Hantaan virus, a prototype Hantavirus, is found in A. agrarius throughout China, Russia, and the ROK [13]. In this study, we conducted RT-N-PCR targeting the L- and S-segments of the hantavirus genome and demonstrated that on the same samples, L- and S-segments had different sensitivities. When we compared the results, there was an almost 10% increase in positive PCR results when using the L-segment. In our previous ROK study, rodents were captured in Gwangju (Buk-gu and Gwangsan-gu). These samples had a high rate of infection, with 46% of lung samples testing positive for Hantaan virus using RT-N-PCR targeting the L-segment [14]. Another group of researchers from the ROK reported a detection rate of 3.3% for hantaviruses in A. agrarius using a partial S-segment in multiplex RT-PCR [3]. Different rodent trapping sites, geographical distributions, climatic variations, and differences in molecular techniques used in these studies may have resulted in the variance observed in the results. To study whether geographical distribution can be used as a surrogate for genetic divergence, we collected rodents from different geographical locations and compared their sequences.
The prevalence of HFRS depends on pathogen viability; this includes adaptation to the climate, human activity, landscape, and seasonality in various regions [15]. A previous study conducted on hantaviruses prevalence provided epidemiological data on human HFRS cases (reported between 2001 and 2010) in the ROK. This study analyzed cases by season, geography, and the residential area of affected individuals. In this study, the majority of the HFRS cases were reported in the last quarter of the calendar year (October, November, and December). Presumably, the higher incidence of HFRS in these months can be attributed to the higher numbers of hantavirus-infected rodents in the ROK during this period [16]. Here, we found that a significantly (P < 0.01) higher proportion of HFRS-positive rodents was collected in Jeju during May (85.7%) and October (50%). Table 6 shows the nucleotide sequence similarity and amino acid identity between the Hantaan virus isolates collected at the three geographical locations shown in Figure 1. The distance between Gwangju and Boseong-gun is only 47.8 km, while the distance from Jeju to Boseong-gun and Gwangju is 150 km and 187 km, respectively. We were able to show that the nucleotide and amino acid identity among study locations mirrored the geographical distance between them. Although the study locations are not far apart, geographical clustering was shown by the isolates. Relatively high nucleotide similarity (95.3%) and amino acid identity of 98.3% was observed between isolates collected at the geographically closer locations of Boseong-gun and Gwangju. On the contrary, while comparing Jeju isolates with isolates from relatively distant locations—Boseong-gun and Gwangju—a lower nucleotide similarity of 82.2% (amino acid identity of 85.6%) and 85.2% (amino acid identity of 86.6%) was recorded, respectively. Given that the climate in Jeju differs from the rest of the Korean Peninsula, it may have contributed to the increased nucleotide and amino acid divergence and the difference in seasonality. Previous studies have suggested that climate change can affect rodent distributions in a given area and consequently, the spread of zoonotic diseases [17]. Further studies are needed to evaluate this aspect of our study.
Additionally, serological results were obtained for 5 (11%) of the A. agrarius serum samples collected from Jeju. Interestingly, until 2013, only a single human HFRS case was documented from Jeju Island, amongst a total of 3,953 HFRS cases reported over almost a decade in the ROK [16, 18]. Another report, published in 2017, referenced five human HFRS cases from Jeju over a period from 2001 to 2009 [15]. The high hantavirus incidence among A. agrarius captured from Jeju Island along with the season variation suggests that this region may be at risk for a viral outbreak in the future. Further investigations are needed to better understand the hantavirus prevalence and seasonal variation in Jeju.
One of the major findings of our study was the geographical clustering of the isolates. These isolates grouped with the known Hantaan viruses but appeared to be distinct from the other Hantaan viruses isolated in different regions of the ROK. Genetic reassortment may explain this observation and amino acid divergence. Reassortment appears to occur frequently within similar hantavirus strains, especially those that share the same rodent host. On the contrary, reassortment between genetically distinct hantaviruses seems to occur infrequently even if they occasionally infect the same rodent host. It has been suggested that the reassortment between closely related strains of the same hantavirus group could lead to the emergence of unique strains with new virulence characteristics or host ranges [19]. A study conducted at Guizhou, China, presented a phylogenetic analysis of the S-, M-, and L-segments of hantavirus isolates and stated that although the S-segment of the two viruses belonged to the Hantaan virus group, both the M and L sequences were more similar to those of SEOV, indicating that a reassortment had occurred spontaneously at some point in their evolution [8]. Similarly, a Korean group reported spontaneous genetic exchanges in Hantaan virus genomes. This study reported that Hantaan virus isolates from Gangwon and Gyeonggi provinces of the ROK (areas where HFRS is highly endemic) show a high molecular diversity with geographically distinct clustering. Moreover, reassortment analysis demonstrated that these Hantaan virus isolates were heterogeneous for the L-segment but homogeneous for the M- and S-segments [20]. In our study, we compared the Gangwon and Gyeonggi Hantaan virus isolates with our isolates from south Jeolla and Jeju. Hantaan virus L-segment sequences obtained from the Jeju isolates demonstrated 70–83.9% nucleotide similarity and 96.2–97.5% amino acid identity. Gwangju isolates shared 78.1–81.9% nucleotide similarity and 95.8– 96.6% amino acid identity. Boseong-gun, Jellanamdo Province isolates shared a 77.2–81.9% nucleotide similarity and 94.9–96.6% amino acid identity with the isolates from Gangwon and Gyeonggi provinces (Table 5). When we compared S-segment sequences of Hantaan viruses from this study, we observed an amino acid identity of 95.3–96.3% when compared to the isolates from Gangwon and Gyeonggi provinces (Table 4). Moreover, phylogenetic analysis using these isolates as comparators based on both the partial S- and L-segments demonstrated distinct geographical clustering of isolates from different regions of the ROK. Figures 2 and 3 show that Hantaan virus isolates from Jeju formed a separate cluster from that of Boseong-gun, and similarly, the Gwangju isolates formed another distinct cluster. These three clusters were distinct from the Hantaan isolates reported in previous studies from Gyeonggi province (Hantaan virus/76-118–a Korean prototype Hantaan virus isolated in Uijeongbu, Yeoncheon, Pocheon, Paju) and Gangwon province (Hwacheon, Yanggu, Cheorwon). All of our Hantaan virus strains also clustered separately from isolates from neighboring countries including China and Russia. Although our results were based on partial segments of the viral genome, they suggest the emergence of a new hantavirus genotype in the southwest region of the ROK. This suggestion is supported by the high degree of reassortments shown to take place within Hantaan virus strains. Further studies based on full genome sequences are required to confirm the emergence of a new genotype of hantavirus.
This study has a few limitations, and the genetic distances were computed for only partial segments of the genome. Additionally, we did not perform serological tests on the Gwangju and Boseong-gun samples, as they were not collected in these regions. Further large-scale studies are required to understand the geographical clustering and seasonal variation of hantaviruses.
Environmental and genetic factors that mold the diverse evolutionary patterns observed in RNA viruses and illustrate the complexity of these systems can make it difficult to predict future viral disease emergence [21]. Therefore, timely surveillance of these pathogens in their hosts and carriers is essential.