Isolation and Characterization of a Distinct Influenza A Virus from Egyptian Bats

Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had an affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species, including domestic birds, mammals, and, possibly, humans.

ABSTRACT

Recently, two genetically distinct influenza viruses were detected in bats in Guatemala and Peru. We conducted influenza A virus surveillance among four bat species in Egypt. Out of 1,202 swab specimens, 105 were positive by real-time PCR. A virus was successfully isolated in eggs and propagated in MDCK cells in the presence of N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin. Genomic analysis revealed that the virus was phylogenetically distinct from all other influenza A viruses. Analysis of the hemagglutinin gene suggested a common ancestry with other H9 viruses, and the virus showed a low level of cross-reactivity with serum raised against H9N2 viruses. Bats were seropositive for the isolated viruses. The virus replicated in the lungs of experimentally infected mice. While it is genetically distinct, this virus shares several avian influenza virus characteristics suggesting a more recent avian host origin.

IMPORTANCE Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had an affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species, including domestic birds, mammals, and, possibly, humans.

INTRODUCTION

Bats are reservoirs for a wide range of zoonotic viruses, such as rabies, Ebola, Marburg, Hendra, Nipah, and severe acute respiratory syndrome (SARS) viruses (1). Furthermore, bats were found to be the major evolutionary reservoir for coronaviruses, including the Middle East respiratory syndrome coronavirus (2, 3). The global distribution, wide species diversity, and high population densities of bats as well as the opportunity for direct and indirect contact with humans and other animals emphasize the need to better understand the ecology of bat-borne viruses.

Until recently, 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A viruses (IAVs) have been detected in shorebirds and waterfowl, which were known to be the only natural reservoirs for IAVs. RNA from two influenza viruses, A(H17N10) and A(H18N11), was detected in the little yellow-shouldered bat (Sturnira lilium) in Guatemala and the flat-faced fruit-eating bat (Artibeus jamaicensis) in Peru, respectively (4, 5). Attempts to isolate the bat influenza viruses were not successful, and the viruses did not appear to utilize the canonical influenza A virus receptor sialic acid (5). Seroprevalence studies of H17/H18 viruses in Central and South American bat species indicated that bats might constitute a vast reservoir of novel influenza viruses (5). In Ghana, 30% of tested frugivorous bat serum specimens contained antibodies that recognized H9 avian influenza viruses and, to a lesser extent, H8 and H12 viruses (6). Surveillance in European bats failed to detect IAVs, indicating that not all bat species may act as reservoirs of influenza viruses (7). Since evidence showed that bats may be a reservoir for novel lineages of IAV, we conducted surveillance among four bat species in Egypt.

RESULTS

Detection and isolation of influenza A virus in bats.

A total of 601 bats were captured from abandoned houses and caves in Egypt during 2016 and 2017. Out of 1,202 oral and rectal swab specimens, 105 samples were positive for influenza A virus by real-time (RT)-PCR; all positive samples were collected from the same site. The positive samples were collected from captured Rousettus aegyptiacus bats from an abandoned mudbrick house in a village in the Nile Delta region in March 2017. The rate of IAV detection in oral swabs (n = 71) was higher than that in rectal swabs (n = 44). Virus was detected in both oral and anal swab samples from 33 animals. BLASTN analysis of the obtained partial PB1 sequences (which were 99.9 to 100% identical to each other) showed that they had 83% similarity with A/black-headed gull/Netherlands/1/2009(H13N2) virus. Unlike the H17 and H18 viruses, the Egypt bat virus was successfully isolated in chicken eggs and propagated in MDCK cells in the presence of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. No plaques were formed in the absence of TPCK-treated trypsin, indicating that the newly detected bat influenza virus requires an extrinsic source of trypsin for replication.

Hemagglutination titration using different types of erythrocytes (RBCs) showed that chicken, turkey, rat, and bat erythrocytes gave the same titer (7 log₂ HA titer), while human erythrocytes yielded lower HA titers (5 log₂ HA titer).

Genome features and sequence identity.

The sequenced segments (GenBank accession numbers MH376902 to MH376909) of the purified bat influenza virus egg isolate were aligned with known IAV genomic segments. The eight RNA segments of the virus consisted of four monocistronic segments that encode PB2, HA, NP, and NA viral proteins and four segments (PB1, PA, M, and NS) containing additional open reading frames to express the PB1, PB1-F2, PA, PA-X, M1, M2, NS1, and NS2 viral proteins.

Except for the HA, M1, and NA viral proteins, insertions or deletions were not detected in the alignment of the bat influenza viral proteins with known influenza A viral proteins. One deletion and one insertion were observed in the BLASTP analysis of HA and M1, respectively. The N terminus of the NA viral protein had 14 amino acid differences consisting of 11 deletions and 3 insertions (Table 1).

TABLE 1.

Highest nucleotide and amino acid similarity of the newly characterized bat influenza virus to different influenza A subtypes

Segment	Viral protein	Influenza A virus with highest similarity to bat isolate	Nucleotide similarity (%)	No. of insertions or deletions in nucleotide alignmenta	Amino acid similarity (%)	No. of insertions or deletions in amino acid alignment
PB2	PB2	A/duck/Hunan/S11682/2015(H7N9)	80	0	91	0
PB1	PB1	A/black-headed gull/Netherlands/1/2009(H13N2)	82	2 (1I and 1D)	92	0
PB1	PB1-F2	A/shorebird/Delaware Bay/3/1994(H3N2)	87		67	0
PA	PA	A/equine/Lexington/1/1966(H7N7)	81	1 (D)	90	0
HA	HA	A/mallard/Ohio/13OS3856/2013(H9N2)	73	3 (D)	73	1 (D)
NP	NP	A/blue-winged teal/Guatemala/CIP049H112-60/2012(H3N8)	81	0	88	0
NA	NA	A/blue-winged teal/Guatemala/CIP049H108-67/2012(H3N2)	72	24 (D)	67	14 (11D and 3I)
M	M1	A/laughing gull/NY/2455/2000(H7N3)	82	4 (I)	91	1
M	M2	A/laughing gull/NY/2455/2000(H7N3)	82	1 (D)	91	0
NS	NS1	A/common pochard/XiangHai/420/2010(H7N1)	78	5I	75	8
NS	NS2	A/common pochard/XiangHai/420/2010(H7N1)	78	6D	77	0

^aI, insertions; D, deletions.

Multiple mutations associated with mammalian transmission (S199 and N701 in PB2; 13P in PB1; N55, Y241, and S404 in PA; K357 and E455 in NP; N20 in M2) and virulence (V504 and N701 in PB2; I317 in PB1; V127, R100, L550, and L672 in PA; 69P in M2; 42S in NS1) were present.

Analysis of the HA gene suggested that the HA of the bat isolate is more closely related to group 1 HAs (subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) than to those of group 2 (subtypes H3, H4, H7, H10, H14, and H15) (Fig. 1A). The bat isolate’s HA had 73% amino acid sequence identity to HA from the A/mallard/Ohio/13OS3856/2013(H9N2) virus (Table 1). Analysis of the HA of the current virus using the SignalP server showed the predicted signal peptide 1-MEVKIFIILLIIQISRG-17 and the N-terminal amino acid sequence of the mature protein (18-DKICIGYQSNNS . . .).

FIG 1.

Phylogenetic tree of the nucleotide sequences of the eight genome segments of the characterized bat influenza virus in Egypt. Maximum likelihood trees were generated in RAxML (v8.0.0) for the eight gene segments with the GTR substitution model and the gamma distribution of rate heterogeneity. Bootstrapping analyses were conducted with the extended majority rule consensus tree criterion (the autoMRE option in RAxML). Trees were midpoint rooted and visualized in FigTree (v1.4.2; http://tree.bio.ed.ac.uk/software/figtree/). Bootstrapping values are shown on the tree nodes. The red star represents the Egyptian bat influenza virus. Orange branches represent H17/H18 bat influenza viruses. The scale bars represent 0.2 substitution per site for each associated tree.

Critical sequence motifs in the HA of the bat virus, such as the sialic acid receptor-binding site (RBS), glycosylation sites, and cleavage site, were identified (Table 2). The monobasic (R) amino acid at the cleavage site of the HA of the currently characterized virus may indicate low pathogenicity. Analysis of the N-XT/S motif (where X can be any amino acid except proline) revealed that the Egyptian bat virus has five potential glycosylation sites at positions 29, 141, 298, 305, and 492 within the HA molecule (Table 2). Within the RBS, the Egyptian bat isolate had Q234 (H9 numbering), indicating preferential binding to sialic acid linked to galactose via 2,3-α linkages (Sia 2,3-α-Gal).

TABLE 2.

Comparison of amino acid sequences of HA of the newly characterized virus isolated from bats in Egypt with ancestor H9N2 viruses at RBS, cleavage site, and glycosylation sites

Virus	Amino acid at RBS position (H9 numbering):									Cleavage site	Sequence at the following glycosylation site:
	166	191	197	198	232	234	235	236	399		29	105	141	218	298	305	492
A/Hong Kong/1073/99 (H9N2)	S	H	T	E	N	L	Q	G	K	PARSSRGLF	NSTE	NGTC	NVTY	NRTF	NSTL	NISK
A/chicken/Egypt/7100/2013 (H9N2)	N	H	T	A	N	L	I	G	K	PARSSRGLF	NSTE	NGTC	NVTY		NSTL	NISK	NGTY
Bat isolate	N	H	E	Q	G	Q	G	R	Q	PAIQTRGLF	NSTD		NVTY		NTSL	NISK	NGTY
A/Duck/Hong Kong/365/78 (H4N6)	D	H	T	E	R	Q	S	G	E

The NA of the bat isolate had 72% amino acid sequence identity to the NA of the A/blue-winged teal/Guatemala/CIP049H108-67/2012(H3N2) virus (Fig. 1B). The protein encoded by the NA gene showed extensive divergence from known influenza virus NAs in amino acid residues.

The PB2 segment of the bat isolate had the highest pairwise similarity with the PB2 of A/gray teal/Australia/2/1979(H4N4) virus (80% nucleotide similarity), while the PB1 gene was related to the PB1 genes of the influenza A/black-headed gull/Netherlands/1/2009(H13N2) and A/duck/Memphis/546/1974(H11N9) viruses with 82% nucleotide sequence identity. The PA genes of the newly characterized virus had higher similarity to A/equine/Lexington/1/1966(H7N7) (81%). The NP, M, and NS genes of the virus isolated from bats had the highest identity of 81, 82, and 78% with A/blue-winged teal/Guatemala/CIP049H112-60/2012(H3N8), A/laughing gull/NY/2455/2000(H7N3), and A/common pochard/XiangHai/420/2010(H7N1), respectively (Fig. 1C to H).

Comparative genomic analysis.

Phylogenetic analysis of all eight gene segments (Fig. 1) supported the possibility that the Egyptian bat influenza virus is a distinct lineage and clustered with avian influenza viruses rather than H17/H18 bat influenza viruses. Specifically, the HA tree (Fig. 1A) showed that the Egyptian bat influenza virus clustered within the group 1 HA. Even though the virus shared ancestry with the avian H9 lineage (bootstrap support = 99%), this virus was of a distinct lineage. The NA tree (Fig. 1B) also revealed that the Egyptian bat NA was distinct from other NA subtypes, sharing ancestry with avian/mammalian N2 (bootstrap value = 100%). All trees of internal genes (Fig. 1C to H) demonstrated that the Egyptian bat virus is distinct from other bat influenza viruses but is related to other IAVs (bootstrap values = 98 to 100%), including avian and mammalian influenza viruses.

Antigenic characterization and seroprevalence of antibodies in bat serum.

The Egyptian bat influenza virus reacted weakly with reference antisera raised against the North American A/turkey/Wisconsin/1/66(H9N2) virus (titer, 1:16) and A/chicken/Egypt/S4456B/2011(H9N2) (titer, 1:8) but did not react with antisera against other subtypes.

Figure 2 shows the serological results for the tested bat sera. Sera collected from bats on 19 March 2017 at the site where the virus was detected showed a low seroprevalence of antibodies against the bat virus, with only two serum samples having titers of ≤1:32. However, about 63% of the serum samples showed antibodies against an Egyptian H9N2 virus, with titers ranging from 1:8 to 1:512. Sera collected 2 months following the detection of the virus from the same brood showed antibodies against the bat influenza virus. About 97% of the tested bat serum samples had an antibody titer ranging from 1:8 to 1:512, with the majority having a titer of 1:64. Cross-reactivity with the H9N2 viruses was also noted. Sera collected from a different roost 7 km away from the roost where the virus was detected also showed evidence of antibodies against the bat virus and cross-reactivity with the avian H9N2 viruses. None of the serum samples reacted against the avian H4N6 virus.

FIG 2.

Distribution of antibodies against the bat influenza virus (bat flu) as well as avian H9N2 and H4N6 viruses.

Receptor binding and antigenic properties.

As predicted by sequence analysis, the Egyptian bat virus had higher binding preference for 3′-sialyllactose (α2,3-SL) receptors than 6′-sialyllactose (α2,6-SL) receptors (Fig. 3). The control H4N6 virus showed a higher binding preference for α2,3-SL, while the A/chicken/Egypt/7100/2013(H9N2) and A/Hong Kong/1073/99 (H9N2) viruses showed a preference for α2,6-SL.

FIG 3.

Receptor-binding specificity of Egyptian bat influenza virus. The direct binding of Egyptian bat influenza virus to biotinylated sialylglycopolymers containing 3′-sialyllactose (α2,3-SL) or 6′-sialyllactose (α2,6-SL) was measured. Influenza A/chicken/Egypt/7100/2013 (H9N2), A/Hong Kong/1073/99 (H9N2), and A/duck/Hong Kong/365/78 (H4N6) were used as controls for the binding assay. O.D., optical density.

Antiviral sensitivity assays.

None of the five amantadine resistance genotypic markers (L26F, V27A/G/I, A30T, S31N, and G34E) in the M2 ion channel protein of the current isolate were identified. The Egyptian bat virus was sensitive to amantadine, showing no HA titer at an amantadine concentration of 0.2 μg/ml. In agreement with NA gene sequence analysis, the Egyptian bat influenza virus was sensitive to zanamivir with a 50% inhibitory concentration (IC₅₀) of <0.005 μg. Similarly, the virus was sensitive to oseltamivir (IC₅₀ < 0.005 μM).

Pathogenicity in chickens and mice.

C57BL/6 mice infected with the bat virus did not show symptoms or weight loss. However, virus was detected in the lungs of those mice at 2 and 7 days postinfection (dpi) (Table 3). BALB/c mice showed no symptoms of infection, but the amount of weight lost in those mice was similar to that lost in mice infected with the avian H9N2 virus. Significant weight loss (t test P values < 0.05) was detected as of 5 dpi (10% weight loss) and reached about 27% by 10 dpi (Fig. 4). Virus was detected in the lungs of those mice at 2 and 7 dpi (Table 3).

TABLE 3.

Viral titers from lungs of sacrificed mice infected with the H9N2 and bat influenza viruses at 2 and 7 dpi

dpi	Mouse strain	Bat influenza virus		H9N2 virus
		No. of EID50/mla	No. of PFU/mlb	No. of EID50/ml	No. of PFU/ml
2	C57BL/6	2.1 ± 0.28 (3/3)	2.99 ± 0.3 (2/3)	4 ± 2.5 (3/3)	5.01 ± 0.01 (2/3)
7	C57BL/6	3.6 ± 0.5 (3/3)	3.2 (1/3)	5 ± 0.5 (3/3)	3.7 ± 0.5 (3/3)
2	BALB/c	3.8 ± 0.2 (3/3)	4 (1/3)	3.5 ± 0.5 (3/3)	4 ± 0.4 (2/3)
7	BALB/c	2.6 ± 1.05 (3/3)	3.07 (1/3)	2 ± 0.1 (2/3)	(0/3)c

^aData are the mean number of log₁₀ EID₅₀ per milliliter ± standard deviation for positive samples. Values in parentheses are the number of positive mice/total number of mice in each group.

^bData are the mean number of log₁₀ PFU per milliliter ± standard deviation for positive samples. Values in parentheses are the number of positive mice/total number of mice in each group.

^cThe titer was below the limit of detection (<1 log₁₀ PFU/ml).

FIG 4.

Weight change of mice infected with A/bat/Egypt/381OP/2017 and avian H9N2 virus. Female C57BL/6 and BALB/c mice were intranasally infected. The virus-infected mice were monitored for 10 days, and the weight was determined daily. Results from each group and each time point are expressed as the means and standard deviations (SD) for infected mice. Analysis and visualization were performed using GraphPad Prism (v5) software.

Chickens infected with the bat influenza virus showed no morbidity or mortality. The virus was not detected in swabs or collected organs.

DISCUSSION

Bats have been shown to be natural reservoirs for several viral families, including IAVs. Recently, two novel subtypes of IAV were detected in bats in Central and South America (4). Here we detected, isolated, and sequenced a new IAV from Egyptian fruit bats. This virus was detected in a densely inhabited agricultural area, a village in the Nile Delta region. The bat roost in which this virus was detected was an abandoned house within the village, and the bats likely feed on fruits of the village orchards. The virus was detected in oral and rectal swabs, indicating potential shedding in saliva and feces. Hence, direct and indirect transmission routes are available if the virus can cross the species barrier to infect humans or other animal species.

At the time of sampling, no evident signs of disease were noticed in the roost. This is concordant with other work showing asymptomatic infection of bats infected with different viruses (8). The detection of the virus in oral and rectal swabs as well as the detection of antibodies against the virus in the same roost a few weeks after the detection of the virus suggests that the virus was causing infection in the bats rather than transiently passing through the digestive track. Due to the nonlethal sampling techniques used for this study, organs were not collected from the sampled bats; hence, the anatomical sites of infection were not identified. The previously identified bat IAVs were detected in lung, kidney, liver, and intestine tissues as well as oral swabs (4). It is unlikely that the virus circulates only at the sampled roost, as serological evidence of infection was detected in another roost 7 km away.

Phylogenetic analyses of the eight gene segments showed that the isolated bat IAV forms a distinct lineage, as bootstrap values to the nearest branch were 98 to 100%. We inferred that this virus is not close to two other bat lineages (H17N10 and H18N11) but was introduced and adapted from avian IAVs. Analysis of the HA tree showed that the novel virus is much closer to avian H9 viruses than other avian subtypes but remains distinct, as the bootstrap value indicated. This relation to H9 was supported by the fact that the virus cross-reacted with sera raised against avian H9N2 viruses. With its biological capability to infect and replicate in chicken eggs, we suggest that this virus may share common origins with avian H9 virus. Analysis of the NA tree showed that the bat virus is a separate lineage from N2, its closest relative, with a bootstrap value of 100% on the node. Hence, the potential ancestor of this virus is an avian H9N2 virus or an unidentified virus from another host.

The isolated virus had an affinity to α2,3-sialic acid receptors and grew efficiently in embryonated chicken eggs, further supporting the hypothesis that this virus has an avian origin. However, the virus did not replicate in experimentally infected chickens, suggesting that the virus would require further changes to cross the species barrier and adapt to domestic poultry. Infection in ducks may be a prerequirement to infection of domestic chickens. The virus was capable of infecting MDCK cells, C57BL/6 mice, and BALB/c mice, indicating its capacity to infect other mammalian species. However, the receptors that the virus used to infect mice and cells require further investigation. Even though we were unable to ascertain the receptor distribution of the Egyptian fruit bat, another species of bat was shown to possess both avian- and human-like sialic acid receptors (9). It is possible that cells of the Egyptian fruit bats may also support α2,3-sialic acid receptors. Taken the findings together, our study suggests that the bat IAV A/bat/Egypt/381OP/2017 from Egyptian fruit bats may have originated from an avian host. Further surveillance for IAVs in bats is recommended to understand the distribution, diversity, disease threat, and other subtypes circulating in fruit bats.

Here, we isolated and characterized a bat IAV, A/bat/Egypt/381OP/2017, from Egyptian fruit bats that has a distant lineage from previously identified IAVs. This virus may have originated from an avian host, as suggested by its genetic and antigenic relationship to avian H9N2 and its affinity to avian-like sialic acid receptors. However, phylogenetic analysis suggests that this may be a novel subtype virus, but more studies are required to validate this. Further surveillance for IAVs in bats is recommended to understand the distribution, diversity, and potential risks of these viruses.

MATERIALS AND METHODS

Sample collection.

A total of 1,202 rectal and oral swab samples were collected from four different bat species (Rousettus aegyptiacus, n = 473; Pipistrellus aegyptius, n = 92; Nycteris thebaica, n = 25; and Taphozous perforatus, n = 11) from 22 August 2016 until 20 May 2017. The protocol for animal capture and sample collection was approved by the ethical committee at the National Research Centre (Cairo, Egypt) and the Institutional Animal Care and Use Committee (IACUC) of the Univeristy of California at Berkeley. A swab was inserted into the rectal or oral cavity of bats and left in place for a few seconds before being slowly withdrawn using a rotating motion. The tip of the swab was placed into a collection vial containing 1 ml transport medium (Dulbecco’s modified Eagle medium [DMEM] supplemented with 2% fetal bovinse serum and 2% antibiotic-antimycotic mixture [BioWhittaker, Walkersville, MD, USA]).

Detection and sequencing of influenza A virus.

RNA extraction was performed using a Zymo Research Direct-zol RNA MiniPrep kit according to the manufacturer's protocol. RT-PCR targeting the PB1 gene was performed for influenza A virus detection first using a Qiagen one-step RT-PCR kit and primers FLUAPB1-F (ATGATGATGGGNATGTTYAAYATG) and FLUAPB1-R (CNGGNCCNAKDTCRYTRTTDATCAT) in a 25-μl reaction mixture (5 μl of 5× reaction buffer, 1 μl deoxynucleoside triphosphates [10 mM], 1 μl enzyme mix, 1 μl [10 μM] forward primer, 1 μl [10 μM] reverse primer, 10 μl double-distilled H₂O, and 5 μl of RNA sample). The RT-PCR cycling conditions were 95°C for 5 min and then 14 cycles of 96°C for 5 s, 65°C for 8 s (−1°C/cycle), and 68°C for 15 s, followed by 35 cycles of 96°C for 5 s, 50°C for 8 s, and 68°C for 15 s. The final elongation was for 4 min at 72°C. The PCR products were subjected to a second round of PCR using the primers FLUAPB1-F and FLUAPB1-R and then analyzed by agarose gel electrophoresis for the presence of a 402-bp amplicon band. The final PCR product was gel purified and sequenced using primers FLUAPB1-NF (GATGGGNATGTTYAAYATGYTDAGYAC) and FLUAPB1-R.

Virus propagation and plaque purification.

Three of the PCR-positive samples that had the strongest band intensity were inoculated in the allantoic fluid cavities of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs and incubated for 2 days. At the end of incubation, 100 μl of the allantoic fluids underwent the HA test with 0.5% human (blood group O), bat, rat, chicken, and turkey red blood cells (10). Positive allantoic fluids were aliquoted and stored at −80°C. The HA-positive samples were subjected to viral RNA extraction and then confirmed to be positive for influenza A virus by M gene testing using real-time PCR (RT-PCR) (11).

Plaque purification was performed as follows. A volume of 100 μl of HA-positive egg harvest and its corresponding 10-fold serial dilutions were inoculated into 6-well plates containing confluent MDCK cells with 400 μl serum-free medium. The plates were incubated at 37°C for 1 h. The wells were aspirated to remove residual viral solution. Each well was then immediately covered with 2 ml 1× agarose overlay mixture (final concentrations, 1% agarose type 1, 1× DMEM, 1% antibiotic-antimycotic solution, 4% bovine serum albumin [BSA]) with and without TPCK-treated trypsin. The plates were then incubated at 37°C under 5% CO₂ for 3 days. Plaques were picked, and each plaque was inoculated into specific-pathogen-free embryonated chicken eggs for propagation of purified plaques. The previous purification was performed twice.

Amplification of full genome and sequencing.

Viral RNA was extracted from harvested allantoic fluid, using a QIAamp viral minikit (Qiagen, Germany) according to the manufacturer’s protocol. The first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) and the Uni-12 primer (5′-AGCAAAAGCAGG-3′) per the manufacturer’s protocol. Using a Phusion master mix kit (Thermo Scientific, Wilmington, DE, USA), the desired genes of the new isolate were amplified using universal primers (12). Briefly, using gene-specific primers, 2 μl of each reverse transcription reaction mixture was subjected to PCR by an initial denaturation step (98°C for 30 s), followed by 40 cycles each consisting of 98°C for 10 s, 57°C for 30 s, and 72°C for 3 min and a final elongation step (72°C for 10 min). Amplicons of the appropriate sizes were subsequently gel purified using a Qiagen gel extraction kit (Qiagen, Germany). The purified PCR products were directly used for sequencing reactions. Sequences were assembled using SeqManDNA Lasergene (v7) software (DNAStar, Madison, WI, USA).

Sequence analysis and phylogenetic tree construction.

The assembled sequences were subjected to NCBI BLAST analysis. The BioEdit (v7.0) program was used for multiple-sequence alignment (13). The nucleotide and amino acid homologies were further assessed by the ClustalW method with MegAlign software (DNAStar).

A representative whole-genome data set of all 18 subtypes of influenza A viruses was downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database). Taxa were chosen to represent each HA (H1 to H16) and NA (N1 to N9) subtype, which was based on the availability of sequences in the database (with H1N1 being the most abundant and H14 being the least) and their divergence, as well as the completeness of the genomic sequence and associated metadata and geographical origin. This data set was comparable to that of Tong et al. (4). All available bat-origin sequences (H17N10 and H18N11) were included to test the hypothesis of common origins among circulating bat-borne influenza A virus. Together with the newly sequenced bat-origin influenza A virus, all eight individual gene segments were aligned using the MUSCLE (v3.8.31) program (14). Gene-specific alignments were manually optimized in the Seqotron (v1.0.1) sequence editor (15). After removing sequences that introduced frameshift errors, the final data set contained 70 isolates with full genomes, 1 isolate with HA and NA only, and another isolate without PB1 and PA.

Maximum likelihood trees were generated in the RAxML (v8.0.0) program (16) for the eight gene segments with the general time reversible (GTR) substitution model and gamma distribution of rate heterogeneity. To evaluate the reliability of tree topologies, bootstrapping analyses were conducted with the extended majority rule consensus tree criterion (the autoMRE option in RAxML), which automatically determines the sufficient bootstrap replicates for getting stable support values. Trees were rooted at the midpoint and visualized in FigTree (v1.4.2) software (http://tree.bio.ed.ac.uk/software/figtree/). Bootstrapping values are shown on the tree nodes.

Antigenic analysis.

Antigenic analyses were performed by the hemagglutination inhibition (HI) test using chicken antiserum generated by vaccination of several chicken groups with different representative H9N2 viruses isolated from Egypt and Lebanon and 17 reference antiserum samples raised against (H1 to H11 and H14) IAVs (10).

Seroprevalence of antibodies against the detected bat influenza virus.

Sera from 124 fruit bats were collected over two sampling trips on 19 March 2017 (n = 87) and 20 May 2017 (n = 37) at the site where the bat influenza virus was detected. Another batch of serum samples was collected from 48 fruit bats on 19 April 2017 at another site about 7 km away. Sera were tested for antibodies against A/bat/Egypt/381OP/2017, A/duck/Hong Kong/365/78(H4N6), A/chicken/Egypt/7100/2013(H9N2), and A/Hong Kong/1073/1999(H9N2) viruses using a hemagglutination inhibition assay with 0.5% turkey RBCs in V-shape 96-microwell plates (10).

Receptor specificity assay.

The virus receptor specificity of the isolated bat influenza virus was determined as previously described (17). In addition, two avian influenza isolates [A/duck/Hong Kong/365/78(H4N6) and A/chicken/Egypt/7100/2013(H9N2)] and one human isolate [A/Hong Kong/1073/1999(H9N2)], which had a α2,3-SL and α2,6-SL binding preference, respectively, were used as a point of comparison in this assay. Ninety-six-well fetuin (10 μg/ml)-coated plates were washed with ice-cold washing buffer (0.01% Tween 80 in 0.23× phosphate-buffered saline [PBS]), blocked with PBS containing 1% bovine serum albumin (BSA), and incubated overnight with 32 hemagglutination (HA) units of influenza viruses at 4°C. The plates were washed with washing buffer four times. Biotinylated sialylglycopolymers, 3′-sialyllactose (α2,3-SL; Neu5Acα2-3Galβ1-4Glc) and 6′-sialyllactose (α2,6-SL; Neu5Acα2-6Galβ1-4Glc) (Glycotech, Gaithersburg, MD), were serially diluted in reaction buffer (0.02% Tween 80, 0.02% BSA, 1 μM sialidase inhibitor [zanamivir], 1× PBS) and added to the plates, and the plates were incubated at 4°C for 2 h. The plates were washed (4 times) and incubated with 100 μl of horseradish peroxidase-conjugated streptavidin (1:2,000) at 4°C for 1 h. After a final wash, 50 μl of the o-phenylenediamine (OPD) substrate was added and the plates were incubated for 10 min at room temperature. The reaction was stopped with 1 N sulfuric acid, and the absorbance was measured at 490 nm.

Antiviral sensitivity assays.

An assay to determine the sensitivity of the isolated Egyptian bat influenza virus to amantadine was performed as previously described (18). An amantadine-sensitive virus [A/chicken/Egypt/M2583D/2010(H5N1)] and a resistant virus [reverse genetics A/chicken/Egypt/D10552B/2015(H5N1)] were used as controls. Briefly, stocks of amantadine hydrochloride (amantadine; Sigma-Aldrich, St. Louis, MO) were prepared in infection medium. Monolayers of MDCK cells in a 12-well tissue culture plate were pretreated with 300 μl of DMEM supplemented with 0.2% BSA containing 0, 0.2, 2.0, or 20 μg amantadine for 60 min at 37°C in 5% CO₂. The cells were then washed with PBS and were infected at a multiplicity of infection (MOI) of a 50% tissue culture infective dose of 0.01 (for control viruses) or 0.1 (for bat virus) for 1 h at 37°C. Culture medium (1.5 ml) containing the respective concentration of amantadine was added to each well. The plates were incubated at 37°C with 5% CO₂ for 36 h. Virus replication was assessed by measuring the hemagglutinin titers in the supernatant.

The resistance of the currently characterized virus to oseltamivir (Toronto Research Chemicals, Canada) and zanamivir (Sigma-Aldrich) was determined in MDCK cells by plaque reduction assay as described previously (19). Cultured MDCK cells in six-well plates were inoculated with the current isolated virus diluted in infection medium to give 30 to 100 plaques per well. The Egyptian viruses were tested for sensitivity to different antiviral concentrations. Cells were incubated for 1 h at 37°C and then overlaid with DMEM overlay medium containing 1% agarose, 4% BSA, 1% antibiotic-antimycotic mixture (Worthington Diagnostics, Freehold, NJ), 1 μg/ml TPCK-treated trypsin, and the respective concentration of antiviral drug. After 3 days of incubation at 37°C, the plaques were visualized by staining the fixed cells with 0.1% crystal violet. The percentage of viral inhibition relative to the level of inhibition of the untreated control viruses was calculated at each concentration.

Experimental infection of mice and chickens.

Three groups of 12 C57BL/6 mice and three groups of BALB/c mice (6 to 8 weeks old) were infected with 100 μl of 10⁶ 50% egg infective doses (EID₅₀; 1.2 × 10⁵ PFU) of the bat virus, 100 μl of 10⁶ EID₅₀ of A/chicken/Egypt/7100/2013(H9N2), or 100 μl of PBS intranasally and monitored daily for body weight loss. Student's t test was used for statistical comparison. At 3 and 7 days postinfection (dpi), three mice from each group were sacrificed and lungs were collected for virus detection and titration.

To determine the pathogenicity of the current new virus in chickens, two groups of 12 4-week-old SPF White Leghorn chickens were infected with 0.5 ml containing 10⁶ EID₅₀/ml of the bat virus or A/chicken/Egypt/7100/2013(H9N2). Another group was left as an uninfected control group. Viral dilutions were prepared in 1× PBS. Infection was done through natural routes (intranasal, intraocular, and intratracheal infection).

Blood, oral and cloacal swab samples, and organs (liver, lung, trachea, kidney, spleen, brain, and intestine) were collected from three chickens per group at 3 and 7 dpi. Swabs and homogenates of organs were subjected to virus detection through egg infection.

Data availability.

The nucleotide sequences obtained in this study are available from GenBank under accession numbers MH376902 for HA, MH376903 for M, MH376904 for NA, MH376905 for NP, MH376906 for NS, MH376907 for PA, MH376908 for PB1, and MH376909 for PB2.