Bat Influenza A(HL18NL11) Virus in Fruit Bats, Brazil

Screening of 533 bats for influenza A viruses showed subtype HL18NL11 in intestines of 2 great fruit-eating bats (Artibeus lituratus). High concentrations suggested fecal shedding. Genomic characterizations revealed conservation of viral genes across different host species, countries, and sampling years, suggesting a conserved cellular receptor and wide-ranging occurrence of bat influenza A viruses.

I nfluenza A viruses are major causes of human disease and are predominantly maintained in avian reservoirs (1). The segmented influenza A genome facilitates reassortment events in birds or intermediate hosts, such as swine and horses, leading to emergence of new variants potentially capable of causing zoonotic infections (2). Bats are major sources of zoonotic pathogens (3). In pioneering studies from 2012 and 2013, the first bat influenza A viruses, termed H17N10 and H18N11, were discovered in 2 bat species, Sturnira lilium (little yellow-shouldered bat) and Artibeus planirostris (flat-faced fruit-eating bat) (4,5).
Bat-associated influenza A viruses are phylogenetically highly divergent from avian-associated influenza A viruses in their hemagglutinin (HA) and neuraminidase (NA) genes, suggesting these viruses represent ancient influenza A strains (2). Consistent with their genetic divergence, batassociated influenza A surface proteins lack typical hemagglutination and neuraminidase activities (6), leading to the terminology HA-like (HL) and neuraminidase-like (NL) for bat-associated influenza surface proteins.
So far, only 4 individual bat specimens yielded influenza A genomic sequences during the pivotal investigations (4,5). HL18NL11 has only been found in 1 A. planirostris bat captured in Peru in 2010 (5), challenging definite host assessments. To investigate bat influenza A virus epidemiology, we investigated bats in southern Brazil during 2010-2014.

The Study
For this study, we sampled 533 individual bats representing 26 species and 3 families across 28 sampling sites (Table  1). Bats were captured using mist nets, euthanized, and necropsied and were identified on the basis of morphological criteria by trained field biologists as described previously (7). Only intestine samples were available for virological analyses. The Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais (21748-1), Instituto Ambiental do Paraná (235/10), and the ethics committee of the Institute of Biomedical Science from the University of São Paulo (56-18-03/2014) authorized sampling.
We tested intestine specimens from all bats using 2 highly sensitive, broadly reactive nested reverse transcription PCRs targeting different regions of the influenza A polymerase basic (PB) 1 gene (5,8 A. lituratus bats were the most abundantly sampled species ( Table 1).
Sanger sequencing of the screening PCR amplicons suggested close genetic relatedness of the strains circulating in Brazil with the HL18NL11 strain circulating in Peru. Virus concentrations in the positive intestine specimens as determined by strain-specific quantitative real-time reverse transcription RT-PCR (Appendix Table 1, https://wwwnc. cdc.gov/EID/article/25/2/18-1246-App.pdf) were high (1.5 × 10 9 and 4.9 × 10 10 RNA copies/g of tissue). High HL18NL11 concentrations in intestinal specimens are consistent with qualitative data from the pioneering study on HL18NL11 (5) and may suggest intestinal tropism and potential fecal shedding into the environment.
We determined the full coding sequence of all 8 segments of the viral genomes using primers aiming at amplifying overlapping regions of bat influenza A virus genomes (GenBank accession nos. MH682200-15) (Appendix Table 1). The 2 HL18NL11 variants in Brazil differed by 15 nt from each other across the combined 8 genomic segments. Four of those substitutions were nonsynonymous, causing amino acid exchanges in the PB2 (V203I), PB1 (R334K), nucleoprotein (G484S), and NA (V191I) genes (Table 2; Figure 1, panel B). This finding suggests recent common ancestry of the HL18NL11 variants identified in the 2 positive bats and was consistent with their detection in the same site 5 days apart. Comparison of the full coding sequence of the novel HL18NL11 variants revealed high sequence identity between the Peru and the Brazil strains, 93.5%-96.9% nucleotide identity across all 8 genomic segments ( Table 2). The genomic relatedness of Peru and Brazil HL18NL11 strains was surprising given a time span of 2 years, a geographic distance exceeding 2,000 km, and 2 different bat species that tested positive in our study and the previous study (5).
All critical amino acid residues associated with influenza A virus replication and entry (4,5) were conserved between the Brazil and the Peru HL18NL11 strains, including the HA monobasic cleavage site motif PIKETR/GLF (5). Thermodynamic modeling revealed that the amino acid exchanges observed between the Brazil and Peru HL18NL11 strains did not alter the tridimensional structure of the HL and NL proteins, and neither mapped to the putative receptor binding site of the HL protein ( Figure 1, panel C), nor to the putative active site of the NL protein ( Figure 1, panel D) (6). This result suggests preservation of the biologic activity of these glycoproteins in different bat species and supported a broadly conserved cellular receptor of bat influenza A viruses that differs from sialic acid receptors used by avian-associated influenza A viruses (10). Significantly fewer amino acid exchanges were observed between the HL proteins of Brazil and Peru bat influenza virus than between the respective NL proteins (p = 0.007 by Fisher exact test) (    N20T, K350R, L357M, I380L, I387V  N20T, K350R, L357M, I380L, I387V, G484S  NL  93.5%  I11V, I15L, V82I, V200I, L254I, A264T, V284I,  D332E, V378I, G382E   I11V, I15L, V82I, V191I, V200I, L254I, A264T,  V284I,  low rate of nonsynonymous substitutions in the HLencoding genes of bat influenza A virus variants was reminiscent of strong purifying selection acting on the hemagglutinin genes in avian-specific influenza A virus strains (11). This finding may suggest comparable evolutionary dynamics between chiropteran and avian reservoirs. Definite assessments will require considerably larger datasets of bat influenza A virus strains.
A. lituratus bats and A. planirostris bats, in which HL18NL11 was originally detected in Peru, represent closely related, yet genetically and morphologically clearly distinct bat species (12). The distribution of these bat species overlaps (Figure 1, panel A), potentially facilitating virus exchange across the populations. Phylogenetic analyses confirmed the close genetic relationship between Peru and Brazil HL18NL11 variants across all 8 segments (Figure 2; Appendix Table 2), suggesting lack of reassortment events according to the available data. Our data thus suggest host associations of HL18NL11 beyond the species level, comparable to genus-level host associations of other batborne RNA viruses such as coronaviruses (13).

Conclusions
The zoonotic potential of HL18NL11 is unclear, yet humanderived cell lines were susceptible to infection by chimeric vesicular stomatitis virus pseudotyped with HL18 (14). The abundance of A. lituratus bats within Latin America (Figure 1  Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. The hypothesis is that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals.