To validate the STLV-1 prevalence in JMs, we first examined for positivity of the anti-STLV-1 antibody in plasma of 280 JMs from five independent troops originating from inhabitants of different areas in Japan. We found that 180 of 280 macaques (64%) were seropositive for STLV-1 (Table 1), which was generally consistent with previous reports [41, 43, 44, 52]. We then determined the variation in the seroprevalence among the troops. The numbers of seropositive individuals were 59, 17, 36, 34, and 34, with a frequency of 68%, 55%, 63%, 56%, and 77%, respectively for each troop (Table 1). In addition, the rearing population density in the free-range facility differed in each troop but was not correlated with the seroprevalence, suggesting that the density did not influence the high STLV-1 prevalence (Table 1).
We hypothesized that a substantial proportion of STLV-1-infected JMs might play a critical role as viral super-spreaders for frequent horizontal transmission and eventual high prevalence of STLV-1, possibly because of their abnormally high viral loads and incidence of poor humoral immune response against STLV-1. In fact, our recent incidence of an outbreak of infectious malignant thrombocytopenia in JMs by simian retrovirus type 4 (SRV-4) demonstrated that some of the monkeys developed asymptomatic SRV-4 infection with persistent viremia in the absence of SRV-4-specific antibody response and became viral super-spreaders [54], [55]. Taking this unexpected result into account, we evaluated ABTs and PVLs in the JM cohort. We found that the ABTs among 180 seropositive macaques were normally distributed with a geometric mean of 4076 and an ABT of 8192 at the maximum number of individuals (Fig. 1A, Additional file 1: Figure S1). We observed no obvious difference in the titers among the five troops (Fig. 1B). We also examined the STLV-1 PVLs in PBMC samples and found that the PVLs of 171 proviral DNA-positive macaques were normally distributed and ranged from 0.01–20% with a geometric mean of 0.62% and PVLs from 0.64–1.28% at the maximum number of individuals (Fig. 2A, Additional file 2: Figure S2). Again, we observed no obvious difference in the PVLs among the troops (Fig. 2B). The data regarding ABTs and PVLs from the 183 macaques positive for either value (herein tentatively regarded as ‘STLV-1-infected’) were plotted as shown in Fig. 3. Among the JMs, 168 were positive for both values, whereas three were negative for ABTs but positive for PVLs and 12 were positive for ABTs but negative for PVLs. Among the STLV-1-infected macaques, we did not observe any individuals with abnormally high PVLs and poor ABTs (Fig. 3). It is notable that the three ABT−PVL+ monkeys belonged to either of two troops (two macaques in troop C and one in troop D), and their PVLs were comparable or less than the mean PVLs. It is therefore unlikely that only three monkeys caused the high prevalence in all the independent troops. Rather, we observed positive correlation between ABTs and PVLs (R = 0.50, p < 0.0001) (Fig. 3), suggesting that humoral immunity was properly induced in response to the increasing proviral loads in these macaques.
We then sought the possible route(s) of transmission by which this high prevalence occurred. It was previously reported the age-associated increase of STLV-1 prevalence in JMs, suggesting the frequent horizontal transmission whereas the mode of transmission remains to be elucidated [43, 44]. If mother-to-child transmission (MTCT) were the main route, the infection rate should drastically increase at around one year of age, followed by a gradual increase with age. On the other hand, if horizontal transmission were the main route, the infection rate should be low in younger ages, followed by a steep increase with age. To verify these possibilities, we examined the age-dependent change of seroprevalence in the cohort. The frequencies of seropositive individuals in each age group were 17%, 33%, 58%, 79%, 93%, 100%, and 95% at age groups of 0, 1, 2, 3–5, 6–8, 9–11, and ≥ 12 years, respectively (Table 2). We also analyzed the age-dependent change of proviral DNA prevalence and found that the frequencies of proviral DNA-positive individuals in each age group were 11%, 31%, 58%, 75%, 89%, 98%, and 91% for the respective age groups (Table 2). The infection rate was over 30% at one year of age and was dramatically increased over 50% at the age of 2 years, over 70% at the age of 3–5 years, followed by infection among almost all of them over 9 years of age, irrespective of the either value of positivity. Considering that the rate of MTCT in the case of long-term breastfeeding is approx. 20% when the children of 3 years old and over of HTLV-1 carrier mothers were tested [56], these results appeared that STLV-1 may be frequently transmitted via both maternal and horizontal routes. Of note, large numbers of younger individuals (i.e., 0–1 years of age) whose STLV-1 prevalence was relatively low reduced apparent prevalence rate of the entire cohort until 64% (Table 2). In addition, significant differences in either parameter among troops were not observed (data not shown). We also examined the dynamics of the ABTs and PVLs with age among the STLV-1-infected JMs. It was found that gradual increases of the ABTs and PVLs were observed from 0 to 2 years of age, followed by a slight increase of ABTs and mostly stable PVLs with age (Table 2).
Results described above suggested that STLV-1 was frequently transmitted via both maternal and horizontal routes. However, it is still possible to speculate that gradual conversion among a part of high frequency of MTCT with age after long-term (e.g. more than 3 years old) latent infection might lead to the results as shown in Table 2. In order to validate this possibility, we conducted a retrospective study of the STLV-1 seroprevalence in this cohort (Table 3). We selected 139 monkeys whose plasma samples in both 2011 and 2015 were available (PBMC samples in 2011 were not available). In 2011, 111 of 139 monkeys were seropositive, whereas 28 were seronegative. It was found that among the 28 seronegative monkeys in 2011, 24 were seroconverted for the antibody within four years from 2011 to 2015. Remarkably, among ten seronegative monkeys of four years of age and older in 2011, eight were seroconverted within four years interval (80%), which was comparable with the monkeys of three years of age and younger in 2011 (16/18, 89%). The frequent seroconversion observed among the seronegative younger and older monkeys was consistent with the results shown in Table 2, which strongly suggests frequent STLV-1 transmission occurring among JMs via horizontal and maternal transmission routes.
It has been shown that a certain degree of heterogeneity of HTLV-1 genome was present among the virus-infected individuals in the same community [57] while the heterogeneity of the viral genome between mother and child was minimal [58, 59]. If it is the case with STLV-1-infected JMs, then it could be possible to differentiate whether the STLV-1 infected in a monkey is derived from mother through MTCT or from any other monkeys through horizontal transmission. In order to examine the possibility, we compared the nucleotide sequences of STLV-1 LTR and tax regions from randomized 12 JMs in a troop. It was found that almost all the sequences of 3’LTR region were identical in all the monkeys except for only a unique heterogeneity in 2 monkeys (C333G for A1671; G346A for A2594, respectively) (Additional file 3: Fig. S3a). Furthermore, no heterogeneity was observed in the tax region of all monkeys (Additional file 3: Fig. S3b). These results indicated that STLV-1 genome was highly conserved among the JMs in the troop, with consistent results in terms of other troops (data not shown). Consequently, it was not possible to determine the transmission route of STLV-1 on the basis of the heterogeneity of the virus genome, which was unlike HTLV-1.