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). Positive results on both tests came from only 2 samples, from Artibeus lituratus great fruit-eating bats captured on March 7 and March 12, 2012, at 2 locations separated by 12 km in an Atlantic rainforest patch. No other sample was positive, yielding a 10.0% (2/20) overall detection rate in this site and 16.7% (2/12) detection in A. lituratus bats from this site (Table 1; Figure 1, panel A). Neither bat testing positive for influenza A virus showed signs of disease. A. lituratus bats were the most abundantly sampled species (Table 1).

The low overall influenza virus detection rate in this study (0.4%, 95% CI 0.0%–1.5%) was not significantly different by Fisher exact test from the previous 2 studies (1/110 bats for HL18NL11 [0.9%, 95% CI 0.0%–5.5%; p = 0.43]; 3/316 bats for HL17NL10 [1.0%, 95% CI 0.0%–2.9%; p = 0.37]). Apparently low rates of acute influenza A virus infection in bats are not consistent with high seroprevalence of 72% in different bat species according to a preliminary investigation (5) and may hint at seasonal variation in bat influenza virus infections, comparable to other batborne RNA viruses (9).

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) were high (1.5 × 10⁹ and 4.9 × 10¹⁰ 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) (Table 2). The apparently low rate of nonsynonymous substitutions in the HL-encoding 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).

The zoonotic potential of HL18NL11 is unclear, yet human-derived 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, panel A) may thus facilitate spillover infections into other vertebrates across an underrecognized geographic and host range. Finally, Artibeus spp. bats have been used previously for infection studies including viruses with evolutionary origins in bats, such as Middle East respiratory syndrome coronavirus (15). The relatively large body size of A. lituratus bats (≈65 g) and ease of keeping these bats under laboratory conditions may thus facilitate experimental infection studies for HL18NL11 to elucidate the exact sites of HL18NL11 replication, receptor usage, and mode of transmission.

Additional information related to bat influenza A(HL18NL11) virus in Brazil, 2010–2014.