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CORD-19:12d9952ba3cf8410eff072c9ec3b62447457122d JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-124 Sentence denotes Immunoglobulin heavy chain diversity in Pteropid bats: evidence for a diverse and highly specific antigen binding repertoire
T2 126-134 Sentence denotes Abstract
T3 135-350 Sentence denotes Bats are the natural host reservoir for range of emerging and re-emerging viruses, many of which cause significant morbidity and mortality in other mammals, yet appear to result in no clinical consequences for bats.
T4 351-594 Sentence denotes The ability of bats to coexist with a variety of viruses presents an interesting immunological problem that has not been examined in any detail but which could provide significant insights into the evolution of antiviral mechanisms in mammals.
T5 595-769 Sentence denotes Towards a better understanding of the bat immune system, we analysed the expressed heavy chain variable (VH) regions of antibodies from the black flying fox, Pteropus alecto.
T6 770-925 Sentence denotes The germline repertoire of the closely related Pteropid bat, Pteropus vampyrus, whose genome has been sequenced was also examined for comparative purposes.
T7 926-1098 Sentence denotes Representative VH genes were found in all three mammalian VH clans (I, II and III) in both the expressed P. alecto VH repertoire and the germline P. vampyrus VH repertoire.
T8 1099-1301 Sentence denotes Evidence for the use of multiple heavy chain diversity (DH) and joining (JH) segments for the generation of diverse VDJ rearrangements was also present in the expressed antibody repertoire of P. alecto.
T9 1302-1569 Sentence denotes The long period of co-evolutionary history of bats with viruses may have resulted in a variety of highly specific VH segments being hardwired into the genomes of bats and may have implications for their ability to successfully cope with a diversity of viral antigens.
T10 1571-1756 Sentence denotes Immunoglobulin genes are assembled by recombination of germline-encoded gene segments: variable (V), diversity (D) and joining (J) for heavy (H) chains and V and J for light (L) chains.
T11 1757-1945 Sentence denotes Variation in the amino acid residues at the N terminal ends that are encoded by the V regions of both H and L chains contributes to antibody diversity and establishes antibody specificity.
T12 1946-2119 Sentence denotes There are three mechanisms for generating antibody diversity that are used to varying degrees by different species: germline diversity, somatic mutation and gene conversion.
T13 2120-2356 Sentence denotes Germline diversity refers to the presence of a significant number of different germline-encoded segments that contribute to generating diversity in the primary antibody repertoire through the random combination of V, (D) and J segments.
T14 2357-2489 Sentence denotes Humans and mice each have a large number of diverse germline-encoded VH segments that generate a highly diverse antibody repertoire.
T15 2490-2696 Sentence denotes However, this seems to be more the exception rather than the rule, with most mammals having germline VH segments of high sequence similarity to each other and relying on post-V(D)J recombination mechanisms.
T16 2697-2958 Sentence denotes In these species, additional diversity may be contributed by more extensive addition of N and P nucleotides during the recombination process, the addition of single base pair mutations through somatic hypermutations or gene conversion mechanisms (Butler 1997) .
T17 2959-3133 Sentence denotes Vertebrate VH genes are grouped into families based on the criteria of ≥75% nucleotide identity, and these families are further grouped into clans (Brodeur and Riblet 1984) .
T18 3134-3269 Sentence denotes Mammalian VH genes can be classified into groups A, B and C (or clans I, II and III) based on their sequence similarity (Kirkham et al.
T19 3270-3277 Sentence denotes 1992) .
T20 3278-3397 Sentence denotes Species such as primates and rodents have multiple VH families with representatives of all three clans (van Dijk et al.
T21 3398-3420 Sentence denotes 1993; Mainville et al.
T22 3421-3428 Sentence denotes 1996) .
T23 3429-3569 Sentence denotes In contrast, most other mammals (e.g., rabbits, cattle, swine, sheep, platypus and opossum) and chickens have limited germline VH diversity.
T24 3570-3753 Sentence denotes In these cases, the germline VH within a species typically share ∼80% nucleotide identity and may be only a single VH family or a set of recently derived VH families (Johansson et al.
T25 3754-3785 Sentence denotes 2002; Butler 1997; Baker et al.
T26 3786-3793 Sentence denotes 2005) .
T27 3794-3850 Sentence denotes The numbers of functional VH genes vary between species.
T28 3851-3999 Sentence denotes In humans and mice, there are approximately 44 and 96 functional VH genes, respectively, in addition to a large number of VH pseudogenes (Das et al.
T29 4000-4007 Sentence denotes 2008) .
T30 4008-4177 Sentence denotes In contrast, rabbits have ≥100 VH genes present in the genome, but only one VH segment is used for generating 70-90% of the expressed antibody repertoire (Knight 1992) .
T31 4178-4300 Sentence denotes A more extreme example is the chicken which has only one functional VH gene and a number of VH pseudogenes (Reynaud et al.
T32 4301-4323 Sentence denotes 1989; McCormack et al.
T33 4324-4331 Sentence denotes 1991) .
T34 4332-4554 Sentence denotes Co-evolution with viruses and other pathogens plays an important role in shaping the diversity and specificity of the antibody repertoire and can act at both the somatic and genomic level (Ota and Nei 1994; Rajewsky et al.
T35 4555-4575 Sentence denotes 1987; Kirkham et al.
T36 4576-4594 Sentence denotes 1992; Roost et al.
T37 4595-4602 Sentence denotes 1995) .
T38 4603-4778 Sentence denotes At the somatic level, hypermutation results in the introduction of single base pair mutations that increase antibody specificity, thus shaping the secondary antibody response.
T39 4779-5059 Sentence denotes Positive selection on germline-encoded V segments is also believed to result in certain V segments being fixed in the germline to ensure efficient protection against common pathogens encountered over a long period of host pathogen co-evolutionary history (Langman and Cohn 1987) .
T40 5060-5230 Sentence denotes Bats have been identified as a natural reservoir for a variety of viruses with RNA viruses accounting for the overwhelming majority of emerging pathogens (Calisher et al.
T41 5231-5248 Sentence denotes 2006; Wong et al.
T42 5249-5256 Sentence denotes 2006) .
T43 5257-5375 Sentence denotes Although bats may be persistently infected with many viruses, they rarely display clinical symptoms (Williamson et al.
T44 5376-5419 Sentence denotes 1998 (Williamson et al. , 2000 Leroy et al.
T45 5420-5442 Sentence denotes 2005; Swanepoel et al.
T46 5443-5461 Sentence denotes 1996; Leroy et al.
T47 5462-5481 Sentence denotes 2009; Sulkin et al.
T48 5482-5489 Sentence denotes 1966) .
T49 5490-5661 Sentence denotes The only viruses that have been demonstrated to cause clinical symptoms of disease in bats are rabies virus and the closely related Australian bat lyssavirus (Field et al.
T50 5662-5681 Sentence denotes 1999; McKoll et al.
T51 5682-5689 Sentence denotes 2002) .
T52 5690-5827 Sentence denotes However, results of experimental infections are inconsistent, with only a small proportion of bats succumbing to infection (McKoll et al.
T53 5828-5835 Sentence denotes 2002) .
T54 5836-5904 Sentence denotes Viruses such as rabies have a long history of association with bats.
T55 5905-6157 Sentence denotes However, as humans and other animals continue to encroach on bat habitats, more viruses are being discovered, many of which, such as SARS coronavirus and Hendra and Nipah viruses, lead to lethal consequences in humans and other animals (Calisher et al.
T56 6158-6175 Sentence denotes 2006; Wong et al.
T57 6176-6183 Sentence denotes 2006) .
T58 6184-6318 Sentence denotes Whilst being the second most species-rich mammalian group after rodents, they are also the least studied of any group (Simmons 2005) .
T59 6319-6431 Sentence denotes How bats remain asymptomatic is not known, and few studies have examined the immune response of any bat species.
T60 6432-6601 Sentence denotes Evidence that antibody responses in bats are both qualitatively and quantitatively lower than other mammals has been reported by a number of investigators (Hatten et al.
T61 6602-6623 Sentence denotes 1968; Wellehan et al.
T62 6624-6665 Sentence denotes 2009; Chakraborty and Chakravarty 1984) .
T63 6666-6788 Sentence denotes In addition, experimental virus infections have demonstrated the simultaneous presence of virus and antibody in bat blood.
T64 6789-6989 Sentence denotes Sulkin et al. (1966) speculated that this could be due to the formation of a virus-antibody complex which dissociates readily or antibody which combines with virus but fails to neutralise infectivity.
T65 6990-7203 Sentence denotes However, the nature of the antibody repertoire in bats has not been investigated, and no information currently exists on other antiviral mechanisms that may be responsible for inhibiting viral replication in bats.
T66 7204-7481 Sentence denotes In this paper, we report the first molecular characterisation of the expressed antibody repertoire of a member of the Chiroptera family, the black flying fox, Pteropus alecto, with comparison to the germline VH repertoire of the closely related Pteropid bat, Pteropus vampyrus.
T67 7482-7721 Sentence denotes Our results demonstrate that similar to primates and rodents, Pteropid bats have VH genes distributed in all three mammalian VH clans, consistent with the maintenance of V genes with the capacity to recognise a range of antigenic epitopes.
T68 7722-7870 Sentence denotes The antigen-binding region of these genes also displays characteristics typically associated with weak antigen binding but high antigen specificity.
T69 7871-7960 Sentence denotes The P. alecto used in this study was a wild-caught individual from Queensland, Australia.
T70 7961-8045 Sentence denotes The spleen from this adult male was collected in RNAlater (Ambion, Austin, TX, USA).
T71 8046-8229 Sentence denotes Total RNA, with the concomitant removal of genomic DNA with DNaseI, was extracted using RNeasy mini kit following the manufacturer's recommended protocols (Qiagen, Valencia, CA, USA).
T72 8230-8489 Sentence denotes Tissue homogenisation and disruption was carried out using a modification of the method described by Bossart et al. (2008) with homogenisation performed for 2 × 20 s at 6,800 rpm with a Precellys 24 tissue homogenizer (Bertin Technologies, Carlsbad, CA, USA).
T73 8490-8589 Sentence denotes All experiments were approved by the Australian Animal Health Laboratories animal ethics committee.
T74 8590-8959 Sentence denotes Full-length coding sequences for IgM, IgG and IgA were obtained using 5′ and 3′ rapid amplification of cDNA ends (RACE) polymerase chain reactions (PCRs) using the GeneRacer Kit (Invitrogen, Carlsbad, CA, USA) with GoTaq polymerase (Promega, Madison, WI, USA) and the manufacturer's recommended PCR conditions on total spleen RNA extracted from an adult male P. alecto.
T75 8960-9108 Sentence denotes Primers were designed based on sequences obtained from the P. vampyrus whole genome sequence deposited in the Ensembl Genome Browser (Hubbard et al.
T76 9109-9116 Sentence denotes 2009 ).
T77 9117-9314 Sentence denotes 5′ RACE was performed as a single-step PCR using IgM, IgG and IgA constant regionspecific primers (5′-GCAGGAGGTTCTCACAGGAGACAA-3′, 5′-GACCGAGGGTCCTCCCAGGAGAT-3′ and 5′-GAGAAGTTCTCTCCGCGGTTCCAT-3′).
T78 9315-9510 Sentence denotes 3′ RACE was performed as a single step with IgM, IgG and IgA variable region-specific primers (5′-GTCCACCGGTAAACCCACCCTGTA-3′, 5′-GACAGGACTTCACGTGTACGGTGAT-3′ and 5′-GTCATGGAAGAGTGGGGACAGCTA-3′).
T79 9511-9681 Sentence denotes To obtain additional sequences for the 5′ end which contains the VH region, multiple clones were sequenced from RACE PCRs performed using 5′ IgM-and IgG-specific primers.
T80 9682-9970 Sentence denotes P. alecto germline JH segments were amplified using a primer specific for the recombination signal sequence (5′-GGGTTTTTGTGAGGGAGA-3′) of a JH segment identified in the P. vampyrus genome in combination with JH-specific primers (5′-TGAGGAGACGGTGACCACGG-3′ and 5′-TGAGGAGACGGTGACTGGCG-3′).
T81 9971-10101 Sentence denotes P. alecto genomic DNA was used as a target in PCR with an annealing temperature of 50°C using GoTaq Hotstart Polymerase (Promega).
T82 10102-10224 Sentence denotes PCR and RACE-PCR products were cloned into the pCR4-TOPO vector using the TOPO TA Cloning Kit for sequencing (Invitrogen).
T83 10225-10546 Sentence denotes Primers for the T3 and T7 promoters were employed for sequencing using BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Foster City, CA, USA) with non-isotopic dye terminators in 10-μl reactions, according to the manufacturer's instructions, and analysed on an Applied Biosystems 3130 XL Genetic Analyser.
T84 10547-10760 Sentence denotes Chromatograms were edited manually using the Sequencher 4.6 software (Gene Codes Corporation, Ann Arbor, MI, USA) and were compared with sequences in the GenBank database using the BLAST algorithm (Altschul et al.
T85 10761-10768 Sentence denotes 1990 ).
T86 10769-10841 Sentence denotes All sequences were aligned using the ClustalX programme (Thompson et al.
T87 10842-10849 Sentence denotes 1994) .
T88 10850-10989 Sentence denotes Nucleotide sequences were aligned and gapped manually using the Bioedit programme based on the protein alignment to retain codon positions.
T89 10990-11154 Sentence denotes Alignments were made using sequences corresponding to the framework regions (FR) 1-3 of the V domains, inclusive of complementary determining regions (CDR) 1 and 2.
T90 11155-11380 Sentence denotes Based on the nucleotide alignments, phylogenetic trees were constructed by the neighbour joining (NJ) method of Saitou and Nei (1987) , maximum parsimony (MP) and minimum evolution (ME) using the MEGA4 programme (Kumar et al.
T91 11381-11388 Sentence denotes 2004 ).
T92 11389-11541 Sentence denotes NJ and MP analyses using MEGA4 were performed using 1,000 bootstrap replicates and the Kimura two-parameter and min-mini heuristic search, respectively.
T93 11542-11610 Sentence denotes Previously published VH sequences from other species are as follows:
T94 11611-11707 Sentence denotes Human, Homo sapiens (Hosa), VH sequences were obtained from the VBASE database (Tomlinson et al.
T95 11708-11715 Sentence denotes 1996) .
T96 11716-11786 Sentence denotes Mouse, Mus musculus (Mumu), VH family representatives were as follows:
T97 11787-11943 Sentence denotes 7183, U04227; 3660, K01569; 3609 N, X55935; DNA4, M20829; J558, Z37145; J606, X03398; Q52, M27021; S107, J00538; SM7, M31285; VH11, Y00743; and X24, X00163.
T98 11944-11983 Sentence denotes Cow, Bos taurus (Bota) VH was AF015505.
T99 11984-12023 Sentence denotes Sheep, Ovis aries (Ovar) VH was Z49180.
T100 12024-12061 Sentence denotes Pig, Sus scrofa (Susc) VH was U15194.
T101 12062-12115 Sentence denotes Possum, Trichosurus vulpecula (Trvu) VH was AAL87470.
T102 12116-12163 Sentence denotes Opossum, Monodelphis domestica VH was AF012122.
T103 12164-12222 Sentence denotes Platypus, Ornithorhynchus anatinus (Oran) VH was AF381291.
T104 12223-12321 Sentence denotes Echidna, Tachyglossus aculeatus (Taac) VH was AAM61760, AAM60783, AY101439, AY101442 and AY101445.
T105 12322-12379 Sentence denotes Horned shark, Heterodontus fransciscii (Hefr) VH, X13449.
T106 12380-12628 Sentence denotes For comparative purposes, VH segments were identified in the whole genome sequence of the Malaysian flying fox P. vampyrus available in the Ensembl database (assembly pteVam1, 2.63× coverage, July 2008) using the BLAT, BLASTN and BLASTX algorithms.
T107 12629-12872 Sentence denotes The constant regions of IgG, IgA and IgM were identified in the whole genome sequence of P. vampyrus and used to design oligonucleotide primers to amplify full-length sequences from P. alecto spleen cDNA using a combination of RACE and RT-PCR.
T108 12873-13010 Sentence denotes Full-length IgG, IgM and IgA sequences containing the VH, DH, JH and CH regions homologous to sequences from other mammals were obtained.
T109 13011-13073 Sentence denotes These sequences have been submitted to Genbank (accession nos.
T110 13074-13093 Sentence denotes GQ427150-GQ427152).
T111 13094-13307 Sentence denotes To sample the expressed VH repertoire in P. alecto, additional 5′ RACE PCRs were performed using the Cγ and Cμ primers paired with the forward Generacer adaptor primer so as not to bias the VH sequences amplified.
T112 13308-13443 Sentence denotes An additional 26 clones containing full VH sequences were isolated and sequenced which contained 20 unique VH sequences (accession nos.
T113 13444-13463 Sentence denotes GQ427153-GQ427172).
T114 13464-13545 Sentence denotes Unique sequences were identified based on the presence of differences in FR1-FR3.
T115 13546-13709 Sentence denotes A total of 23 unique sequences including these 20 sequences along with the VH regions obtained from full-length IgG, IgM and IgA were included in further analyses.
T116 13710-13884 Sentence denotes An alignment of the deduced amino acid sequences of the 23 unique P. alecto VH cDNAs is shown in Fig. 1 , with sequences aligned based on sequence similarity in the V region.
T117 13885-14024 Sentence denotes Pairwise analysis of the 23 P. alecto VH sequences revealed that these sequences shared 50-96% nucleotide identity to each other (Fig. 2) .
T118 14025-14321 Sentence denotes The presence of five distinct families designated VH1 (clones 19g, 20g, 21g, 26g, 30g, 31g, 35g, 36g, 1m and 2m) ; VH2 (IgA, 23g, 25g, 32g, 34g and 18g); VH3 (13g, 12g, 28g and 33g); VH4 (IgG and 10g) and VH5 (IgM) was apparent based on sharing ≥75% nucleotide identity within a family (Fig. 2) .
T119 14322-14505 Sentence denotes The same analysis performed excluding CDRs to limit the impact of somatic mutations on family assignment did not change the assignment of sequences to particular families (not shown).
T120 14506-14668 Sentence denotes Conserved alanine and arginine residues are present in the CDR3 region of VH genes of most species and form the base of the antigen-binding site (Schelonka et al.
T121 14669-14676 Sentence denotes 2007 ).
T122 14677-14759 Sentence denotes These residues were not conserved in the P. alecto group 3 VH sequences (Fig. 1) .
T123 14760-14915 Sentence denotes It is possible that the lack of conservation of these residues may result in the formation of a different range of structures in this subset of antibodies.
T124 14916-15043 Sentence denotes As expected, most of the variation in the P. alecto VH sequences was found in the CDRs, particularly the CDR3 region (Fig. 1) .
T125 15044-15164 Sentence denotes Sequences encoding the heavy chain CDR3 consist of the DH segment, N and P nucleotides and the 5′ end of the JH segment.
T126 15165-15291 Sentence denotes The CDR3 region plays an important role in determining the fine specificity of the antibody binding site (Xu and Davis 2000) .
T127 15292-15405 Sentence denotes The P. alecto CDR3 sequences ranged from 18 to 54 bp in length, corresponding to 6-18 amino acid residues ( Figs.
T128 15406-15416 Sentence denotes 1 and 3) .
T129 15417-15545 Sentence denotes The diversity of the P. alecto CDR3 sequences is consistent with the use of multiple different DH segments during recombination.
T130 15546-15724 Sentence denotes Short stretches of similarity can be recognised between some CDR3 regions (20g and 30g; 36g and 33g) which may correspond to common germline DH segments, or closely related ones.
T131 15725-15854 Sentence denotes However, most of the DH regions appear quite different from each other, consistent with their origin from a diverse DH gene pool.
T132 15855-16073 Sentence denotes Also evident in these sequences is a high GC content which often reflects extensive N nucleotide addition which in turn may contribute to changes in the amino acid composition within the antigen-binding site (Fig. 3) .
T133 16074-16296 Sentence denotes The JH segment encodes the 3′ part of the CDR3 and the whole of the FR4 region, and in most species, there are multiple JH segments in the germline that can be randomly recombined during V(D)J recombination (Butler 1997) .
T134 16297-16392 Sentence denotes A nucleotide alignment of the CDR3 to FR4 regions of 23 unique VH segments is shown in Fig. 3 .
T135 16393-16595 Sentence denotes Based on distinct patterns shared by multiple sequences, we identified at least five JH segments among the 23 sequences analysed, consistent with the presence of multiple germline JH segments (Fig. 3) .
T136 16596-16721 Sentence denotes Included in the alignment are two germline JH segments which correspond to JHI and JHII of the P. alecto cDNAs (accession no.
T137 16722-16732 Sentence denotes GU354017).
T138 16733-16972 Sentence denotes Comparison of the cDNA sequences and germline segments provides evidence for nucleotide substitutions, some of which may be the result of allelic variation, but some of these changes are also likely to be a consequence of somatic mutation.
T139 16973-17226 Sentence denotes Additional germline JH segments were also identified which did not correspond to any of the cDNAs (not shown), thus providing additional evidence that P. alecto may have an even more diverse JH repertoire than is represented by the cDNAs sequenced here.
T140 17227-17451 Sentence denotes As the amino acid composition of the CDR3 region plays an important role in antigen specificity, the predicted residues in the CDR3 regions of the P. alecto VH genes were compared to those of mouse CDR3 regions (Kabat et al.
T141 17452-17459 Sentence denotes 1991) .
T142 17460-17653 Sentence denotes The per cent amino acid residue composition of the CDR3 regions of P. alecto and mice, excluding the three C-terminal positions which are highly conserved in most species, is shown in Table 1 .
T143 17654-17829 Sentence denotes The most abundant amino acid residues in the P. alecto CDR3 regions were alanine (A, 14.9%), glycine (G, 13.6%), arginine (R, 10.9%), tyrosine (Y, 10.3%) and serine (S, 7.5%).
T144 17830-18018 Sentence denotes Compared with the mouse sequences, A and R made up a larger proportion of the amino acid residues in the P. alecto CDR3 regions, whereas Y residues made up a smaller percentage (Table 1 ).
T145 18019-18164 Sentence denotes In all species examined, from shark to human, the germline composition of the DH segment is enriched for Y, G and S in one of the reading frames.
T146 18165-18330 Sentence denotes The amino terminus end of the JH segment also contributes to the CDR3 region, and Y is the most highly used amino acid in JH segments in other species (Ivanov et al.
T147 18331-18338 Sentence denotes 2002) .
T148 18339-18531 Sentence denotes Identification of several germline JH sequences of P. alecto revealed that most sequences contained few Y residues similar to that observed in the P. alecto cDNAs (Fig. 3 and data not shown) .
T149 18532-18685 Sentence denotes DH segments identified in the genome of the closely related Pteropid bat, P. vampyrus, also appeared to display a paucity of Y residues (data not shown).
T150 18686-18877 Sentence denotes This result is consistent with the lack of Y in the P. alecto sequences being the result of the amino acid composition of the DH and JH regions rather than a consequence of somatic mutations.
T151 18878-19050 Sentence denotes Although no genome sequence is available from P. alecto as yet, a low coverage whole genome sequence is publicly available from a closely related Pteropid bat, P. vampyrus.
T152 19051-19252 Sentence denotes VH sequences from P. alecto and other mammals were used to search the P. vampyrus whole genome sequence in the publicly available Ensembl database to examine the germline VH repertoire of this species.
T153 19253-19399 Sentence denotes Thirty-three scaffolds ranging from 1.22 to 54.06 Kb contained VH segments, resulting in the identification of 74 unique P. vampyrus VH sequences.
T154 19400-19466 Sentence denotes Of these, 11 contained stop codons and are presumably pseudogenes.
T155 19467-19633 Sentence denotes Fifty-one sequences had a leader peptide, open reading frame (ORF) and an identifiable recombination signal sequence (RSS) and are potentially functional VH segments.
T156 19634-19732 Sentence denotes Of the remaining 12 VHs with ORFs, no leader was identified in five and no RSS was found in seven.
T157 19733-19956 Sentence denotes Pairwise analysis was performed using 61 of the most complete P. vampyrus sequences with ORFs (51 potentially functional VHs, five with an RSS but no signal sequence and five with a signal sequence but no identifiable RSS).
T158 19957-20162 Sentence denotes The results of this analysis demonstrated that these sequences share 49-99% nucleotide identify and belong to five different VH families based on sharing ≥75% nucleotide identity within a family (Fig. 4) .
T159 20163-20308 Sentence denotes However, due to the fragmentary nature of the P. vampyrus assembly, it is likely that additional VH segments exist in the genome of this species.
T160 20309-20475 Sentence denotes Since P. alecto and P. vampyrus are closely related species of Pteropid bats, we might expect P. alecto to have similar numbers of germline VH segments in its genome.
T161 20476-20626 Sentence denotes Phylogenetic analysis was performed using a nucleotide alignment of the P. alecto VH sequences with VH sequences from several other mammalian species.
T162 20627-20793 Sentence denotes Included in the analysis were 11 mouse, six human, one pig, one sheep, one cow, one opossum, one possum, one platypus, five echidna and five P. vampyrus VH sequences.
T163 20794-20989 Sentence denotes Horned shark, H. fransciscii, was used as an outgroup to the mammalian VH because elasmobranch VH genes have been shown to be the most ancient of the vertebrate VH genes (Sitnikova and Su 1998) .
T164 20990-21129 Sentence denotes The tree shown in Fig. 5 was reconstructed using the NJ method; however, identical results were found when MP and ME were used (not shown).
T165 21130-21262 Sentence denotes The mammalian VH cluster into three groups (I, II and III in Fig. 5) , sometimes referred to as A, B and C, respectively (Nei et al.
T166 21263-21270 Sentence denotes 1997 ).
T167 21271-21355 Sentence denotes The P. alecto and P. vampyrus VH are clustered within clans I, II and III (Fig. 5) .
T168 21356-21592 Sentence denotes The clustering of sequences is consistent with the pairwise analysis described above and demonstrates that the P. alecto sequences that are members of the same VH family cluster in a species-specific manner within the phylogenetic tree.
T169 21593-21708 Sentence denotes Phylogenetic analysis was also performed using 61 P. vampyrus VH sequences with the most complete ORFs (not shown).
T170 21709-21857 Sentence denotes As with P. alecto, the P. vampyrus VH segments clustered in all three known mammalian VH clans, (16 in group I, 16 in group II and 29 in group III).
T171 21858-22021 Sentence denotes Of these 61 P. vampyrus sequences, only one representative of each of the five VH families identified in the pairwise analysis described above is shown in Fig. 5 .
T172 22022-22207 Sentence denotes The P. vampyrus VH sequences were interspersed with the P. alecto sequences, with the exception of members of family 3 which formed their own clade in each species within the VHII clan.
T173 22208-22410 Sentence denotes The clustering of P. alecto and P. vampyrus sequences together in the phylogenetic tree is consistent with the duplication of these genes prior to the divergence of Pteropid bats from a common ancestor.
T174 22411-22620 Sentence denotes Overall, our phylogenetic and pairwise analyses described above demonstrate that P. alecto and P. vampyrus have a diverse set of VH genes distributed in multiple families across each of the mammalian VH clans.
T175 22621-22758 Sentence denotes Bats are asymptomatic carriers of a variety of viruses but have been poorly studied in terms of their immune response to viral infection.
T176 22759-22994 Sentence denotes The few studies that have examined immune responses of bats have reported lower antibody responses compared to other mammals and evidence for inconsistent formation of neutralising antibody in response to viral infection (Sulkin et al.
T177 22995-23014 Sentence denotes 1966; Hatten et al.
T178 23015-23036 Sentence denotes 1968; Wellehan et al.
T179 23037-23078 Sentence denotes 2009; Chakraborty and Chakravarty 1984) .
T180 23079-23339 Sentence denotes Although it is possible that other immune mechanisms such as those involved in innate immunity are responsible for controlling viral replication prior to the development of the antibody response, little information is available on any aspect of bat immunology.
T181 23340-23560 Sentence denotes Defining the antibody repertoire of this unique group of mammals is an important step in understanding the nature of antiviral responses and the mechanisms involved in the asymptomatic nature of viral infections in bats.
T182 23561-23861 Sentence denotes Towards understanding the antibody responses of bats and the evolution of the antibody repertoire in Chiroptera, we analysed the level of expressed VH diversity in the black flying fox, P. alecto, and compared that to the level of germline VH diversity in the closely related flying fox, P. vampyrus.
T183 23862-24036 Sentence denotes Both P. alecto and P. vampyrus are old-world fruit bats and members of the Pteropodidae family which forms a monophyletic clade within the Megachiropteran order (Jones et al.
T184 24037-24044 Sentence denotes 2002) .
T185 24045-24260 Sentence denotes P. alecto has a geographic distribution that extends from Australia into Papua New Guinea and Indonesia, whilst P. vampyrus is found predominantly in Malaysia, Indonesia, Thailand and the Philippines (Nowak, 1994) .
T186 24261-24430 Sentence denotes Although their geographic range is not the same, they are host reservoirs of a variety of closely related viruses, the most important of which include the Henipaviruses:
T187 24431-24488 Sentence denotes Hendra in P. alecto and Nipah in P. vampyrus (Wong et al.
T188 24489-24496 Sentence denotes 2006 ).
T189 24497-24743 Sentence denotes VH genes evolve by the birth and death process with the duplication and deletion of VH segments, resulting in some species having retained a high level of germline diversity whereas others having lost it (Ota and Nei 1994; Butler 1997; Das et al.
T190 24744-24751 Sentence denotes 2008) .
T191 24752-24841 Sentence denotes The VH genes of a range of tetrapods are distributed in three main clans (I, II and III).
T192 24842-25033 Sentence denotes VH segments corresponding to all three VH clans are found in the amphibian Xenopus laevis, consistent with the appearance of VH groups I, II, and III early in tetrapod evolution (Haire et al.
T193 25034-25041 Sentence denotes 1990 ).
T194 25042-25216 Sentence denotes Species such as sheep and cattle have deleted much of the ancestral repertoire and only have representatives of clan II, whereas pigs only have clan III genes (Butler 1997) .
T195 25217-25390 Sentence denotes Among mammalian lineages, primates and rodents have the most diverse VH repertoires, with a large number of VH genes distributed in all three clans (Butler, 2006; Das et al.
T196 25391-25398 Sentence denotes 2008) .
T197 25399-25784 Sentence denotes A recent analysis of the genomes of cats and dogs has revealed that these species also have representative VH genes in all three mammalian VH clans, but the majority of their VH genes belong to clan III and they have only limited diversity in clans I and II (cats have three VHI genes and a single VHII gene and dogs have only a single member of both the VHI and VHII clans; Das et al.
T198 25785-25792 Sentence denotes 2008) .
T199 25793-25921 Sentence denotes The monotreme, the echidna, also has VH genes in all three mammalian clades, but the platypus has only VHIII genes (Baker et al.
T200 25922-25929 Sentence denotes 2005) .
T201 25930-26184 Sentence denotes The expressed VH repertoire of P. alecto and the germline repertoire of P. vampyrus provide evidence for a diverse VH repertoire within the Pteropid bats, with the expressed antibody diversity of P. alecto being derived from all three mammalian VH clans.
T202 26185-26396 Sentence denotes The two members of the Chiroptera family described here represent the only eutherian species other than primates and rodents that are known to have retained a high level of the ancestral VH diversity (Das et al.
T203 26397-26404 Sentence denotes 2008) .
T204 26405-26650 Sentence denotes As the H chain has been demonstrated to make the most important contribution to antigen binding in humans and mice, evidence of a diverse VH repertoire in bats may be consistent with this also being the case in bats (Wilson and Stanfield 1993) .
T205 26651-26798 Sentence denotes Although the fossil record for bat evolution is incomplete, bats are believed to have originated approximately 50 million years ago (Teeling et al.
T206 26799-26806 Sentence denotes 2005) .
T207 26807-26981 Sentence denotes The correspondingly ancient origin of many of the viruses maintained in bats is consistent with a long period of co-evolution of bats and viruses (reviewed in Calisher et al.
T208 26982-26989 Sentence denotes 2006) .
T209 26990-27240 Sentence denotes The presence of a large pool of V segments from which to recombine the expressed VH repertoire might be expected to provide bats with a large number of antigenic specificities that may be important in a species exposed to a variety of viral antigens.
T210 27241-27493 Sentence denotes The co-evolution of hosts and pathogens has the ability to shape antibody diversity, resulting in some V region genes being selectively retained in the germline due to their capacity to accommodate particular antigens (Ota and Nei 1994; Rajewsky et al.
T211 27494-27514 Sentence denotes 1987; Kirkham et al.
T212 27515-27533 Sentence denotes 1992; Roost et al.
T213 27534-27541 Sentence denotes 1995) .
T214 27542-27805 Sentence denotes It is possible that the high VH diversity seen in bats is a consequence of the co-evolution of bats with viruses, resulting in certain V regions from each of the three mammalian VH clans being hardwired into the genome to maintain important binding specificities.
T215 27806-27998 Sentence denotes In addition to a diverse repertoire of VH segments, the expressed VH genes of P. alecto also displayed evidence of being derived from a number of different germline-encoded DH and JH segments.
T216 27999-28152 Sentence denotes The DH segment is encoded within the CDR3 region which is responsible for much of the fine specificity of the antibody binding site (Xu and Davis 2000) .
T217 28153-28401 Sentence denotes Long CDR3 regions are found in species with only a single VH family such as platypus, cattle and chickens whose CDR3 regions are up to 19, 27 and 30 amino acid residues, respectively, and may compensate for limited germline diversity (Berens et al.
T218 28402-28452 Sentence denotes 1997; Mansikka and Toivanen 1991; Johansson et al.
T219 28453-28460 Sentence denotes 2002) .
T220 28461-28610 Sentence denotes The P. alecto CDR3 regions ranged from 6 to 18 amino acid residues in length and are therefore comparable to those of humans and mice (Stenvik et al.
T221 28611-28618 Sentence denotes 2000) .
T222 28619-28775 Sentence denotes The Pteropid bats therefore appear to fit the same pattern as humans and mice, with high germline diversity reducing the need to diversify the CDR3 regions.
T223 28776-28942 Sentence denotes The JH regions identified in the expressed VH repertoire also appeared to correspond to multiple germline segments which displayed some evidence for somatic mutation.
T224 28943-29062 Sentence denotes The number of functional JH segments in other species ranges from only one in chickens to six in humans (Reynaud et al.
T225 29063-29083 Sentence denotes 1989; Ravetch et al.
T226 29084-29091 Sentence denotes 1981) .
T227 29092-29199 Sentence denotes The five different JH regions that were identified in our cDNAs are therefore comparable to that of humans.
T228 29200-29325 Sentence denotes Overall, our data are consistent with P. alecto deriving its antibody repertoire from a large pool of VH, DH and JH segments.
T229 29326-29431 Sentence denotes A curious aspect of P. alecto VH sequences was evident in the amino acid composition of the CDR3 regions.
T230 29432-29679 Sentence denotes The amino acid composition of the CDR3 region plays an important role in the ability of antibodies to bind antigen, affecting both the specificity of the antigen-antibody interaction and the ability of antibodies to accommodate different antigens.
T231 29680-29832 Sentence denotes Antibodies from a variety of species ranging from amphibians to humans and mice have CDR3 regions that are rich in tyrosines and glycines (Berens et al.
T232 29833-29851 Sentence denotes 1997; Golub et al.
T233 29852-29872 Sentence denotes 1997; Reynaud et al.
T234 29873-29894 Sentence denotes 1989; Friedman et al.
T235 29895-29902 Sentence denotes 1994) .
T236 29903-30125 Sentence denotes The CDR3 regions of polyreactive antibodies are rich in aromatic residues such as tyrosine and tryptophan which interact with structurally diverse antigens and form a "sticky" antigen-binding site (Padlan 1994; Mian et al.
T237 30126-30133 Sentence denotes 1991) .
T238 30134-30302 Sentence denotes The CDR3 regions of the P. alecto VH repertoire contained fewer tyrosine residues compared with other mammals and a greater proportion of arginine and alanine residues.
T239 30303-30530 Sentence denotes Tyrosine residues appear to be directly involved in antigen binding as they can interact with relatively distant structures by hydrogen bonding through their aromatic ring or through polar atoms in their side chain (Mian et al.
T240 30531-30538 Sentence denotes 1991) .
T241 30539-30744 Sentence denotes The presence of tyrosines in the CDR3 also confers structural diversity, allowing for many forms of intermolecular and intramolecular bonding which in turn can lead to greater polyreactivity (Zemlin et al.
T242 30745-30752 Sentence denotes 2003) .
T243 30753-30919 Sentence denotes In contrast, arginine residues have been reported to be detrimental to antigen binding even in small doses and may even contribute to self-reactivity (Birtalan et al.
T244 30920-30938 Sentence denotes 2008; Radic et al.
T245 30939-30946 Sentence denotes 1993) .
T246 30947-31187 Sentence denotes The combination of fewer tyrosines and a higher number of arginines in the bat CDR3 regions compared to other species may have resulted in the evolution of antibodies with lower polyreactivity that form only a weak association with antigen.
T247 31188-31416 Sentence denotes Although such antibodies would be unable to bind with the same avidity as those with a "sticky" antigen-binding site, they may be less promiscuous and therefore have greater specificity for particular antigens once they do bind.
T248 31417-31610 Sentence denotes Observations that bats fail to produce neutralising antibody following infection with certain viruses may also be due to the weak association of antigen-antibody complexes formed (Sulkin et al.
T249 31611-31618 Sentence denotes 1966) .
T250 31619-31787 Sentence denotes However, whether this is really the case remains to be experimentally determined as do the other immune mechanisms responsible for inhibiting viral replication in bats.
T251 31788-31879 Sentence denotes In mice, distinct populations of B cells exist within different compartments of the spleen.
T252 31880-32077 Sentence denotes During development, B cells with CDR3 regions that are enriched in arginine and hydrophobic amino acids are sorted away from the follicles but remain abundant in the marginal zone (Schelonka et al.
T253 32078-32085 Sentence denotes 2007 ).
T254 32086-32299 Sentence denotes As unsorted spleen cells were used in the present study, it is possible that bats have a larger marginal zone population that may have biased the population of VH regions to those enriched in arginine and alanine.
T255 32300-32388 Sentence denotes Other factors that shape the antibody repertoire include age and prior antigen exposure.
T256 32389-32550 Sentence denotes The sequences obtained in the present study were from the spleen from a mature wild-caught male flying fox and therefore represent the adult antibody repertoire.
T257 32551-32755 Sentence denotes However, as the history of antigen exposure of this individual is unknown, it is possible that previous viral infections have contributed to the selection of antibodies that encode particular amino acids.
T258 32756-32891 Sentence denotes Clearly, more work remains to be performed to examine the nature of antibody development in bats and the influence of antigen exposure.
T259 32892-33105 Sentence denotes However, the identification of sequences with a similar bias in amino acid composition in the genome of the closely related Pteropid bat, P. vampyrus, is consistent with bats having an unusual antibody repertoire.
T260 33106-33212 Sentence denotes In summary, this study represents the first analysis of the antibody repertoire in any Chiroptera species.
T261 33213-33409 Sentence denotes Our results demonstrate that at least in Pteropid bats, there is evidence for a diverse antigen-binding repertoire with antibodies that may form weak but highly specific antigenantibody complexes.
T262 33410-33681 Sentence denotes The evolution of antibodies of this nature is likely due to the long coevolutionary history of bats with viruses, providing a mechanism to rapidly recognise common viruses without the need for antibodies to undergo extensive further hypermutation to increase specificity.
T263 33682-33921 Sentence denotes Further work will examine the nature of the antibody response to viral infection, the antibody repertoire of infected bats and determine whether post-V(D)J recombination mechanisms also play a role in generating antibody diversity in bats.
T264 33922-33984 Sentence denotes Alignment of deduced amino acid sequences of 23 P. alecto VHs.
T265 33985-34100 Sentence denotes The designation following the clone number refers to isolation by RACE-PCR using Cμ (m) or Cγ (g) specific primers.
T266 34101-34178 Sentence denotes IgM, IgG and IgA clones refer to VH regions obtained from full-length clones.
T267 34179-34247 Sentence denotes Sequences are aligned based on sequence similarity in the VH region.
T268 34248-34452 Sentence denotes Dashes indicate similarity and dots indicate gaps Range of per cent nucleotide identity between 23 P. alecto VH sequences based on an alignment of the region corresponding to FR1 to FR3 inclusive of CDRs.
T269 34453-34678 Sentence denotes Numbers in parentheses indicate number of clones isolated from each family Nucleotide and amino acid translation from CD3 to FR4 of 23 different P. alecto VH sequences and two germline JH segments (germline3-2 and germline1).
T270 34679-34744 Sentence denotes Roman numerals indicate the five different groups of JH segments.
T271 34745-34837 Sentence denotes Regions of homology within the CDR3 region of different clones are shown underlined in bold.
T272 34838-35021 Sentence denotes Dashes indicate similarity Range of per cent nucleotide identity between 61 P. vampyrus VH sequences based on an alignment of the region corresponding to FR1 to FR3 inclusive of CDRs.
T273 35022-35240 Sentence denotes Numbers in parentheses indicate number of sequences identified from each family Phylogenetic tree based on alignments of VH sequences from P. alecto, P. vampyrus and representatives of other mammals and one non-mammal.
T274 35241-35335 Sentence denotes All P. alecto sequences are shown in blue and P. vampyrus sequences are shown red in the tree.
T275 35336-35404 Sentence denotes The tree was constructed using the NJ method (Saitou and Nei 1987) .
T276 35405-35489 Sentence denotes The numbers on the branch nodes indicate bootstrap values based on 1,000 replicates.
T277 35490-35572 Sentence denotes Roman numerals on the right indicate the three VH clans (Tutter and Riblet 1989) .
T278 35573-35697 Sentence denotes Numbers within the three clans indicate the P. alecto VH family identified by pairwise analysis (Fig. 2) a From Kabat et al.
T279 35698-35702 Sentence denotes 1991