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Phylogenetic and Structural Diversity in the Feline Leukemia Virus Env Gene

Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1–7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.

Introduction
Feline leukemia virus (FeLV) is an exogenous retrovirus, belonging to the genus Gammaretrovirus. FeLV has been shown to induce many diseases in cats, such as thymic lymphoma, multicentric lymphoma, myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), aplastic anemia, and immunodeficiency [1], [2]. The outcome of natural FeLV infection is variable, and little is known about the mechanisms of pathogenesis. Variants of FeLV that induce specific types of disease have been described, and several characteristic genetic changes in the long terminal repeat (LTR), as well as the env gene in the viral genomes appear to be responsible for pathogenicity. For example, FeLV proviruses molecularly cloned from lymphomas typically contain two or three tandem direct repeats of enhancer elements in the LTR [3][4][5]. On the other hand, LTRs of FeLVs derived from non-neoplastic disease or weakly pathogenic strains contain only a single copy of the enhancer [4], [6][7][8][9], but may contain other repeated elements such as the upstream region of the enhancer (URE) in MDS [1] and AML [10], [11], and the 21-bp triplication in non-T-cell disease [12], [13].
In addition to characteristic LTR structures, env genes also appear to play a role in pathogenicity. For example, the env gene of an anemia-inducing FeLV variant (Sarma strain) contains mutations and recombination, and its pathogenicity results from the Env protein binding to and disrupting the cellular function of FLVCR1, which acts as a receptor for the strain [14][15][16]. Mutations of the env gene and unique receptor usage have also been identified in FeLV variants that cause immunosuppression in cats (feline acquired immunodeficiency syndrome) [7], [17][18], and several other studies have similarly linked disease outcome to determinants located in the env region [19], [20].
The primary translation product of the FeLV env gene is processed through proteolytic cleavage into two functional units: the surface protein (SU; gp70) and the transmembrane protein (TM; p15E). The entry of retroviruses into target cells is governed by the interaction of glycoproteins on the retroviral SU with specific cell surface receptors [21]. FeLV can be categorized into several FeLV subgroups based on their interference and host range properties: FeLV-A, FeLV-B, FeLV-C, FeLV-AC and FeLV-T [14], [16], [22][23][24]. In addition to these FeLV subgroups, our laboratory recently identified a novel FeLV subgroup (FeLV-D) which was generated by ERV-DC env transduction [25]. FeLV-A is the most common subtype, and other subtypes may have arisen from this variant. For instance, it has been shown that FeLV-B arose through recombination in the env region between FeLV-A and endogenous FeLV sequences (enFeLV) present in the feline genome [9], [26], and FeLV-C apparently also arose through deletion and mutation of the FeLV-A env gene [2], [27]. Since the initial discovery of FeLV in domestic cats in 1964 [28][29], the virus has also been isolated from wild cats such as the Florida panther [30] and the Iberian lynx [31]. Preventing FeLV infection in both domestic and wild cats is of considerable interest. In the present study, we investigated patterns of FeLV genetic diversity based on entire env gene sequences sampled from FeLV isolates throughout Japan, as well as env gene sequences of several additional FeLV isolates from Europe and the Americas. We identify genotypes encompassing the full range of FeLV structural diversity, which we consider to be reliable for evaluating the phylogenetic relationships of the virus. Overall, the findings of this study extend the understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats. Our results may also provide insights into disease alteration caused by mutations of the env gene.

Epidemiological Survey of the Prevalence of FeLV Infection
From March to October 2008, a total of 1770 EDTAanticoagulated blood samples were collected from cats admitted at 47 private veterinary hospitals, one located in each prefecture of Japan. The samples were voluntarily submitted from veterinarians. For each cat, the age, sex, and primary complaint were recorded. The health profile of these cats has been partly described previously [32]. Blood samples were stored between 220 and 230uC prior to DNA extraction, and each of the 1770 samples was tested for serological evidence of FeLV infection by screening for the FeLV Gag antigen using a commercially-available test kit (SNAP FeLV/FIV combo kit; IDEXX Laboratories Inc., USA). A summary of the FeLV status, age, gender and other basic background data for the sample of cats is provided in Table 1. Cats that had outdoor access at least once a week were included in the study.

Detection and Amplification of FeLV Proviral DNA
Genomic DNA was isolated from blood samples testing positive for the FeLV antigen, using the QIAamp DNA Blood kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions. PCR primers for a full-length FeLV env amplification were designed from the conserved regions of pol, env, and LTR sequences of FeLV strains. PCR primers used were as follows: PCR was performed using approximately 100 ng genomic DNA in 50 mL reactions, with PrimeSTAR HS DNA Polymerase enzyme (Takara, Japan). PCR cycling conditions were as follows: 98uC for 10 s, 62uC for 5 s, 72uC for 1.5 min, for 30 cycles, followed by 5 min at 72uC. PCR products were directly cloned into the pCR4Blunt-TOPO vector (Invitrogen) or the pUC118 vector (Mighty cloning kit, Takara), and these recombinants were also used as a plasmid library to isolate full-length FeLV-B env genes and env deletion mutants. For detection of FeLV-B env, PCR was performed as described above, using primers PRB1 and Fe-3R. This primer pair is specific for FeLV-B, and a full-length FeLV-B env was isolated by screening the env plasmid library with colony PCR methods using these two primers. Based on the entire env gene, non-recombinant virus, recombinant virus including FeLV-B (identified by PCR) and non-FeLV-B (identified by recombination analysis), and deletion type and/or insertion type virus were isolated from the plasmid libraries using primer pairs Fe-8S/4S and Fe-3R. Alternatively, the entire env gene was amplified with primers Fe-9S and Fe-7R (specific for pol and exogenous LTR U3 regions), and these fragments were cloned into the pCR4Blunt-TOPO vector (Invitrogen). PCR was performed on a TaKaRa PCR Thermal Cycler Dice (Takara, Japan), and plasmids were sequenced with an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, USA).

Recombination Analysis
The Simplot software (version 3.5.1) was initially used to perform similarity and boot scanning analyses of a query sequence against a set of other sequences [34], [35]. This method measures the similarity/dissimilarity of the query sequence to a set of reference sequences.

Phylogenetic Analysis
The env sequences were aligned using Muscle [36] and these initial alignments were manually edited by eye in SEAVIEW [37]. FeLV env sequences from the NCBI database (see Table S1) were also included in the alignments. Subsequent phylogenetic analyses were based on virtually complete sequences of the SU and TM regions within the env gene (position 6080-7885 of FeLV-A 61E). Recombinant env genes, including FeLV-B identified by recombination analyses, were usually excluded from the data set for the purposes of phylogenetic reconstruction. However, partial sequences from some recombinants were included in the analyses after excluding their recombinant regions to determine FeLV genotype from cats (ST19, IT10, WY24, SA15 and IT38), from which non-recombinants were not identified. Partial sequences from representatives of the FeLV-C and FeLV-B subgroups were also used for the analyses. Phylogenetic trees were estimated using both the maximum likelihood method (ML) [38] and the neighbor joining approach (NJ) [39], and sequences from endogenous FeLVs were used as outgroups to root the trees. ML reconstructions were performed in PhyML [40], applying the general timereversible (GTR) model of nucleotide substitution. Nodal support was assessed via a likelihood-ratio test (LRT) [41], [42], as implemented in PhyML. NJ trees were constructed using CLUSTALW [43], [44], and distances were calculated using the Kimura two-parameter (K2P) method [45]. To assess the support for nodes in the NJ trees, a bootstrap analysis was performed using 1000 pseudoreplicates. Phylogenetic trees were visualized using MEGA5 [46], [47].

Analysis of Branch-specific Selection Forces
For these phylogenetic analyses, we utilized env gene sequences that meet the following criteria: a) have no recombination with enFeLV; b) are longer than 1800-bp; c) do not contain any frame shift mutations or undetermined nucleotides. We then translated these sequences and computed a multiple alignment at the amino acid level using the L-INS-i module of the MAFFT program [52]. Applying this amino acid alignment, we generated a corresponding nucleotide sequence alignment using TranslatorX [53]. Based on the multiple alignment of nucleotide sequences, we performed phylogenetic tree reconstruction using ML methods as implemented in RAxML v. 7.2.6 [54], applying the GTR model with a discrete gamma distribution to account for heterogeneity in evolutionary rates among sites (+G), and including an estimation of the proportion of invariant sites (+I). The robustness of the phylogenetic tree was evaluated by rapid bootstrapping [55] with 1000 pseudoreplicate datasets. To detect branch-specific positive selections, we used ML methods implemented in the codeml program of the PAML v. 4.5 package [56] to calculate the ratio of substitution rates at non-synonymous and synonymous sites (dN/ dS) for the each branch of the phylogenetic tree. We applied the F364 codon frequency model in which the average nucleotide frequencies are calculated separately at the three codon positions [57].

Nucleotide Sequence Accession Numbers
The nucleotide sequences reported in this paper are available in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the following accession numbers: AB635483-AB635582, AB635586-AB635685, AB635687-AB635763, AB635764-AB635863 and AB672612.

Prevalence of FeLV in Japan
A clinical and molecular-based epidemiological survey of FeLV infection covering the whole of Japan was performed in this study. FeLV Gag antigen-positive reactions were detected in 216 (12.2%) of the 1770 cats from which blood samples were collected (Table 1), and infected cats were identified in 45 of the 47 private veterinary hospitals visited. As shown in Fig. 1 and Table S2, The incidence of FeLV was higher in southern areas of Japan, such as parts of the Kyūshū area (Fukuoka, Saga, Nagasaki, Kumamoto, Miyazaki and Kagoshima prefectures; .20% positive) and parts of the Shikoku area (Ehime, Tokushima, and Kōchi prefectures; .15% positive). The seroprevalence of FeLV infection in Iwate, Yamagata, Chiba, Toyama, Wakayama, and Shimane prefectures was also high (.15% positive) in comparison to the average FeLV infection rates throughout Japan (12.2%). Although all owned cats we tested had outdoor access at least once a week, there was no clear correlation between FeLV infection and the number of days of outdoor access (Table 1). Interestingly, of the 286 cats vaccinated against FeLV, 18 tested positive for the virus (6.3%) (Table 1). Overall, these results indicate that FeLV infection is observed widely in Japan, and the control and prevention of this infectious disease should thus be a focal point for Japanese veterinary medicine.

Detection of Proviral env Fragments by PCR
To evaluate the divergence of the FeLV env gene across Japan, we established a PCR method to specifically detect exogenous FeLV env genes ( Fig. 2A, B). PCR amplification using primer pairs Fe-8S/Fe-4S and Fe-3R encompassed the entire FeLV env gene, and resulted in a fragment of predicted size (approximately 1.9 kb) when applied to AH927 cells infected with FeLV-A Glasgow-1 (GA5), FeLV-B Gardner-Arnstein (GB) or FeLV-C Sarma (SC) (Fig. 2B), and when applied to FT-1 cell lines [5] derived from the thymic lymphoma of a FeLV-positive cat, and samples of FeLV antigen-positive blood (SN5, MZ29). However, the 1.9 kb fragment was not detected in genomic DNA derived from uninfected AH927 cells, or in FeLV antigen-negative feline blood (FS23). This indicates that the primers did not amplify detectable levels of enFeLV, and that these two primer pairs are specific for env sequences of known FeLV-A, -B, and -C types (Fig. 2B). The sensitivity of this PCR method for detection of the env gene was investigated by application to the FeLV-A clone33 plasmid, and the limit of detection was determined to be 10 fg per reaction in this system (data not shown).
FeLV env PCR fragments were detected in 167 of the 216 samples of genomic DNA from FeLV antigen-positive cats. We were unable to amplify FeLV env fragments in the remaining seropositive samples, and in these samples we also failed to amplify LTR fragments despite attempts with different primer combina-tions, increased DNA concentration, lower annealing temperature and different DNA polymerase enzymes. These negative results may reflect divergent proviral sequences in primer sites for viral detection, low viral load, or other factors.
In addition to the fragments discussed above, several smaller fragments were amplified by the PCR primer pairs Fe-8S/Fe-4S and Fe-3R. For example, several smaller bands in addition to the expected 1.9-kb band were detected in FeLV-positive blood (MZ29) (Fig. 2B), and such bands were seen in 18 of the 167 PCRpositive samples. Eight of these 18 samples were from cats with neoplastic disease, including five lymphomas and three suspected cases of lymphoma. These fragments were cloned and their identities confirmed by sequencing.
A FeLV-B-specific detection system was established by PCR amplification using the enFeLV-specific PRB1 primer paired with an exogenous FeLV-specific antisense primer, Fe3R. These PCR reactions resulted in fragments with the predicted size of approximately 1.4 kb when applied to AH927 cells infected with FeLV-B, and FeLV antigen-positive blood (SN22), but not when applied to FeLV-A Glasgow-1 (GA5), FeLV-C Sarma (SC), FeLV antigen-positive (SN5) or negative blood (FS23) ( Fig. 2A, B). Sequencing was used to confirm the identity of PCR fragments that were amplified with FeLV-B-specific primers, indicating that the primers did not amplify detectable levels of enFeLV, and that these primer pairs detected at least env sequences from the known FeLV-B types. FeLV-B env was present in 74 of the 167 (44.3%) samples of genomic DNA from FeLV antigen-positive cats in which the env gene had been detected via PCR. Our results regarding the incidence of FeLV-B infection were similar to results previously reported [2], [58], [59]. Our PCR method did not Figure 1. Incidence of FeLV in blood samples collected from private veterinary hospitals located in each prefecture of Japan. The incidence of samples testing positive for the FeLV antigen was divided into six color-coded groups in increments of 5%. A two-letter code was assigned to each prefecture as described in Table S2.

Phylogenetic Analysis of FeLV env Gene Sequences
We isolated 261 clones of non-recombinant full-length env genes having an approximately 1.9 kb env gene. From the cases positive for FeLV-B and having an approximately 1.9 kb env gene, we isolated 76 FeLV-B specific clones, which were FeLV-B-positive, as determined by detecting a 1.4-kb fragment of partial FeLV-B env gene by our PCR method. In addition, from 18 cats we isolated 39 clones having several deletions of variable sizes. All of these clones were sequenced and analyzed. For the purposes of phylogenetic analysis, recombinant env genes such as FeLV-B, and env genes with large deletions or insertions, were identified by recombination analyses and excluded from the alignment (Fig. 2C). However, sequences from cats ST19, IT10, WY24, SA15 and IT38, (FeLV-C Sarma strain and FeLV-B GA strain), which are recombinant env genes, were included in the multiple sequence alignment after removal of their recombinant regions (Fig. 2D). The results from the phylogenetic analysis of the alignment using the ML method are shown in Fig. 3. ML and NJ methods both retrieved similar phylogenetic topologies (data not shown), in which all isolates (with the exception of GM35) grouped into three major, strongly supported clades: Genotype I (GI), Genotype II (GII), and Genotype III (GIII). Bootstrapping was employed to assess the support for these major groups and their component subclades, and we generally considered clades with bootstrap support values of .90% to be robust. Within Genotype I, seven clades were recognized as separate strains (see Fig. 3): GI/Clade1 (41 sequences), GI/Clade2 (37 sequences), GI/Clade3 (51 sequences), GI/Clade4 (10 sequences), GI/Clade5 (11 sequences), GI/Clade6 (3 sequences), and GI/Clade7 (4 sequences). Of these, all but one had bootstrap values ,90%; Clade 4 was supported by a bootstrap value of only 76%, and this clade may thus be reclassified based on future analyses. FeLV-A clone33 [10] and pJ7E2 [5] previously isolated in Japan (obtained from the NCBI database) clearly belong within Genotype I. Several clones with unusually long branches were identified in the various clades (e.g., MZ17, IK10, NN6-1, SA15-2, SI31-3). These viruses appear to possess different evolutionary dynamics from the other samples. In addition to having long branches, IT12-3 and NG19-1 did not fit into any of the clades in Genotype I. Sequence GF37-1, though not possessing a long branch, was somewhat divergent from the Genotype I clades and also did not fit into any of these groupings.
Genotype II consisted of samples from only seven cats, and future large-scale analyses are likely to provide additional resolution of relationships in this group. Genotypes I and II were derived exclusively from Japan and were newly characterized in this study, while all FeLV strains from Europe, South America and the USA (obtained from the NCBI database) were classified as Group III. Genotype III corresponds to known FeLV strains such as FeLV-A clone 945 [60], FeLV-A 61E [6], FeLV-T 61C [7], FeLV-A Glasgow-1 [9], FeLV-A Richard (pFRA) [61], FeLV-AC FY981 [62], FeLVs derived from Iberian lynx [31], Brazilian FeLVs [58], and the exogenous FeLV components of the FeLV-C Sarma and FeLV-B GA strains. Most Genotype III samples were included in two strongly supported groups, GIII/Clade1 and GIII/Clade2. Sequences from a single cat (ON33) collected in Okinawa (ON) prefecture, Japan were classified as GIII/Clade2. Interestingly, GM35 sequences from Gunma (GM) prefecture did not fall within any of the major genotypes. In this analysis, there were no GI or GII viruses detected outside of Japan, suggesting that our genotypic classification may be in some way dependent on geographical distribution.
We did not find any cases of superinfection with multiple FeLV groups in any cats; all clones obtained from a given individual belonged to the same clade. Furthermore, mutant env containing insertions or deletions, isolated from individual cats, had a high level of homology with full-length env derived from the same individual, suggesting that such mutants possibly occur de novo in these individuals. Our results suggest that FeLV has perhaps undergone extensive evolutionary diversification within Japan, and the genetic distinctness of Japanese FeLV strains may indicate a long history of geographical isolation in the country.

Receptor Usage of Representative FeLV env Genes from Japan
Next, to delineate FeLV subgroups on the basis of receptor usage, we conducted a viral interference assay. Env genes from each genotype and clade were inserted into the mammalian expression vector, which was introduced into GPLac cells [25] to produce FeLV env pseudotype viruses carrying the LacZ marker gene. HEK293T cells persistently infected with FeLV-A clone33, FeLV-B GA and FeLV-C Sarma were superinfected with each pseudotype virus. As shown in Table 2, the superinfection of 12 clones (two from GI/1, three from GI/2, one from GI/4, two from GI/5, and one each from GI/6, GI/7, GI/unclassified and GII) was completely interfered by FeLV-A clone33, but not by FeLV-B and FeLV-C receptor subgroups. This indicates that these Japanese isolates belong in the FeLV-A subgroup and possess the same receptor usage as FeLVs in other countries despite their different genotypic classification. Furthermore, pseudotype viruses from NS33-4 (GI/3) and KC18-6 (GI/2) were unable to infect uninfected HEK293T cells, HEK293T cells persistently infected with FeLV-A clone33 (HEK293T/FeLV-A clone33) or cells infected with FeLV-C Sarma (HEK293T/FeLV-C). However, Figure 2. Detection of FeLV env genes by PCR, and strategies for analysis of these genes. (A) Strategy used for generating PCR products. Schematics of coding sequences for the FeLV env gene are shown. FeLV proviral env sequences were amplified by PCR with the primer pairs Fe-8S/Fe-4S and Fe-3R, PRB-1 and Fe-3R, Fe-9S and Fe-7R. The lengths of the expected products from amplifications using each primer pair are shown. The abbreviations s.p., SU and TM represent, respectively, signal peptide, surface glycoprotein, and transmembrane subunit. (B) The DNA templates for PCR amplifications were as follows: neg. (genomic DNA isolated from FeLV-negative AH927 cells), GA5 (DNA from FeLV-A Glasgow-1-infected AH927 cells), GB (DNA from Gardner-Arnstein FeLV-B-infected AH927 cells), SC (DNA from FeLV-C Sarma-infected AH927 cells), DNA from FT-1 cell line, DNA from sample SN5, DNA from sample MZ29, DNA from a FeLV-positive cat (SN22), and DNA from a FeLV-negative cat (FS23). c-myc was amplified as a positive control [25]. PCR products were electrophoresed and stained with ethidium bromide. Asterisk indicates atypical bands of env gene. (C) Each detected PCR fragment was cloned into a cloning plasmid, and full-length env gene libraries were constructed. Several unique env genes were isolated from these full-length env gene libraries using information on fragment size, or by screening using FeLV-B specific PCR. In addition to nonrecombinant env genes, FeLV-B-type and other recombinants were isolated and analyzed. Furthermore, env genes smaller than full length were also analyzed. N indicates negative and P positive for FeLV-B detection or recombination detection. (D) For the most part, only non-recombinant sequences were used for phylogenetic analysis, but where recombinant sequences were included, their endogenous-derived regions were removed from the alignment. However, partial sequences from some recombinants as well as representatives of the FeLV-C and FeLV-B subgroups were included in the analyses after excluding their recombinant regions, to determine the FeLV genotypes from cats from which non-recombinants were not identified. doi:10.1371/journal.pone.0061009.g002 these pseudotype viruses could infected HEK293T cells infected with FeLV-B GA (HEK293T/FeLV-B) ( Table 2). It has previously been reported that the FeLV-T subgroup can infect cells infected with FeLV-B [17]. In order to determine whether NS33-4 and KC18-6 viruses belong to the FeLV-T subgroup, the supernatant of 3201 cells as a source of a co-factor, FeLIX, for FeLV-T was used in an additional viral infection assay. As shown in Table 3, both KC18-6 and NS33-4 were able to infect AH927 cells and HEK293T cells in the presence of the conditioned medium, but not in its absence. These FeLV clones had mutations within their SPHQ motifs which are related to the unique receptor usage of FeLV-T [17], suggesting that both KC18-6 and NS33-4 belong to the FeLV-T subgroup. Taken together, our results suggest that most of the FeLV isolates distributed in Japan may show the same receptor usage as FeLVs in other countries despite their different genotypes.

Geographic Distribution of Each FeLV Clade
We plotted Japanese viral isolates on a map to investigate the relationship between geographical distribution and reconstructed phylogenetic patterns. As shown in Fig. 4 and Table S3, GI/ Clade1 and GI/Clade2 are broadly distributed in Japan, while GI/Clade1 is more concentrated in the Tōhoku and Kantō regions, and GI/Clade2 is more concentrated in the Kinki and Chūgoku regions. GI/Clade3 tends to be distributed in the Kyūshū region. GI/Clade4 samples are found predominantly in Kōchi (KC) prefecture (Shikoku region) and GI/Clade5 occur in the prefectures of Toyama (TY)(Chūbu region) and Ehime (EH) (Shikoku region) as they appear in Fig. 4 and Table S3. Most phylogenetic groupings are thus broadly consistent with geographic distribution, indicating that the major genotypes and clades may have arisen in allopatry within the areas of their current occupancy, or that FeLVs were introduced several times into Japan via distinct transmission routes. Interestingly, GI/Clade1 representatives in the Wakayama (WY) prefecture (Kinki region) and the Kyūshū region are substantially isolated from each other geographically despite their close phylogenetic relationship. This could plausibly be the result of an artificial transmission via anthropogenic transportation of infected cats. We also analyzed the fixation and purifying selection of each genotype and clade by calculating the branch-specific ratios of substitutions at nonsynonymous and synonymous sites (dN/dS) (see Table S4 for a list of the sequences used). The dN/dS ratio for the root branch of each genotype and clade is shown on a schematic representation of the best phylogenetic tree from ML analysis of the complete nucleotide alignment (see Fig. S1 and Fig.S2). In most cases dN/ dS values were less than 1, providing no evidence of positive selection. However the clade containing IT12-3 and NG19-1 had a dN/dS ratio of 1.0322, suggesting that selection may be in operation in this lineage.

Recombination Analysis
FeLV-B env sequences contain enFeLV env sequences as a result of recombination. To determine structural diversity of FeLV-B, full-length FeLV-B-like env sequences were isolated from plasmid libraries constructed using Fe-8S and Fe-3R primers, which amplify exogenous FeLV env genes (Fig. 2C). FeLV-B and other recombinant sequences isolated from PCR were subjected to similarity plot analyses following boot scanning analyses to identify recombination events between exogenous FeLV and enFeLV sequences (Fig. 2C, D). The recombinant env sequences were compared with non-recombinant exogenous FeLV env sequences derived from an identical sample or from the same clade, as well as with enFeLV-AGTT (endogenous FeLV), using FeLV-A clone33 or FeLV-A 61E as an outgroup. Similarity plot analysis (Fig. 5A) showed that all of FeLV-B-like env sequences arose from the recombination of exogenous FeLV env sequences and the endogenous FeLV-AGTT. According to the boot scanning analyses, recombination apparently produced the FeLV-B Gardner-Arnstein (GA) strain by uniting a section of 59 endogenous FeLV env sequence and a section of 39 exogenous FeLV env sequence. Furthermore, the FeLV-C Sarma strain was observed to be a product of recombination events in two regions of the env gene. Fig. 5B shows representations of the recombination patterns that were observed in the present study. Many cases of recombination shared similarities with the recombination pattern in the FeLV-B GA strain (Fig. 5B). However, some unique recombination patterns were also observed. For example, clones FO36-5B, KG20-5B and KM16-3B contain both the VRA and VRB motifs from endogenous FeLV env sequences. Clones KG20-5B and KM16-3B possess endogenous FeLV sequences within the Figure 3. The best maximum-likelihood (ML) tree from phylogenetic analysis of near-full-length env nucleotide sequences generated in this study and obtained from the NCBI database. FeLV sequence information obtained from the NCBI database is listed in Table  S1. doi:10.1371/journal.pone.0061009.g003  5B and data not shown). Some env sequences (e.g. MZ40-5B) were almost entirely derived from endogenous FeLV env, and in these cases the boot scanning analysis did not detect recombination breakpoints between exogenous and endogenous FeLV sequences. To further confirm recombination in our virus samples, we isolated the 4B-MZ40 clone that possesses a portion of 39 pol, a full-length endogenous env gene, and an exogenous LTR U3 section, by PCR amplification with specific primers, Fe-9S and Fe-7R ( Fig. 2A), indicating with certainty that the recombinant virus was derived from exogenous FeLV (Fig. 5A, 5B). Interestingly, VRA recombination was often coupled with VRB recombination, as shown in Fig. 5B. When 59 and 39 recombination breakpoints were marked on the corresponding positions of the FeLV 61E env sequence, some recombination hot spots were observed and breakpoints tended towards regions of high homology between endogenous and exogenous FeLV sequences    (Fig. 5C). However, recombination junctions varied substantially between different recombinant clones, indicating that multiple recombination events have occurred, producing appreciable structural diversity of the FeLV env gene. Furthermore, many recombinant viruses have apparently arisen de novo in individual cats. In order to determine whether recombinant viruses isolated in Japan belong to the FeLV-B receptor-usage phenotype, we conducted a viral interference assay using pseudotype viruses. Four of these (MZ40-5B, FO36-5B, IT38-5RC and WY24-1RC) were able to infect AH927 cells, and we tested whether they could also infect AH927/FeLV-A cells, AH927/FeLV-B cells and AH927/ FeLV-C cells. As shown in Table 4, MZ40-5B and FO36-5B isolates could not infect AH927/FeLV-B cells, but were able to infect AH927/FeLV-A and AH927/FeLV-C cells, indicating that these two viruses belong to the FeLV-B receptor-usage subgroup.
On the other hand, IT38-5RC and WY24-1RC isolates (which contain enFeLV sequences in the TM region and/or the Cdomain) were unable to infect AH927/FeLV-A cells, but could infect AH927/FeLV-B and AH927/FeLV-C cells. These isolates must thus belong to the FeLV-A subgroup despite the fact that they are clearly recombinant in nature.

Analysis of Deletion Mutants of the env Gene
In samples from 18 cats, several smaller fragments were amplified by primer pairs Fe-8S/Fe-4S and Fe-3R, and were cloned and sequenced. Deletion regions within these env gene sequences were determined by comparison with full-length env sequences isolated from the same individual cats. The 59 and 39 positions of deleted regions were shown on the FeLV-A 61E env sequence (Fig. 6A), and several deletion patterns were evident from these plots. Multiple mutant sequences were isolated from one particular cat (MZ29), each with a different region deleted (Fig. 6B), but these sequences were all very similar to the nonmutated MZ29 env gene, suggesting that the various deletions could have occurred de novo in this particular cat. Furthermore, in the case of NR9 (see Fig. S3A), one deletion mutant of the env gene was isolated that had an inversion of 95-bp in addition to its deletion, and another that had a 95-bp inversion as well as an insertion of 28-bp from the cat genome sequence (see NCBI accession numbers ACBE0152516 and AANG01802278).
In addition to inversions and insertions, deletion mutants may also contain enFeLV sequences acquired by recombination, as in the case of ST17 (Fig. S3B). Overall, these results indicate that deletion events within env genes may often occur de novo, and such deletions contribute to the structural diversity of the FeLV env gene.

Analysis of Structural Diversity in Small Regions of the env Gene
All known full-length env genes observed in other countries belong to the GIII group and are 1929 bp in length, whilst FeLV env genes isolated from Japanese cats are variable in size. The env genes from GI/Clade1 are generally 1938 bp in length and env genes from GI/Clade2 and GI/Clade3 are typically 1932 bp in length. We searched for defining sequence characteristics for the various genotype and clade groupings, and found a 3-bp insertion (AGT/AAT) (Fig. 7A, box a) in the FeLV env gene of most Genotype I sequences, between positions 807 and 808 of the FeLV-A 61E sequence (Genotype III). This is absent from Genotypes II and III, and from the GM35 FeLV env sequence (genotype unclassified), so it seems logical to assume that the insertion must have originated in the common ancestor of Genotype I. Some clones from GI/4 have a 12-bp insertion (AATACAAGCAGT) between positions 798 and 799 of the  FeLV-A 61E sequence (Fig. 7A, box b). In GI/5, a 6-bp insertion (CCCCAC) is present in isolates from TY24 and TY10, between positions 827 and 828 of the FeLV-A 61E sequence (Fig. 7A, box c), and a 6-bp insertion (ACTACT) is present in some GI/1 isolates between positions 528 and 529 of the FeLV-A 61E sequence (Fig. 7B, box d). Some GI/5 isolates include a 6-bp insertion (CAGGGC) between positions 534 and 535 of the FeLV-A 61E sequence (Fig. 7B, box e), whilst others have a 3-bp deletion (AAT) encompassing positions 535-537 of the FeLV-A 61E sequence (Fig. 7B, box f). In Genotype II, FeLVs from cats AT34 and AT37 have a 3-bp insertion (CTT) between positions 214 and 215 of the FeLV-A 61E sequence (Fig. 7C, box g). The location on the phylogenetic map (indicated as a and d in Fig. 7D, b,c,e and f in Fig. 7E, and g in Fig. 7F) of these small structural changes in the env gene of exogenous FeLVs indicates that these genetic traits were established in viruses and transmitted to cats via viral infection.

Discussion and Conclusion
FeLV infection in Japan is frequently observed amongst cats that are free to venture outside. It has been shown that FeLV transmission is related to close contact between cats, such as mutual grooming or sharing food or water containers. The incidence of FeLV infection is clearly dependent in part on the region, indicating that infection may be associated with specific habits or lifestyles of either (or both) the animals or the people.
The FeLV genome was detected in the PBMC of 77.7% of the cats which tested positive for the FeLV antigen. Failure to amplify viral sequences in the remainder of infected cats indicates a certain level of inconsistency between the antigen-screening procedure and our PCR-based method. Although nested PCR amplification was carried out in these samples, we did not detect any viral genome. PBMCs infected with FeLV may not always be circulating or present in the blood, even though the cats show viremia. It may depend on the timing of collecting the blood from the cats. It has been reported that in domestic cats with FeLV-FAIDS, the virus variant FeLV/61C was mainly found in the intestines and was not present in the bone marrow [7]. Thus, different strains of the virus may target different cells or tissues, and it would be informative to determine the copy number of FeLV provirus in each tissue type in naturally infected cats. We further established a PCR method for detecting FeLV-B. This PCR method can be used as diagnostic test because the previous studies reported higher incidence of the FeLV-B variant in cats with malignant tumors or hematopoietic disorders [33], [63]. It could be used to establish the clinical stage of such diseases.
One important goal of the present study was to use sequence comparisons to investigate the structural and genetic diversity of FeLV env genes derived from cats that had been naturally infected with FeLV. We have previously determined the nucleotide sequences of two Japanese FeLV strains: FeLV-A clone33 isolated from cats with AML, and pJ7E2 isolated from cats with thymic lymphoma. On the basis of sequence homology, these env sequences were highly divergent from FeLV-A Glasgow-1 and FeLV-A 61E sequences, which prompted us to investigate the genetic diversity of the FeLV env gene in detail. This is the first report of a comprehensive analysis of FeLV env genes isolated from naturally infected cats.
The strongly supported phylogenetic structuring of the FeLV sequences analyzed in this study was unexpected. We identified three major genotypic groups, GI (containing clades 1-7), GII, and GIII (containing clades 1-2), and we found that most FeLV strains in Japan are highly associated with geographical distribution, while GI/3, GI/4 and GI5 are strongly associated. It is thus possible that these FeLV strains may have arisen by isolation in  their respective areas of occurrence over a long period of time, producing a clear phylogenetic structure consistent with geographic distribution. Japanese FeLV strains are genetically distinct from variants of the virus occurring in other parts of the world, and have apparently originated and diversified within Japan. Detection of the GI/Clade 1 genotype in the geographically disjunct Kyoto, Wakayama and Oita prefectures, and detection of the GI/Clade2 genotype in the Kagoshima prefecture (geographically distant from other populations of this clade) are thought to be due to anthropogenic translocation of infected cats. With the exception of a single cat from Okinawa, FeLV sequences belonging to the GIII group were not detected in Japan, indicating that the GI and GII groupings are likely to be the dominant virus genotypes present in Japanese domestic cats. In a study of purifying selection, we did not observe significant branch-specific positive selection in Japanese FeLV strains, suggesting that adaptive evolutionary change (driven by variation in host cats, extrinsic environmental pressures, etc.) is an unlikely explanation for the observed variation in the viral env gene. In addition, there is no evidence that genetic diversification has led to altered receptor usage in the various strains comprising the FeLV Genotype I grouping; most epidemic FeLV strains analyzed in this study appear to belong to the FeLV-A receptor subgroup, which is thus the most prevalent subgroup among Japanese cats. In addition to genetic diversity, substantial structural diversity of the FeLV env gene was detected in our samples, including insertions, deletions, inversions and recombination. Similar types of structural diversity in the env gene (point mutations, insertions, deletions and recombination) were observed in cats that had been experimentally infected with FeLV [64]. Several FeLV env recombinants have also been reported in cats with naturally occurring lymphomas [33]. Retroviral recombination, such as transduction of cellular oncogenes and circulating recombinant forms (CRF), has been reported to involve 'copy-choice' or 'template switching' via formation of heteroduplexes of the viral RNA genome during reverse transcription [65], [66]. The endogenous FeLV env gene is expressed in normal cat lymphoid organs and FeLV-negative lymphomas [59], [67]. Therefore, exogenous FeLV infections involving these organs may expedite FeLV recombination. Analysis of recombination between exogenous and endogenous FeLVs revealed several recombination hot-spots on the env gene (areas in which multiple recombination breakpoints coincide); recombination tends to occur preferentially in regions of high homology between exogenous and endogenous FeLV sequences. In contrast, whilst deletion events also appear to be relatively common in FeLV strains, there may be fewer constraints on the locations of these deletions and therefore no pronounced hot-spots of deletion breakpoints on env gene sequences. Overall, the combined effect of recombination, insertion and deletion events is likely to result in a proliferation of structural diversity in the FeLV env gene.
In this study we found some unique recombinant env genes that contain both VRA and VRB regions derived from the endogenous FeLV env gene. Particular recombination patterns in env genes as well as point mutations affecting the SPHQ motif of the Env protein can alter viral infectivity and cell type specificity of viruses. For example, we confirmed that certain recombinant FeLV strains belong to receptor subgroups FeLV-B or FeLV-T instead of the more common FeLV-A subgroup. The SPHQ motif is highly conserved within the genus Gammaretrovirus and has been identified as an important determinant for mediating fusion events during virus entry into host cells [68], [69]. The FeLV-T receptor subgroup has a particular mutation (H6P) within the SPHQ motif, and this subgroup has been shown to possess unique receptor usage, involving Pit1 and FeLIX [17]. Eighteen FeLV env clones with mutations in the SPHQ motif were isolated from 11 samples, suggesting that the viruses belonging to the FeLV-T subgroup may be relatively common in Japan and may arise de novo in individual cats via mutation of FeLV-A viruses. These events are also likely to amplify viral diversity. Overall in this study, we show that the structural and genetic divergence of FeLV has probably been brought about by substitution, deletion, insertion, and recombination events within env genes, and these processes are thus likely to be key drivers of FeLV evolution, especially in Japan. We have identified structural and genetic diversity of FeLV env genes that may provide new insights into the prevention of FeLV infection, development of more effective vaccines, the geographical components of FeLV infection, and the evolutionary dynamics of the virus. Furthermore, in keeping with our division of FeLV samples into three major genotype groups and several cohesive clades within these groups, we propose that phylogenetic relationships be used as one of the methods of viral classification. Figure S1 The maximum-likelihood (ML) tree from phylogenetic analysis of near-full-length env nucleotide sequences used for dN/dS analysis. The sequences used were listed in Supplemental Table 4. Duplicated sequences were removed. (TIF) Figure S2 Branch specific selection forces for each genotype and clade. Genotype or clade specific dN/dS ratio is shown above each root branches of the schematic phylogenetic tree. The ratio of dN/dS .1 indicates the force of significant positive selection (colored in red). (TIF) Figure S3 Isolates with atypical structural mutations. Schematic representation of the various structural mutations is shown. The motifs are abbreviated s.p. (signal peptide), SPHQ (SPHQ motif), VRA (variable region A), VRC (variable region C), VRB (variable region B), PRR (proline-rich region) and C-dom. (C-terminal domain). (A) Three clones (NR9-7, NR9-2SH and NR9-4SH) were isolated from cat NR9, and NR9-2SH and NR9-4SH had small inverted sequences (95 bp; pale gray) in addition to 1 kbp deletions. Isolate NR9-4SH also had very small inserted sequence (28 bp; black) which may be originated from a part of cat genomic sequence. (B) Two isolates from cat ST17 had FeLV-Blike recombination as well as deletion. enFeLV derived sequence are colored in black. Asterisk indicates stop codon. (TIF)


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

Background Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail. Methodology/Principal Findings We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina. Conclusion/Significance In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.

Introduction
Functional analysis of the genes expressed in the mammalian retina is essential for understanding the molecular basis for human retinal development and disease. Recent progress in techniques of comprehensive analysis of gene expression using microarray and next generation sequencing make it possible to obtain many candidate genes that are possibly associated with retinal development and disease [1,2]. Although in vivo analysis of candidate genes using transgenic and/or knockout mice is beneficial in revealing the in vivo functions of the genes, it is still expensive, timeconsuming, and requires a great deal of skill. Therefore, a rapid and convenient method of in vivo gene transfer would be beneficial to the field. To transduce a gene into the mouse retina, in vivo electroporation and virus-mediated gene transfer are currently the most available methods. In vivo electroporation is a method in which plasmid DNA is incorporated into retinal tissue by highvoltage pulses. This method efficiently transduces DNA into rod photoreceptor cells, but much less efficiently into bipolar, amacrine, and Müller glial cells. Moreover, cone photoreceptor, horizontal, and ganglion cells are barely transduced by in vivo electroporation [3]. For virus-mediated transduction, retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV) have been developed as vehicles for retinal gene transfer. In particular, AAV has many advantages for retinal gene transfer, including high transduction efficiency in non-dividing cells, long-term transgene expression, and low-toxicity. AAV is a non-pathogenic parvovirus, which consists of single-stranded DNA covered with capsid proteins. Each AAV serotype is different in the capsid structure, which leads to different tropisms and transduction efficiencies. Twelve serotypes have currently been used as a vehicle for in vivo gene transfer (AAV2/1-AAV2/12). AAV tropisms for gene transduction into several murine organs and tissues, including the retina, are different according to developmental stage (neonatal or adult) [4,5]. The previous studies on AAV serotype tropism in subretinal injections into the adult mouse retina revealed that retinal pigment epithelium (RPE) cells are efficiently transduced with AAV2/1, and RPE and photoreceptor cells are efficiently transduced with AAV2/2, AAV2/5 [6,7], and AAV2/8 [7]. However, detailed AAV tropisms for transduction into the developing mouse retina have not been reported.
In the current study, we examined the tropism of seven AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/8, AAV2/9, AAV2/10, and AAV2/11) by subretinal injection into the P0 mouse retina. We revealed that AAV can transduce encoded genes into various retinal cell types in the developing mouse retina. In addition, to validate the usefulness of AAV-mediated gene transfer into the developing mouse retina, we performed AAV-mediated rescue of Crx KO mice. CRX is a transcription factor that is predominantly expressed in photoreceptor cells and is essential for photoreceptor maturation [8,9,10]. We previously reported that Crx KO mice exhibit a total lack of outer segment formation, an absence of both scotopic and photopic electroretinograms (ERG), and progressive photoreceptor degeneration [10]. Our AAVmediated rescue experiment led to a partial restoration of morphological and functional characteristics in the Crx KO retina. In humans, the mutations of Crx are associated with three forms of retinal degeneration, including cone and rod dystrophy (CORD) [11,12,13], retinitis pigmentosa (RP) [13], and Leber congenital amaurosis (LCA) [13,14], all of which can lead to vision loss. Thus, our results also provide a clue to the suitability of gene therapy for development disorders and degeneration of the retina in humans.

Infection Efficiencies of Seven AAV Serotypes
To quantitatively assess the tropism of seven AAV serotypes in the mouse retina, we measured the infection efficiencies for each retinal cell type. Infection efficiency is calculated by the percentage of cells expressing mCherry out of retinal cell-specific markerpositive cells ( Fig. 2A). Photoreceptor and horizontal cells were efficiently transduced with AAV2/1 (Fig. 2B, Rod: 65.563.9% in the middle area, Cone: 80.864.5% and horizontal: 55.669.2%). The AAV2/1-injected retinas also exhibited the highest efficiency for Müller glial cells among seven serotypes (Fig. 2B, 47.965.8%). AAV2/2 and AAV2/8 showed similar transduction patterns with each other, and horizontal and ganglion cells were transduced mainly with these serotypes (

AAV-mediated Rescue Experiment for the Crx KO Retina
To validate the usefulness of AAV-mediated gene transfer into the developing mouse retina, we performed an AAV-mediated rescue experiment for the Crx KO mice. CRX is a transcription factor which plays a crucial role in photoreceptor maturation through photoreceptor gene transactivation [1,8,9,10]. We generated an AAV2/5 vector expressing Flag-tagged Crx cDNA under the control of the Crx 2kb promoter to drive specific expression in photoreceptor cells (AAV2/5-Crx2kb-Flag-Crx = AAV-Crx) [17,18] (Fig. 3A). We injected AAV-Crx subretinally into Crx whole retinas (photoreceptor-side-up) two weeks after subretinal injection with PBS or one of each of the seven serotypes of AAV-CAG-mCherry. (C-I) Fluorescence images of mCherry expression two weeks after subretinal injection into the P0 mouse retina with AAV2/1-(C), AAV2/2-(D), AAV2/5-(E), AAV2/8-(F), AAV2/9 (G), AAV2/10-(H), and AAV2/11-(I), CAG-mCherry. Scale bar represents 1 mm (B) and 50 mm (C-I). ITR: inverted terminal repeat, RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. doi:10.1371/journal.pone.0054146.g001 KO retinas at P0. To confirm the Flag-Crx expression in the retina, we performed an RT-qPCR analysis using RNA from the whole retina at three weeks after injection. We observed significant expression of Flag-Crx mRNA in the Crx KO retinas treated with AAV-Crx (Fig. 3B). We further analyzed FLAG-CRX expression in control and AAV-Crx treated retinas by western blotting using an anti-FLAG antibody. We detected a 38 kDa FLAG-CRX band in the AAV-treated Crx KO retinal lysates (Fig. 3C). To determine whether FLAG-CRX is expressed in photoreceptor cells, we performed an immunostaining of the control Crx KO and AAV-Crx-injected Crx KO retinas with the anti-FLAG antibody. FLAG signals were predominantly detected in photoreceptor cells in AAV-Crx-treated Crx KO retinas (Fig. 3D). Non-specific signals were also detected in the RPE and blood vessels in the INL in both control and AAV-Crx-treated Crx KO retinas. This is very likely due to autofluorescence from the RPE and to the reaction of the anti-mouse secondary antibody to endogenous mouse antibodies in the blood vessels (Fig. 3D). AAV-Crx-mediated FLAG-CRX expression was widely distributed throughout the AAV-Crxtreated Crx KO retinas (Fig. 3E, F).

Immunohistochemistry of Crx KO Retinas Treated with AAV-Crx
We further analyzed the expression of RHODOPSIN, GNAT1, S-OPSIN, and M-OPSIN in AAV-Crx-treated Crx KO retinas by immunostaining. The RHODOPSIN protein level was slightly increased in AAV-Crx-injected Crx KO retinas ( Fig. 4A-D), while GNAT1 signals were markedly increased ( Fig. 4E-H). Similarly, S-OPSIN and M-OPSIN signals were markedly increased ( Fig. 4I-P). Consistent with the results of the RT-qPCR analysis shown in Figure 3G-Q, the levels of these molecules also increased in AAV-Crx-treated Crx KO retinas. In addition, GNAT1, S-OPSIN, and M-OPSIN signals appeared to be localized in outer segments in AAV-Crx-injected Crx KO retinas ( Fig. 4E-P). RHODOPSIN and GNAT1 are localized in the rod outer segment and S-OPSIN and M-OPSIN are localized in the cone outer segment. The Crx KO retina lacks outer segment formation [10,20]. This immunohistochemical data suggests that the defect of outer segment formation in the Crx KO retina was partially restored by the subretinal injection of AAV-Crx into Crx KO retinas.
We further examined the morphology of the outer segment in the control Crx KO and AAV-Crx-injected Crx KO retinas in detail by transmission electron microscopy at fifteen weeks after AAV treatment (Fig. 4Q-S). We observed no photoreceptor cells in control retinas because of severe photoreceptor degeneration in the Crx KO retina (Fig. 4Q). In contrast, outer segments containing disk lamina were observed in AAV-Crx-injected Crx KO retinas (Fig. 4R, S). These data showed that the subretinal delivery of AAV-Crx into P0 Crx KO retinas partially restored outer segment formation in the Crx KO mice.

The Improvement of Retinal Function in the Crx KO Retina Treated with AAV-Crx
To evaluate the effect of the AAV-Crx injection on retinal function, we performed electroretinogram (ERG) recordings. Crx KO mice exhibit flat scotopic and photopic ERG responses at P30, resulting from the defect of formation of outer segments and considerable loss of phototransduction molecules in the Crx KO mice [10]. At 6 and 15 weeks after AAV treatment, we measured ERG responses from three control and three AAV-Crx-treated eyes. The mice used for ERG recordings were independently prepared between the two time points. We first tried to record scotopic ERG responses from the AAV-treated mouse eyes in a conventional way [21,22], but no positive response was detected. Therefore, we then measured photopic ERG responses with one hundred stroboscopic flashes of 1.0 log cd-s/m 2 . The ERG responses obtained under these conditions majorly reflect cone photoreceptor functions. Consistent with our previous observation [10], all of the control eyes exhibited completely flat responses at both 6 and 15 weeks after AAV treatment (Fig. 5A top, Table 1). In contrast, two of three AAV-Crx-treated eyes showed significant photopic a-and b-waves at both 6 and 15 weeks after AAV treatment (Fig. 5A bottom, Table 1). One of the AAV-Crx-treated Crx KO mice did not show a detectable ERG photopic response. Although this mouse eye widely expressed FLAG-CRX, the eye was much smaller than the other two mouse eyes and was severely damaged histologically (data not shown), probably due to subretinal injection damage, resulting in physiological dysfunction. This result shows that the subretinal injection of AAV-Crx into Crx KO retinas partially restored the physiological function of Crx KO photoreceptor cells.
Progressive photoreceptor degeneration is also observed in the Crx KO mice [10]. We examined photoreceptor degeneration in the retinas fifteen weeks after AAV treatment by immunostaining using an anti-FLAG antibody (Fig. 5B). Although there was substantial photoreceptor cell death in both control and AAV-Crx-RHODOPSIN (RHO), Cone: S-OPSIN, Horizontal: CALB1, Bipolar: CHX10, Amacrine: PAX6, Ganglion: BRN3B, Mü ller: S100b). According to retinal cell types, the numbers of marker-positive and marker/mCherry double-positive cells were counted for calculation of infection efficiency (n = 3 from three different mice). Cells were counted in the central area of the retina for calculating the infection efficiencies of cone photoreceptor, bipolar, and Mü ller glial cells, and in the central, middle and peripheral areas of the retina for calculating the infection efficiencies of rod photoreceptor and amacrine cells. Infection efficiency was calculated using the formula indicated in Figure   treated Crx KO retinas, AAV-Crx-treated Crx KO retinas had a thicker outer nuclear layer than that of control retinas (Fig. 5B top  panels). Remarkably, most of the surviving photoreceptor cells in AAV-Crx-treated Crx KO retinas expressed FLAG-CRX (Fig. 5B  bottom panels). This observation suggests that photoreceptor cells without FLAG-CRX expression died and that AAV-Crx inhibited photoreceptor cell death in the Crx KO retinas. To confirm this result, we performed immunostaining with an anti-active-caspase-3 antibody, an apoptosis marker, three weeks after AAV treatment (Fig. 5C, D). We observed a significant reduction of apoptotic cell numbers in AAV-Crx-treated Crx KO retinas (Fig. 5C, D). These results show that subretinal injection of AAV-Crx prevented photoreceptor cell death to some extent in the Crx KO retina.

Discussion
The main goal of this study is to establish a method for AAVmediated retinal gene transfer into developing retinas for in vivo analysis of retinal genes and to apply the method to the rescue of retinal degeneration. Through the transduction of AAV-CAG-mCherry into the developing mouse retina, we showed that various retinal cell types were transduced differently with each of seven serotypes of AAV. We quantitatively analyzed infection efficiencies of these AAV serotypes. In addition, we demonstrated the usefulness of AAV transduction into the developing mouse retina for the rescue of the retinal degeneration of Crx KO mice. These results will be useful to researchers who perform in vivo analysis of retinal genes using AAV.
We subretinally injected seven serotypes of AAV into the P0 mouse retina. We chose P0, because the retina at this stage is still developing and amenable to surgical treatment by subretinal injection. Serotypes AAV2/5 and AAV2/9 exhibited remarkable and efficient infection. Photoreceptor cells were selectively and efficiently transduced with AAV2/5. Retinal cells, except for Müller glial cells and bipolar cells, were efficiently transduced with AAV2/9. We observed efficient transduction into horizontal and/ or ganglion cells with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. These data suggests that in vivo gene transfer by subretinal injection of AAV into the mouse retina enables us to analyze gene function in retinal cell types that are difficult or impossible to transduce by in vivo electroporation (cone photoreceptor, horizontal, and ganglion cells) [3]. To perform an analysis specifically in a certain cell type in the retina, a cell type-specific promoter of relatively short length for each cell type will need to be developed in the future.
In the current study, the infection efficiencies of AAV into bipolar and Müller glial cells were low in all tested serotypes, although AAV2/1 efficiently targeted Müller glial cells. These two retinal cells undergo differentiation at the latest stages during development [21]. In contrast, retinal cells, which are generated at embryonic stages or at early postnatal stages (photoreceptor, horizontal, amacrine and ganglion cells), were transduced with all analyzed serotypes. These results suggest that the AAV serotypes tested in this study are more infectious to differentiated retinal cells than to retinal progenitor or differentiating cells. Since more than 100 AAV serotypes have been isolated [22], AAV serotypes which are capable of efficiently infecting bipolar and Müller glial cells may be found in the future. Our observations on AAV tropism in the developing retina in the current study may expand the application of AAV for in vivo transduction to retinal cell types, such as cone photoreceptors, horizontal cells, and ganglion cells, which are barely transduced by in vivo electroporation.The AAV tropisms shown in the current study were different from those for the adult mouse retina in previous reports. In subretinal transduction into the adult retina AAV2/1 mainly targets RPE cells, and AAV2/2, 2/5, and 2/8 mainly target both RPE and photoreceptor cells [6,7]. However, based on our results using P0 mice, each AAV serotype targeted several retinal cell types. This observation is consistent with the results reported by Surace et al. [5], in which they showed the difference of tropisms for transduction into fetal, neonatal and adult retinas using AAV2/ 1-, 2/2-, and 2/5-CMV-EGFP. There are two possible explanations for the shift of AAV tropism between the developing retina and the mature retina. The first is that the expression or abundance of AAV receptors in host retinal cells may change during retinal development. The second is that injected virus particles may more easily diffuse across the retina due to the smaller size of the P0 retina and dynamic cell migration during retinal development. The differences of AAV tropisms between neonatal and adult mice are also observed in the aorta, liver and kidney [4]. This suggests that the time point of transduction of an AAV vector during development is important to appropriately perform gene transfer to a cell type of interest.
We attached the Crx 2kb promoter to the AAV vector for the rescue experiment on the Crx KO retina. We previously reported that this promoter directs specific gene expression in developing and mature rod and cone photoreceptor cells [17,18]. Expression analysis by RT-qPCR and immunohistochemistry showed improved expression of both rod-specific genes (Rhodopsin and Gnat1) and cone-specific genes (S-opsin and M-opsin) in AAV-Crx-treated Crx KO retinas (Fig. 3G-Q and 4A-P). This indicates that the Crx 2kb promoter successfully drove the Crx expression in both rod and cone photoreceptor cells. Thus the Crx 2kb promoter can be used with AAV to drive expression specifically in photoreceptor cells.
AAV-Crx modestly but significantly improved the photopic ERG responses, but we were not able to detect scotopic ERG responses in AAV-treated eyes. In order to detect relatively weak ERG responses in AAV-Crx-treated Crx KO eyes, we stimulated the mouse eyes with one hundred stroboscopic flashes (1.0 log cds/m 2 ). However, under these conditions, it is very difficult to measure scotopic ERG responses because the intermittently repeated flashing lights abrogate the dark adaptation that is necessary for scotopic ERG recordings. Because of such technical difficulties, we were not practically able to measure scotopic ERG responses in our current study. AAV-Crx also partially restored outer segment formation in the Crx KO mice. This result not only confirms that AAV transduction into the P0 retina is a useful method for in vivo retinal gene transfer but also suggests that gene AAV-treated Crx KO retinas from three different mice respectively. An anti-FLAG antibody was used to detect FLAG-CRX. ACTB (b-actin) was used as a loading control. (D-F) Immunostaining of the Crx KO retinas treated with AAV-Crx or PBS using an anti-FLAG antibody. The distribution of FLAG-CRX expression in control and AAV-Crx treated Crx KO retinas (E, F). Enlarged images in white boxes (a-c and d-f) in Figure 3E and F, respectively. Scale bar represents 50 mm (D) and 1 mm (E, F). (G-Q) Expression analyses of eleven genes related to human retinal diseases three weeks after treatment (Control Crx KO retinas: n = 3 from three different mice and AAV-treated Crx KO retinas: n = 4 from four different mice). Rhodopsin (G), Gnat1 (H), Sopsin (I), M-opsin (J), Pde6g (K), Slc24a1 (L), Rdh12 (M), Rpgrip1 (N), Nrl (O), Cabp4 (P), and Fscn2 (Q). Control retinas were injected with PBS (Vehicle). Rpl4 was used for normalization. Primers for qPCR were listed in Table S1. Error bar represents the SD from the means of three control retinas and four treated retinas. ** p,0.01, * p,0.05. ITR: inverted terminal repeat, RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. doi:10.1371/journal.pone.0054146.g003 In addition, this is the first report on AAV-mediated rescue for mice with mutations of a transcription factor regulating photoreceptor development and related to human photoreceptor degeneration. Nrl, Nr2e3, and Otx2 also play crucial roles in photoreceptor development through transcriptional regulation of photoreceptor genes [1,23,24,25]. The mutations of these genes in humans are associated with several types of retinal degeneration (RetNet: https://sph.uth.tmc.edu/retnet/home.htm). Our current result suggests that human retinal degenerations caused by the mutations in these transcription factors can be restored by AAVmediated gene therapy. This possibility will be examined by AAVmediated rescue of mice with these gene mutations in the future.

Animals
For the evaluation of the tropism of AAVs in vivo, we used ICR mice (Charles River). The Crx KO mice were generated as described in our previous study [10]. All procedures conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, and these procedures were approved by the Institutional Safety Committee on Recombinant DNA Experiments and the Animal Research Committee of Osaka Bioscience Institute (approval ID 10-401) and Institute for Protein Research, Osaka University (approval ID 24-05-0). Mice were housed in a temperature-controlled room with a 12-hour light/dark cycle. Fresh water and rodent diet were available at all times.

AAV Production
AAV was produced by triple transfection of an AAV vector plasmid, an adenovirus helper plasmid, and an AAV helper plasmid (pAAV2/1, pAAV-RC [Agilent technologies], pXR5, pAAV2/8, pAAV2/9, pAAV2/rh10, and pEEV2/11) into AAV-293 cells by the calcium phosphate method. The cells were harvested at 72 hours after transfection, and lysed by four freeze-and-thaw cycles. The supernatant was collected by centrifugation, and treated with Benzonase nuclease (Novagen) to eliminate cellular DNA/RNA and excess plasmid DNAs. This virus preparation was used for subretinal administration. A titer of each AAV (in vector genomes (VG)/mL) was determined by qPCR using SYBR GreenER Q-PCR Super Mix (Invitrogen) and Thermal Cycler Dice Real Time System Single MRQ TP870 (Takara). The primers used for AAV titrations are listed in Table S1. The titers of all serotypes of AAV-CAG-mCherry used in this study were adjusted to approximately 2610 12 VG/mL. The titer of AAV2/5-Crx2kb-Flag-Crx used in the Crx KO rescue experiment is 2.6610 12 VG/mL.

Subretinal Injection
Subretinal injection of AAV was performed as described elsewhere [3,29]. P0 mice were anesthetized by chilling on ice, the eye was opened by cutting along the fused junctional epithelium where the two eyelids come together, and a small incision was made with a 30-gauge needle in the sclera near the junction with the cornea. 0.4 mL of an AAV preparation was injected into the subretinal space through the incision using an Ito micro syringe (Ito Corporation) with a 33-gauge blunt-ended needle under a dissecting microscope. Fast Green dye was added to AAV preparations at a final concentration of 0.1% as a tracer to confirm that the AAV preparations were injected into the subretinal space [29]. For histological analyses, we only used retinas in which the dye in the AAV preparation was confirmed to be evenly distributed and which were without severe damage caused by the injection process.

Immunostaining
For immunohistochemistry, 14 mm thick retina sections were washed twice in phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 (wt/vol) in PBS, then incubated with PBS containing 4% donkey serum (vol/vol) for 1 h to block samples. The samples were incubated with a primary antibody at 4uC overnight. After washing with PBS, these samples were incubated with secondary antibodies at 25uC for 1 hour. In the current study, we also used the following primary antibodies: anti-

Quantification of Infection Efficiency
AAV-CAG-mCherry-injected retinas were co-immunostained with cell type-specific markers for each retinal cell shown above and an anti-mCherry antibody. We counted the number of marker-positive cells and marker/mCherry double-positive cells according to cell types for the calculation of infection efficiencies.

Western Blot Analysis
Western blot analysis was performed as described previously [27]. The membrane was incubated with an anti-FLAG antibody (1:1000, F1804, Sigma). The membrane was then incubated with a horseradish peroxidase-conjugated goat antibody against mouse IgG (1:10000, Zymed). For the secondary immunoreaction, the PVDF membrane was incubated with WB Stripping Solution (Nacalai Tesque) to remove antibodies, and blocked again with 5% skim milk (wt/vol) in TBS. Further immunoblots were performed using a mouse antibody against b-actin (ACTB, 1:5000, Sigma).

RT-qPCR
The mouse retinas were harvested and dissected at 3 weeks after injection. Total RNA (1 mg) from the retina was isolated using TRIzol reagent (Invitrogen) and converted to cDNA using Superscript II Reverse Transcriptase (Invitrogen). Real time PCR was performed using SYBR GreenER Q-PCR Super Mix (Invitrogen) and Thermal Cycler Dice Real Time System Single MRQ TP870 (Takara) according to the manufacturer's instructions. Quantification was performed by Thermal Cycler Dice Real Time System software version 2.0 (Takara). The primer sequences used for qPCR are listed in Table S1.

ERG Recordings
Mice were anesthetized with an intramuscular injection of 80 mg/kg ketamine and 16 mg/kg xylazine. Pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine HCl, and the mice were placed on a heating pad for the duration of the ERG recordings. ERGs were recorded with a gold wire loop placed on the cornea anesthetized with 1% tetracaine. A gold wire electrode was placed on the sclera 1 mm from the temporal limbus as the reference electrode. The mice were placed in a Ganzfeld bowl and 100 stroboscopic stimuli of 1.0 log cd-s/m 2 (PS33 Plus; Grass Telefactor) were averaged with a repetition rate of 1 sec to record the ERGs. Signals were amplified and bandpass filtered between 1 and 1000 Hz (Power Lab; AD Instruments, Castle Hill, mice showed no ERG response, while two of the three treated eyes from different mice showed significant responses. (B) Immunostaining of FLAG-CRX in the retinas at fifteen weeks after AAV-Crx treatment. (C and D) Immunostaining of active-caspase-3 and FLAG-CRX in Crx KO retinas three weeks after AAV-Crx treatment (C). The number of active-caspase-3-positive cells (D, Control retinas: n = 3 from three different mice and AAV-treated Crx KO retinas: n = 4 from four different mice). Arrowheads indicate active-caspase-3-positive cells. Scale bar represents 50 mm. Error bar represents the SD from the means of three control retinas and four treated retinas. * p,0.05. RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. doi:10.1371/journal.pone.0054146.g005 Table 1. Quantitative analysis of ERG amplitudes in control and AAV-Crx treated eyes. Australia). Amplitudes of both a-and b-waves were quantified from photopic ERG responses.

Transmission Electron Microscopy
Specimens for transmission electron microscopy were prepared in the following manner. Eyes were enucleated from anaesthetized mice. Following the removal of anterior segment, each posterior eyecup was fixed with 2% glutaraldehyde and 2% paraformaldehyde in a cacodylate-based buffer adjusted at pH 7.4. After fixation with 1% osmium tetraoxide for 90 min, the retinas were dehydrated through a graded series of ethanol (50%-100%) and nbutylglycidylether. Finally, they were embedded in epoxy resin. Ultrathin sections were cut on an ultramicrotome (Ultracut E, Reichert-Jung, Vienna, Austria), and stained with uranyl acetate and lead citrate. Retinas were observed by transmission electron microscope (1200EX, JEOL, Japan).

Statistical Analysis
Statistical significance was calculated with a Student's t test. A value of p,0.05 was taken to be statistically significant. Data are presented as means 6 SD.