauritius macaques hepatocytes with human HBV, but only with the HBV Mauritian isolate (data not shown), we may suppose that minor changes in the pre-S1 domain of the Mauritian HBV may have possibly been adapted to the Mauritian cynomolgus NCTP receptor.
Detection of HBsAg and HBcAg in liver sections from Mauritius Island's M. fascicularis showed approximately 30% of strongly stained hepatocytes. In addition, to confirm the infectivity of this isolate, 3 naïve M. sylvanus were inoculated with a pool of sera from HBV-positive Mauritius Island macaques. We have used the M. sylvanus macaques for this transmission study becausee most of Mauritius Island's M. fascicularis macaques have either anti-HBsAg Abs or were HBV carriers (data not shown). All 3 M. sylvanus macaques presented a parallel rise in HBsAg levels and HBV DNA with increasing ALT values and histopathological signs of acute hepatitis, which were observed in serum of all these animals for several weeks postinoculation, thus confirming the infectivity and pathogenicity of this inoculum.
The occurrence of HBV zoonosis still remains poorly documented. Zoonotic infection of HBV has been suggested in great apes because their HBV genotypes tend to cocluster according to the environmental geographic distribution of genotypes in Africa and Southeast Asia.[17, 35] The sequence homology between HBV DNA isolated from Mauritius M. fascicularis and human HBV is probably related to the introduction of a few animals approximately 400 years ago by Portuguese sailors from Java to Mauritius island. Since then, animals may have expanded from an initial effective of 10-15 individuals and remained isolated in the island for approximately 80-100 generations.[37, 38] The initial event leading to HBV infection of macaques by humans may have occurred at the time of capture and importation by the Portuguese that may have been infected by HBV genotype D. Genotype D is widespread all over the world, with accounts in India, Asia, Europe, and North America.[25, 39, 40] Whether the existence of this HBV infection among the Mauritius M. fascicularis population could be a potential source of infection transmission to humans who come in contact with them, as demonstrated for simian immunodeficiency virus (SIV), is, at present, speculative and the precise risk remains to be assessed.
An understanding of HBV evolution in humans could greatly benefit from better knowledge of its predecessor, simian HBV, in NHPs. Whereas HBV causes liver disease in humans, this virus generally produces only a benign infection in primates. This avirulence is often attributed to coevolution between the virus and its host, possibly resulting from codivergence over millions of years. We now provide a temporal reference to further test this hypothesis about the evolution of HBV in its natural small primate host.
In addition to data presented here, we have previously reported that M. sylvanus species can be transfected with an HBV-coding plasmid. Moreover, in vitro transduction of primary macaque hepatocyte with baculovirus-HBV constructs secreted HBV particles. Those results had already opened up the possibility of using the macaque model for HBV study, although chronic viral infection was not achieved in these experimental studies.
Our current data support the evidence of chronic HBV infection in some macaques and the possible transmission of the HBV strain to M. sylvanus from Morocco. Moreover, our screening evaluation performed on 120 animals demonstrates, for the first time, that a high proportion of wild-living M. fascicularis in Mauritius Island is naturally infected with HBV. However, to date, most of the HBV infections in M. fascicularis were found to be cryptic, with very low titer or undetectable markers in the absence of liver disease. To date, we have no hypothesis how this prevalence can be maintened in natura, because very low peripheral viral load implies a poor transmission efficiency either within the macaque group or from macaque to human animal-care team. Such natural, low-grade infections in Mauritius macaques are similar to occult HBV infections in humans, which present a major concern in the hepatitis B field[42-45] and may therefore represent an excellent model and unique opportunity for their study and therapy for which no recommendations exist.
The Mauritius M. fascicularis could represent a particularly valuable model for novel anti-HBV immunotherapeutic approaches for evaluation because of their limited genetic diversity, notably in the Mhc class I region, that could influence the immune cellular response as it has been demonstrated for SIV infection.[46-48] Further characterization of this novel small primate close to humans will make it possible, for the first time, to test innovative immunotherapeutical approaches, including therapeutic DNA vaccination, alone or in combination with cytokines, possibly as an adjuvant to antiviral nucleos(t)ides for their ability to cure chronic HBV infection.