Bats are newly identified reservoirs of hantaviruses (HVs) among which very divergent HVs have been discovered in recent years. However, their significance for public health remains unclear since their seroprevalence as well as antigenic relationship with human-infecting HVs have not been investigated. In the present study archived tissues of 1,419 bats of 22 species from 6 families collected in 5 south and southwest provinces in China were screened by pan-HV RT-PCR following viral metagenomic analysis. As a result nine HVs have been identified in two bat species in two provinces and phylogenetically classified into two species, Laibin virus (LAIV, ICTV approved species, 1 strain) and Xuan son virus (XSV, proposed species, 8 strains). Additionally, 709 serum samples of these bats were also analyzed by ELISA to investigate the seroprevalence and cross-reactivity between different HVs using expressed recombinant nucleocapsid proteins (rNPs) of LAIV, XSV and Seoul virus (SEOV). The cross-reactivity of some bat sera were further confirmed by western blot (WB) using three rNPs followed by fluorescent antibody virus neutralization test (FAVNT) against live SEOV. Results showed that the total HV seropositive rate of bat sera was 18.5% (131/709) with many cross reacting with two or all three rNPs and several able to neutralize SEOV. WB analysis using the three rNPs and their specific hyperimmune sera demonstrated cross-reactivity between XSV/SEOV and LAIV/XSV, but not LAIV/SEOV, indicating that XSV is antigenically closer to human-infecting HVs. In addition a study of the distribution of the viruses identified an area covering the region between Chinese Guangxi and North Vietnam, in which XSV and LAIV circulate within different bat colonies with a high seroprevalence. A circulation sphere of bat-borne HVs has therefore been proposed.
Hantaviruses (HVs), members of the genus Orthohantavirus within the family Hantaviridae in the order Bunyavirales, are responsible for two major life-threatening diseases in humans: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas . Every year around 100,000 HFRS cases and 1,000 HCPS cases are reported worldwide . China suffers severely from epidemic HFRS; in 2017 alone, official statistics reported 11,262 cases with 64 deaths .
HVs are predominantly carried and transmitted by rodents, but insectivores and bats have also been reported as hosts. Several bat-borne HVs are presently known, which show large genetic diversities from currently known rodent- and insectivore-borne HVs. The first reported bat-borne HVs, Magboi virus (MGBV) and Mouyassué virus (MOUV), were identified respectively in Sierra Leone and Côte d’Ivoire of Africa in 2012 [4, 5]. Then two bat-borne HVs, Longquan virus (LQUV) and Huangpi virus (HUPV), were reported in China in 2013 , followed by the detection of Xuan son virus (XSV) at three locations in North Vietnam [7, 8]. We reported the first complete genome of a bat-borne HV, Laibin virus (LAIV), identified from a black-bearded tomb bat in Guangxi Province of China in 2015 [9, 10]. Since then three more complete genomes of bat-borne HVs, Makokou virus (MAKV), Quezon virus (QZNV) and Brno virus (BRNV) have been reported sequentially in 2016, in Central Africa (Gabon), Southeast Asia (Philippines) and Central Europe (Czech Republic), respectively [11–13]. Most recently, a sister lineage of MOUV was detected in dried blood samples from bats in Eastern Africa (Ethiopia) in 2017 . Of these HVs only three, Laibin, Longquan and Quezon viruses were approved as bat-borne HV species within genus Orthohantavirus in the 10th report of International Committee on Taxonomy of Viruses (ICTV) released in 2017 . Phylogenetic analysis of bat-borne HVs has indicated that bats might be the natural original hosts of HV: i.e., the viruses first appeared in bats or insectivores, then emerged in rodents [6, 16–19]. However, due to lack of sufficient bat-borne HV genomic sequences, their evolutionary phylogeny and genetic diversity as well as biological features are poorly understood.
HVs are enveloped and spherical in shape although pleomorphic forms are also found with the diameters ranging from 80–120 nm. Within the capsid is a tripartite negative-stranded RNA genome consisting of small (S), medium (M) and large (L) segments with a total length of about 11.8 kb, respectively encoding nucleocapsid protein (NP), glycoprotein (GP, a precursor for two viral surface glycoproteins, Gn and Gc) and RNA-dependent RNA polymerase (RdRp) . The NP is multifunctional and plays an essential role in viral replication, not only binding viral RNA strands to form a ribonucleoprotein (RNP) to prevent RNA from degradation, but also regulating virus replication and assembly [1, 20, 21]. NP is also the main target for the earliest immune response. Its coding gene is much more conserved than the GP gene, and is therefore commonly used as a diagnostic antigen for HV detection [22–25]. Different serotypes of HVs can be determined by an at least four-fold difference in two-way cross neutralization tests, and it has been reported that serotype-specific as well as group-common and genus-common epitopes can be found in the NP. Cross-reactivity has been found between different serotypes of HVs in rodents and insectivores [26–28]. However, the serological and antigenic relationships between bat- and rodent- or insectivore-borne HVs have not yet been studied.
South and southwest China have a high density of bat population consisting of a large number of diverse species. Recently, investigations on bat viruses in this area have revealed many novel viruses, such as coronaviruses [29–34], filoviruses [35, 36] and group A rotaviruses (RVA) [37, 38]. Among these, some bat-borne coronaviruses [29, 30, 33] and RVAs  have been found to cross species, causing outbreaks of emerging infectious diseases in human and pigs. South and southwest China are also the major epidemic areas of HFRS with all transmission events associated with exposure to rodents . Although increasing number of HVs have been identified in bats, no investigation has been shown their seroprevalence and antigenic characters. The implication of bat-borne HVs to public health is still unclear. In present study, we have conducted systematic etiological and serological investigation on bat-borne HVs in south and southwest China, revealing the antigenic relationships between bat-borne and human-infecting HVs and identifying a geographic region between southwest China and north Vietnam in which divergent bat-borne HVs circulate.
To gain genetic insight into the HVs, the full genomes of LAIV BT33, XSV AR18, AR23 and PR15 were sequenced and analyzed using previously reported methods [9, 40]. As shown in Table 2, three gene segments of LAIV BT33 had the same sizes as previously reported LAIV BT20 . Three segments of XSV AR18 and AR23 had exactly the same size (1,753 nt of S, 3,751 nt of M and 6,521 nt of L), while PR15 had similar sized S (1,743 nt) and L (6,522 nt), but its M segment was shorter (3,584 nt) than those of XSV AR18 and AR23, resulting from a 50-aa deletion at the 5’ terminal of the coding sequence, corresponding to 6–55 aa of Gn protein. This deletion was confirmed by repeated RT-PCR and sequencing. Currently the function of Gn is largely unknown and 1–17 aa is the signal peptide of Gn responsible for translocation of Gn to Golgi [41, 42], therefore the deletion may have impact on Gn location. In addition, the highly conserved motif WAASA (polyprotein-recognized pentapeptide) in the M segment of HV was observed in all four strains, but the ORF in the S segment of some HVs (such as Puumala, Tula and Andes viruses) encoding a 7–12 KDa nonstructural protein (NSs) which functioned as an interferon antagonist were not found [43–45]. Sequence comparison of the four strains with other bat-borne HVs available in GenBank (Table 2) showed that LAIV BT33 shared the highest (98.4–98.6% nt and 99.2–100.0% aa) identities with LAIV BT20 in its three genomic (full-length) segments and low identities with other bat-borne HVs, (49.6–75.4% nt and 45.8–87.3% aa identities in full or partial gene segments), indicating that it is a variant of LAIV. XSV AR18 and AR23 shared the highest (91.8–93.4% nt and 99.0–100.0% aa) identities with the XSV strain F42682 (partial gene segments, full-length not available) and XSV PR15 the highest (82.8–84.9% nt and 97.9–99.1% aa) identities with XSV F44601 (partial gene segments, full-length not available), indicating that they are novel variants of XSV. Full-length genomic sequence comparison of the four strains with those of rodent-and insectivore-borne HVs showed that bat-borne HVs of the present study had very low nt (43.3–66.6%) and aa (40.0–67.6%) similarities to rodent-and insectivore-borne HVs (S3 Table).
Results of the serological assay of 709 bat sera by ELISA against the three viruses are shown in S2 Fig with 88 of them being further confirmed by WB (S3 Fig). Since no standard bat sera (either positive or negative) were available, the highest coincidence rate (CR) between WB and ELISA was used to determine OD492 ELISA positive cut-off values: 0.10, 0.10 and 0.11 at the highest CR value for each virus (87.5%, 86.4% and 86.4%, respectively, for LAIV, XSV and SEOV) (see S4 Fig). With such cut-offs, the κ test showed high levels consistence between the two methods with Z values being 7.0862 for LAIV, 6.8255 for XSV, and 6.9270 for SEOV (all p<0.0001), and the high κ values being 0.7260–0.7505. These results indicate that the established ELISA was valid to test the bat sera. Using these cut-offs, 131 of 709 (18.5%) bat sera were found to be HV antibody positive. Fig 3 shows the distribution of OD492 readings of the 131 positive bat sera, with most sera having OD492 readings between the cut-off and 0.30. To further determine antibody titers, the positive sera were 4-fold diluted from 100× to 1,600× and retested by ELISA. Results showed that most positive sera had titers of 100×, yet 18 sera reached 400×, with the H anti-SEOV at 1,600× (Fig 4). Of 131 positive sera, 55 (7.76%) showed cross-reactivity to all three viruses, 19 (2.7%) to both of LAIV and XSV, 9 (1.3%) to both XSV and SEOV, and 7 (1.0%) to both LAIV and SEOV, whereas sera reacted exclusively with one virus were only 9 (1.3%) with LAIV, 10 (1.4%) with XSV and 22 (3.1%) with SEOV. This further showed that seroprevalence of HVs in bats widely existed in four provinces (in Guangdong bat sera were not collected). As shown in Fig 5, among 13 cities with bat serum collection 12 were seropositive with levels from 5.5% to 35.9%. Of 16 bat species tested 13 had seropositive rates ranging from 4.8% to 50.0%.
As bat-borne HVs have only recently been identified, there is insufficient sequence data at present to provide a comprehensive analysis of their genetic diversities. Apart from the four complete genomic sequences reported here (Table 2), the sequences of representative bat-borne HVs published to date in GenBank show that many are not of complete genomes or even of a full-length gene segment [5–7, 11]. Currently Laibin, Longquan and Quezon viruses are the only bat-borne HVs approved by ICTV so far [6, 9, 12]. Among completed genomes, there are the complete coding sequences of the three gene segments of BRNV from the Czech Republic . Here we report the first genomic sequence of XSV and show that XSV and BRNV should be considered new HV species awaiting ICTV consideration. Fortunately, there are partial L gene sequences available for all bat-borne HVs, which allowed construction of a phylogenetic tree (using 314 bp), permitting comparison with rodent- and insectivore-borne HVs (S5 Fig). This showed that current bat-borne HVs can be classified into nine species as listed in Table 2, with almost every one having a specific bat genus as host. Of them XSV is the most notable bat-borne HV, which has been found in different locations in present and previous studies [7, 8], and showing significant nt variation although aa sequences of the genomic segments are conserved. The nt identity between currently identified XSV variants ranges from 76.1%-93.4% (Table 2). Furthermore the NP of XSV showed cross antigenicity with both SEOV and LAIV but the NP of LAIV showed no cross antigenicity with SEOV (Fig 2B), indicating that XSV is antigenically closer than LAIV to SEOV and therefore an ideal focus for gaining insight into the role of bat-borne HVs in public health. Meanwhile the closer relationship of insectivore-borne HVs NVAV and ALTV to bat-borne than to insectivore-borne HVs in the NP and GP trees (Fig 1) indicated that HVs from bats and insectivores could share the common ancestry for evolution [18, 46]. In general our sequence comparisons and phylogenetic analyses show that bat-borne HVs had broad genetic diversities and had evolved worldwide within an independent and diverse phylogroup. In this regard, more extensive studies obtaining more complete sequences in extended areas will undoubtedly identify more novel bat-borne HVs in future.
Although nine bat-borne HVs have been identified worldwide, the virus detection rate is low and in limited locations [5, 7, 9, 11]. The present study investigated bats in 22 cities, but the viral RNA was found in only three bat colonies in three cities, and RNA-positive rates were only 1.4% (1/74) for LAIV in BS, 3.0% (5/168) for XSV in LB and 7.5% (3/40) for XSV in PE (see Fig 5 and Table 1). In contrast, seropositive rates are higher: 28.1% (9/32) for LAIV and 40% (2/5) for XSV in BS and LB respectively (sera were not collected in PE) (Table 1). Low viral RNA detection rates have also been reported in previous publications with 5.6% (1/18) for MGBV in Sierra Leone , 3.1% (1/32) for LAIV BT20 in China , and 0.8% (1/123) for MAKV in Gabon . Unfortunately, the seroprevalence was not reported in these publications. The antibody titers of bat sera against HVs in our study were rather low (the most were 100× and only 18 were 400×) as compared to those of rodent reservoirs which were usually higher and could reach 50,000 [24, 47], this might be ascribed to the higher diversity of VH (especially FR3) in immunoglobulin genes of bats in comparison to those of mouse, swine and human .
The correlation between RT-PCR positivity and antibody positivity about hantavirus infection in rodents or shrews were reported [49, 50]. Song et al. reported that a certain proportion, although not all, of Ussuri white-toothed shrews (Crocidura lasiura) with IgG antibodies against Imjin virus (MJNV, a newly isolated hantavirus) had MJNV RNA detectable by RT-PCR . In our study bats sampled in 2015 and 2016 showed higher seropositive rates, but HV RNA was not detected from either seropositive or seronegative bats. All nine RT-PCR positive samples were collected in between 2012–2014, but their antibody titers were not tested since the sera were not collected during that time. Moreover, serology study on bat-borne HVs was not conducted in previous publications, therefore further study is needed to understand the dynamics of HV infection and its antibody response in bats.
Serological epidemiology is important to uncover the real situation of bat-borne HV prevalence, and is critical for eventual estimation of the potential risk of these viruses to public health. Since bat-borne HVs have never been isolated, their NP is a preferential target for serological investigation and antigenic differentiation. It is the main immunogenic protein which contains both serotype-specific and group common epitopes, and is commonly used as diagnostic antigen for HV detection [22–25, 51]. For these reasons, rNPs of LAIV, XSV and SEOV expressed in E. coli were used to assay all 709 bat serum samples by ELISA, resulting in identification of a large number of seropositive sera (Fig 3), with many likely to cross react with two or three rNPs (see OD492 values in S2 Fig). To confirm this, 88 ELISA sera were further tested by WB against all three rNPs with results showing that, except for sera reacting exclusively with one rNP, some could cross recognize two rNPs, mainly the rNPs of LAIV/XSV, or XSV/SEOV, and seldomly the rNPs of LAIV/SEOV (see S3 Fig). It is notable that 21 sera could cross recognize three rNPs with 10 showing very strong reactivity against all three rNPs. The role of NP in producing this cross antigenicity was further verified by WB using a combination of eukaryotically expressed rNPs and NP-specific antiserum (see Fig 2B). To identify NAb against SEOV, 48 bat sera were analyzed by FAVNT, which identified 9 (18.8%) positives. Fig 4 summarizes the results of 48 bat sera assayed by FAVNT, ELISA and WB. Of the nine NAb-positive sera, four (BN5, ZS7, ZS25, ZS27) neutralized but did not react by ELISA or WB, three (BN64, CZ67, NP6) not only neutralized but also reacted with three rNPs by ELISA and WB. The most interesting bat serum was BN78, which neutralized SEOV and reacted with the rNP of SEOV but not with that of LAIV and XSV. BN78 was collected from a Rousettus leschenaultii bat, of this species 51 of 142 individuals showed anti-HV antibody positive (35.9%), the highest among all bat species (Table 1). Furthermore another Rousettus species (Rousettus amplexicaudatus) was reported to harbor Quezon virus in the Philippines , suggesting that fruit bats in genus Rousettus are likely major reservoirs of HVs. Moreover many sera without neutralizing activity reacted with the three rNPs by ELISA and WB. Altogether, it is interesting to have found multiple patterns of cross-reactivity with three rNPs. Illustration of the complex patterns will be difficult but likely to imply that the bats had been infected with other unknown HVs. The prime example is bat serum BN78. It had the highest neutralization titer against SEOV and exclusively strong reactivity with the rNP of SEOV, indicating that this bat was infected by an unknown HV antigenically very close to SEOV, but not SEOV since both human anti-SEOV convalescent serum (Fig 2A) and SEOV-specific anti-rNP serum (Fig 2B) could also cross react with the rNP of XSV. Altogether, the multiple genetic diversities and different cross-reactivity patterns indicate that more as yet unknown bat-borne HVs circulate in the investigated region, but to uncover them further investigation is needed.
As shown in Fig 5, 12 of the 13 cities in 4 provinces in which serum collections were made had a positive seroprevalence, with Guangxi having the most positive samples and most seropositive locations (6 of 7 sampled cities were seropositive). Furthermore, two HVs were detected in two of its cities, BS and LB. In 2015, LAIV was identified in LB , in which XSV was found in the present study although from another location within the city, indicating that divergent bat-borne HVs co-exist in LB. LAIV was also found in BS this time, several hundred kilometers west of LB (see Fig 5), indicating that LAIV has a broad distribution in Guangxi province. It is notable that XSV has been identified in two north Vietnamese provinces, Tuyên Quang (TQ) and Phú Thọ (PT), as shown in Fig 5, and in the central Vietnamese province Quảng Nam since 2013 [7, 8]. In present study eight strains of XSVs were identified in LB of Guangxi and PE of Yunnan, indicating that XSV circulates in the vast area between Chinese Guangxi/Yunnan and Vietnam. The accumulated serological and molecular data highly support the proposition that a vast area between China and southeast Asia provides a natural focus for bat-borne HV circulation. In this area natural circulation of genetically divergent bat-borne HVs in their hosts would be maintained, and therefore the concept of a bat-borne HV circulation sphere has been introduced to describe the situation. While there is a lack of sufficient serological data in Yunnan Province, a narrow area between southwest Guangxi and north Vietnam likely forms a main circulation sphere of at least two species of bat-borne HVs (Fig 5). With more extensive investigations this area may be extended, particularly to surrounding areas in Laos, Myanmar and even Thailand.
In conclusion, the present study has compiled the first profiling of cross antigenicity between bat-borne and human-infecting HVs as well as among bat-borne HVs. It has also revealed the seroprevalence and wide distribution of bat-borne HVs in south and southwest China. A comprehensive analysis based on genetic diversity, seroprevalence, cross antigenicity and host range of the viruses has helped identify an area between China and Vietnam as a main circulation sphere where at least two bat-borne HVs circulate in the bat population. Given the existence of bat-borne HVs genetically and antigenically close to human-infecting HVs, extensive studies should be emphasized in future to assess the potential risk of bat-borne HVs to public health.
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