Virology 468-470 (2014) 524–531
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
Brief Communication
Identification of a natural recombination in the F and H genes of feline morbillivirus Eun-Sil Park a, Michio Suzuki a, Masanobu Kimura a, Keiji Maruyama b, Hiroshi Mizutani b, Ryuichi Saito b, Nami Kubota b, Tetsuya Furuya c, Tetsuya Mizutani c, Koichi Imaoka a, Shigeru Morikawa a,n a
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan Tokyo Metropolitan Animal Care and Consultation Center Jounanjima Branch Office, Tokyo 143-0002, Japan c Research and education center for prevention of global infectious diseases of animals, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Returned to author for revisions 31 July 2014 Accepted 4 September 2014
Feline morbillivirus (FmoPV) has recently been identified in Hong Kong and Japan. FmoPV is considered to belong to the genus Morbillivirus, in the family Paramyxoviridae. In this study, the complete nucleotide sequences of three strains of FmoPV detected in cats in Japan were determined. Among the six genes in FmoPV; N, P/V/C, M, F, H and L, the P gene showed the highest polymorphism in the nucleotide and putative amino acid sequences among the FmoPV strains. There was no geographical association in terms of the FmoPV phylogeny; however, from extensive phylogenetic and recombination analyses, we found that one Japanese FmoPV strain, MiJP003, was a probable recombinant between two virus strains in the independent lineages found in Japan and Hong Kong, respectively. The recombination was considered to have occurred within the F and H genes. Such recombination is thought to be involved in the evolution of FmoPV. & 2014 Elsevier Inc. All rights reserved.
Keywords: Feline morbillivirus Fusion protein Hemagglutinin protein Recombination
Introduction Morbilliviruses belong to the family Paramyxoviridae and possess a non-segment single-stranded negative RNA genome. Morbilliviruses include the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RV) and peste des petits ruminants virus (PPRV), and these viruses cause similar diseases in their respective host species. They use the SLAM (CD150) and nectin4 of their hosts as common receptors (Baron, 2011; Bevan et al., 2001; Birch et al., 2013; Schneider-Schaulies et al., 2011; Takeda et al., 2012). Recently, feline morbillivirus (FmoPV) has been newly isolated and reported in Hong Kong and Japan (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). FmoPV is phylogenetically related to other morbilliviruses, and thus is considered to belong to the genus Morbillivirus. Similar to other morbilliviruses, the genome of FmoPV encodes eight non-structural and structural proteins, which are the N, P/V/C, M, F, H and L proteins. The complete genome size of FmoPV is 16,050 bases, which is the largest among the morbilliviruses. FmoPV may be associated with
n
Corresponding author. E-mail address: [email protected] (S. Morikawa).
http://dx.doi.org/10.1016/j.virol.2014.09.003 0042-6822/& 2014 Elsevier Inc. All rights reserved.
feline tubulointerstitial nephritis, although more studies are needed to understand the virological and molecular basis of the disease (Furuya et al., 2014; Woo et al., 2012). Recently, newly emerging viruses in the genus Morbillivirus, such as porcine distemper virus (PDV) and cetacean morbillivirus (CMV), were identified (Mazzariol et al., 2013; Rubio-Guerri et al., 2013; Saliki et al., 2002). The CDV has a wide host range, including the felidae (except domestic cats), marine mammals and primates (Roelke-Parker et al., 1996, Sakai et al., 2013a, Sakai et al., 2013b; Yoshikawa et al., 1989). Indeed, PDV, CMV and CDV are reported to be genetically and antigenically related, and they have also been shown to cross-react in serological tests (Saliki et al., 2002). FmoPV is a novel morbillivirus that infects domestic cats, which have not been a natural host of the previously known morbilliviruses. The cluster of this virus is genetically different from that of CDV, but is close in the phylogenetic tree (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). It is uncertain whether FmoPV is a newly emerged virus or whether it has existed, undetected, and it is also uncertain whether it causes diseases in the other felidae. Re-assortment and non-homologous or homologous recombination have been reported to contribute to the evolution and the virulence of negative-strand RNA viruses, although the rates of these processes are controversial (Archer
E.-S. Park et al. / Virology 468-470 (2014) 524–531
and Rico-Hesse, 2002; Chare et al., 2003; Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; Han and Worobey, 2011; He et al., 2008; He et al., 2009; Lukashev, 2005; Wittmann et al., 2007; Zhang et al., 2010). To elucidate the evolution of FmoPV, it is necessary to understand its genome on the genetic and molecular bases. Therefore, in this study, we determined the whole genome sequences of three strains of FmoPV that were detected in Japan, and found a possible recombination event between two FmoPV strains from independent lineages initially isolated in Japan and Hong Kong.
Results We collected cat urine samples in Tokyo and detected FmoPV by RT-PCR (Supplementary Table 1). FmoPV was detected in 13.7% of the cat urine specimens. Among these FmoPV-positive specimens, we amplified the whole genome region by overlapping RTPCR amplicons and sequenced the full genomes of three FmoPV strains, OtJP001 (Accession no. AB924120), MiJP003 (Accession no. AB924121) and ChJP073 (Accession no. AB924122) to compare them with those reported in Hong Kong and Japan (Sakaguchi et al., 2014; Woo et al., 2012). The genome organization was also consistent with that of the previously reported strains. The genome of all three FmoPV strains possessed six genes, N, P/V/C, M, F, H and L. The degree of nucleotide and deduced amino acid identity was obtained by pairwise comparisons among the three Japanese FmoPV strains and four previously reported strains (Table 1). The P gene and the F gene showed the highest rates of nucleotide polymorphism having 88.6% to 99.3% and 88.6% to 99.2% nucleotide identities, respectively. The P (V) gene also showed the highest rate of amino acid polymorphism having 85.5–98.9% amino acid identity. Phylogenetic trees were constructed using the complete genomic and amino acid sequences of FmoPV and other paramyxoviruses deposited in the GenBank (Supplementary Table 2). The three Japanese FmoPV strains were clustered with the four reported strains, and grouped against other viruses, indicating that FmoPVs form a distinct group in the phylogenetic tree (Fig. 1). Interestingly, the phylogenetic grouping of the intragenomic region, F and H genes, of the MiJP003 strain was different from those observed for other genes (Fig. 1). Thus, the complete genome sequences of the three Japanese strains, OtJP001, MiJP003 and ChJP073, the three Hong Kong strains, M252A, 761U and 776U, and the one Japanese strain, SS1, were aligned using the MEGA6 program, and then were screened for any possible recombination events using the SimPlot and Recombination Detection Program (RDP) software platforms. When the standard similarity plots were constructed using all sequences as queries with SimPlot, the sequence of MiJP003 showed high similarity with that of ChJP073 except in the F and H genes which had high similarity with that of 776U, suggesting the presence of recombination (Fig. 2A and B). To obtain a more accurate overview of the recombination, a subsequent bootscanning analysis was carried out with a SimPlot subprogram. Bootstrap values of over 80% are considered to be significant. When the sequences of MiJP003, ChJP073 and 776U were studied as queries, reliable bootstrap values in the bootscanning analysis were produced (Fig. 2B). These results indicated the putative recombination among FmoPV strains. To obtain detailed information about such putative recombination, all sequences were studied using several methods included in the RDP. The results are shown in Fig. 3 and Supplementary Table 3. Similar to the results of the SimPlot analyses, one possible recombination signal was detected. When we further limited the analyses to the three strains, MiJP003, ChJP073 and 776U, MiJP003, a Japanese strain, was identified as the putative recombinant by
525
the RDP (Fig. 3A C). The major parental-like strain was ChJP073, another Japanese strain, and the minor parental-like strain was 776U, a Hong Kong strain, with nucleotide identities of 99.2% and 98.2%, respectively. The break-points were identified using the RDP and MaxChi methods, which are two of the methods in the RDP (Fig. 3D). The beginning break-point was located from position 5600 to 5703 in the alignment (exactly at position 5670, corresponding to the F protein, Po 0.05) and the ending breakpoint was located from position 7951 to 8030 (exactly at position 7983, corresponding to the H protein, P o0.05). The results of the RDP supported those of SimPlot, indicating that there was recombination among the FmoPV strains. To provide strong evidence of the putative recombination event, a further phylogenetic analysis was performed. The phylogenetic trees were constructed from the nucleotide sequences of the seven FmoPV strains from Japan and Hong Kong and two currently available sequences of CDV, 50Con and 011C (as outgroups), with the MEGA6 program. To avoid topological errors resulting from the putative recombination, separate phylogenetic trees were constructed from the sequences on either side of the break-point and the putative recombination region. The phylogenetic trees were delimitated by the break-points (Fig. 3E). Whereas both sides of the break-points in MiJP003, the putative recombinant, were most closely related to ChJP073, it was most closely related to 776U in the putative recombination region (Po 0.01, Shimodaira–Hasegawa test). Conflicting phylogenetic trees for different regions of the sequence supported the results of the SimPlot analysis and RDP, and provided strong evidence of recombination.
Discussion In this study, we investigated the whole genome sequences of FmoPV from cats in Japan, and detected one possible recombination event between strains from Japan and Hong Kong. To date, FmoPV has only been found in Hong Kong and Japan, and the whole genome sequences of these FmoPV strains have been reported (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). At present, it has not been confirmed whether FmoPV belongs to the genus Morbillivirus, although it was closest to the morbillivirus groups in the phylogenetic analysis (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). It was first considered that the Hendra virus belonged to genus Morbillivirus when it had been isolated (Lo et al., 2011). Now, the Hendra virus is included in a novel genus, Henipavirus, with the Nipah virus. Thus, it is necessary to obtain more information on FmoPV to determine whether the virus belongs to the genus Morbillivirus. In this regard, we determined the complete nucleotide sequences of three Japanese FmoPV strains. The FmoPV strains detected in Japan showed high nucleotide and amino acid identities to those from Hong Kong. In the phylogenetic analysis, the FmoPV from Japan and Hong Kong formed the same group apart from other morbilliviruses. Our study and previous studies (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012) showed that FmoPV is distributed in at least Japan and Hong Kong. In addition, when we aligned the nucleotide sequences of FmoPV, we recognized that the phylogenetic grouping of the intragenomic region, the F and H genes, of a Japanese FmoPV strain, MiJP003, was different from that observed for other genes. We could detect one possible recombination event between the F and H gene regions by further recombination detection analyses. The phylogenetic analyses and statistical tests strongly supported these results. The rate of recombination in negative-strand RNA viruses is controversial, due to the possible presence of laboratory artifacts or contamination (Chare et al., 2003; Han, Worobey,
526
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Table 1 Similarities in the FmoPV strains for each protein. N Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 776U 761U M252A
OtJP001
MiJP003
ChJP073
SS1
776U
761U
M252A
0.947
0.949 0.998
0.992 0.951 0.953
0.965 0.965 0.967 0.965
0.965 0.963 0.965 0.965 0.992
0.949 0.984 0.986 0.953 0.961 0.959
0.901 0.901 0.992 0.9 0.9 0.896
0.996 0.9 0.921 0.926 0.981
0.9 0.922 0.927 0.981
0.9 0.901 0.895
0.985 0.917
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.859
0.859 0.989
0.987 0.863 0.863
0.859 0.971 0.973 0.863
0.855 0.896 0.896 0.861 0.885
0.859 0.902 0.902 0.865 0.892 0.973
0.923
P Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.901 0.9 0.993 0.901 0.886 0.888
0.993 0.901 0.984 0.924 0.931
0.901 0.984 0.924 0.931
0.901 0.886 0.888
0.921 0.928
0.982
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.943
0.94 0.997
0.997 0.94 0.937
0.94 0.997 0.994 0.937
0.961 0.967 0.964 0.958 0.964
0.952 0.961 0.958 0.949 0.958 0.988
M Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.895 0.897 0.989 0.898 0.905 0.896
0.993 0.894 0.994 0.923 0.922
0.896 0.993 0.921 0.92
0.897 0.902 0.893
0.922 0.921
0.987
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.926
0.926 0.974
0.994 0.928 0.928
0.922 0.968 0.985 0.924
0.926 0.966 0.963 0.928 0.959
0.93 0.97 0.966 0.931 0.963 0.988
F Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.888 0.888 0.992 0.887 0.886 0.89
0.958 0.89 0.955 0.956 0.957
0.89 0.991 0.931 0.936
0.889 0.887 0.891
0.928 0.933
0.979
H Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 SS2 SS3 M252A 776U 761U
OtJP001
0.898 0.907 0.988 0.906 0.908 0.908 0.898 0.902
MiJP003
ChJP073
SS1
SS2
SS3
M252A
776U
761U
0.932
0.937 0.964
0.986 0.932 0.937
0.936 0.957 0.964 0.936
0.941 0.968 0.993 0.941 0.968
0.941 0.968 0.986 0.941 0.968 0.989
0.932 0.971 0.952 0.932 0.949 0.952 0.952
0.942 0.981 0.963 0.942 0.959 0.963 0.963 0.989
0.952 0.897 0.939 0.954 0.95 0.96 0.965
0.906 0.952 0.994 0.985 0.927 0.93
0.906 0.907 0.906 0.898 0.902
0.954 0.952 0.925 0.927
0.987 0.928 0.932
0.927 0.929
0.988
L Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
OtJP001
0.9 0.901 0.993 0.903 0.896 0.896
MiJP003
ChJP073
SS1
M252A
776U
761U
0.96
0.962 0.992
0.997 0.961 0.962
0.963 0.988 0.991 0.963
0.962 0.972 0.974 0.961 0.973
0.96 0.969 0.971 0.959 0.97 0.993
0.993 0.9 0.983 0.932 0.932
0.901 0.984 0.933 0.932
0.903 0.896 0.896
0.933 0.932
0.985
E.-S. Park et al. / Virology 468-470 (2014) 524–531
527
Fig. 1. Phylogenetic trees based on the N, P, M, F, H and L amino acid sequences of FmoPV. Phylogenetic trees were inferred using the maximum likelihood method in the MEGA6 package. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The trees are drawn to scale, and the scale bars indicate the branch length corresponding to 0.2 substitutions per site. The three strains of FmoPV from Japan were named OtJP001, MiJP003 and ChJP073 (red). The strains from Hong Kong and other morbilliviruses are represented by blue and green, respectively. Information about the strain names and accession numbers is listed in Supplementary Table 2.
528
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Fig. 2. The results of the SimPlot analyses. (A) The genomic organization of FmoPV. The putative recombination region was found within the F and H genes. The break-points are shown with arrowheads. (B) The standard similarity plot was constructed using MiJP003 as a query for comparison with other strains, with a window size of 200 bp and a step size of 20 bp. (C) The bootscan analysis was performed using MiJP003 as a query for comparison with other strains, with a window size of 200 bp and a step size of 20 bp. The bootstrapping tests were performed with 1000 replicates.
2011). We designed new primers and re-sequenced the region including the putative recombination area, and confirmed the identical recombinant sequences (data not shown). Therefore, contamination and artifacts should not have been present. It is thought that the recombination or reassortment of the virus genome affects the evolution and virulence of the virus (Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; He et al., 2008; He et al., 2009; Wittmann et al., 2007; Zhang et al., 2010). In particular, the recombination of envelope genes has been reported in
negative-strand RNA viruses, such as CDV, influenza A virus and Newcastle disease virus (Chare et al., 2003; Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; Zhang et al., 2010). The recombination of genes related to the entry of viruses may affect the host ranges. In the case of CDV, a putative recombination in the H gene region was found in a CDV strain isolated from a giant panda, and the recombination event was thought to confer the ability for CDV to infect different host species (Han et al., 2008b). Therefore, the recombination of FmoPV might result in changes in the host range.
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Genetic typing using partial sequences of viruses has been widely performed as a means of tracing the spread of viruses, such as the Newcastle disease virus, influenza A virus and CDV (Gibbs et
529
al., 2001; Han et al., 2008a; Han et al., 2008b; He et al., 2008; He et al., 2009; Zhang et al., 2010). It is also helpful for epidemiologists to survey the origin and epidemics of the viruses. From
530
E.-S. Park et al. / Virology 468-470 (2014) 524–531
these reports, it has been emphasized that ignoring recombination might severely distort the results of the phylogenetic analysis (Han et al., 2008a; Han et al., 2008b). The present study indicates that this is also the case for FmoPV. Therefore, recombination should be considered when a phylogenetic analysis of FmoPV is performed using partial sequences. In the present study, we showed that the MiJP003 strain was a putative recombinant strain, with the parents being viruses closely related to ChJP073 and 776U. The recombination is considered to have occurred in a cat infected with two different virus strains, and this suggests that an FmoPV strain genetically closely related to the 776U strain is also distributed in Japan. A previous report indicated that FmoPV is associated with tubulointerstitial nephritis, which could be an autoimmune response caused by the infection (Furuya et al., 2014; Woo et al., 2012). However, the association of FmoPV with tubulointerstitial nephritis is not observed (Sakaguchi et al., 2014). A cat infected with MiJP003, a putative recombinant, had mild pathological lesions and clinical characterstics in this study (Supplementary Table 1). This disease could be acute, chronic or persistent. The chronic or persistent infection conditions would be favorable for the recombination of FmoPV. Given the remarkable development of the global pet business, the FmoPV strains may expand their distribution in association with this development. However, further studies are needed to better understand the virulence of FmoPV, including the identification of receptors and a survey of host animal species for FmoPV.
Materials and methods Sample collection Cat urine samples (Supplementary Table 1) were collected from Tokyo Metropolitan Animal Care and Consultation Center and were used under the approval of the Bureau of Social Welfare and Public Health of Tokyo Metropolitan Government in Japan (24-DouSou-4557). The maintenance and handling of cats were performed at the Tokyo Metropolitan Animal Care and Consultation Center (Jounanjima Branch Office) in accordance with the Act on the Welfare and Management of Animals (Article 40) of Japan. Virus RNA was extracted from the urine samples using ISOGEN (Wako, Osaka, Japan) and a precipitation carrier (Ethachin mate, Wako) and was used as the template for RT-PCR. RT-PCR of the L gene of FmoPV and DNA sequencing FmoPV was detected by amplifying a 401 bp fragment of the L gene of FmoPV. Reverse transcription (RT) was performed using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad CA, USA) with random hexamer primers, according to the manufacturer's manual. Then, two-step nested PCR was performed with sets of primers (Furuya et al., 2014). The PCR products were purified from agarose gels using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE healthcare, Buckinghamshire, UK).
Both strands of the extracted PCR products were sequenced with an ABI 3130 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). The sequences were compared with known sequences of the L genes of FmoPV in the GenBank database. Complete genome sequencing and analysis Three complete genomes, OtJP001, MiJP003 and ChJP073, determined in this study, were amplified and sequenced using virus cDNA converted from RNA using random hexamer primers as templates. The primers used to amplify separated fragments of the FmoPV whole genome were designed with reference to the conserved regions of the three FmoPV sequences (Woo et al., 2012) available in the GenBank database. The primers are listed in Supplementary Table 4. The PCR products were purified from agarose gels using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE healthcare). Both strands of the extracted PCR products were sequenced with an ABI 3130 Genetic Analyzer (Applied Biosystems). The sequences were assembled and edited with the MEGA version 6 software package (Tamura et al., 2004; Tamura et al., 2013). The sequences were compared with the sequences of FmoPV (M252A, 776U and 761U, Hong Kong) in the GenBank database, and were aligned by the MEGA6 program. The nucleotide and amino acid sequence identities were determined with the Bioedit version 7.1.11 software program (Hall, 1999). For the phylogenetic analysis, the nucleotide and amino acid sequences of paramyxoviruses were aligned by the clustalW and clustalW (codon) programs included in the MEGA6 software package (Felsenstein, 1985; Tamura et al., 2004; Tamura et al., 2013; Saitou and Nei, 1987). Information about the sequences used in this analysis is shown in Supplementary Table 2. A phylogenetic tree was constructed using the maximum likelihood method with the Kimura two-parameter model included in the MEGA6 package. Analysis of recombination Six complete genomes from the strains isolated in Japan and Hong Kong were aligned with the clustalW program using the MEGA6 package. To detect the probable recombination event(s), the standard similarity plot and bootscanning analysis were performed using all six sequences as queries with the SimPlot v.3.5.1 software program (Lole et al., 1999). To identify the putative recombinant sequence and the parent sequences, further analyses were implemented in the Recombination Detection Program (RDP) v.4.27 using the putative recombinants as queries (Boni et al., 2007; Gibbs et al., 2000; Holmes et al., 1999; Lemey et al., 2009; Martin and Rybicki, 2000; Martin et al., 2005; McGuire and Wright, 2000; Padidam et al., 1999; Posada, Crandall, 2001; Smith, 1992; Weiller, 1998). The sliding window size was 200 base pairs, and the step size was 20 nucleotides. For this purpose, bootstrapping tests were performed with 1000 replicates. To analyze the break-points, the maximization of ϰ2 using the MaxChi method in RDP was performed (Smith, 1992). Phylogenetic trees using the sequences on either side of the break-point and the putative recombination region were constructed with the max-
Fig. 3. The results of the RDP analyses. (A) The genomic organization of FmoPV. The putative recombination region was found within the F and H genes. The break-points are shown with arrowheads. (B) The pairwise identity was determined using MiJP003, the putative recombinant, ChJP073, the major parent, and 776U, the minor parent. The number in the upper region of the graph (black) indicates the average P value (*) calculated by the RDP. The numbers on the bottom of the graph (red) were the positions of the break-points identified by the RDP. The χ2 value of each break-point is shown in the middle part of the graph. (C) A bootscan analysis was performed using MiJP003, ChJP073 and 776U. The black dashed line indicates the cut-off of the bootstrapping test ( 4 80%). (D) The break-point distribution test was performed using the RDP. The beginning and ending break-points on either side of the break-point (red) were identified with the MaxChi program in the RDP, and are shown at the bottom of the graph, with the 99% CI. (E) Phylogenetic trees were inferred from the sequences on either side of the break-point and the putative recombinant region using six strains of FmoPV (red indicates strains from Japan and blue indicates those from Hong Kong), and two strains of CDV were used as outgroups. Phylogenetic trees were constructed by using the maximum likelihood method based on the Kimura 2-parameter model. The trees with the highest log likelihood ( 19850.9468 left, 8010.0924 middle, 25071.1108 right) are shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The trees are drawn to scale, and the scale bars indicate the branch length corresponding to 0.1 substitutions per site.
E.-S. Park et al. / Virology 468-470 (2014) 524–531
imum likelihood method based on the Kimura 2-parameter model with the MEGA6 package (Kimura, 1980). The sequences of two CDV strains were used as outgroups in the phylogenetic analysis. These trees were tested by bootstrapping with 1000 replicates. All branches supported by 480% bootstrap values were considered to be in the same group in the trees. The Shimodaira–Hasegawa test was implemented to prove whether the phylogenetic trees inferred from the different regions were significantly different using the PHYLIP 3.6a2.1 software program (Felsenstein, 1989). Acknowledgments This work was supported by a grant-in-aid from the Ministry of Health, Labor and Welfare of Japan (grants H25-shinkou-ippan008). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.09.003. References Archer, A.M., Rico-Hesse, R., 2002. High genetic divergence and recombination in arenaviruses from the Americas. Virology 304, 274–281. Baron, M.D., 2011. Rinderpest and Peste des Petits Ruminants viruses. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 293–327. Bevan, S., Sebastien, D., Veronika, von M., 2001. Canine distemper virus. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 275–284. Birch, J., Juleff, N., Heaton, M.P., Kalbfleisch, T., Kijas, J., Bailey, D., 2013. Characterization of ovine Nectin-4, a novel peste des petits ruminants virus receptor. J. Virol. 87, 4756–4761. Boni, M.F., Posada, D., Feldman, M.W., 2007. An exact nonparametric method for inferring mosaic structure in sequence triplets. Genetics 176, 1035–1047. Chare, E.R., Gould, E.A., Holmes, E.C., 2003. Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J. Gen. Virol. 84, 2691–2703. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Felsenstein, J., 1989. PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166. Furuya, T., Sassa, Y., Omatsu, T., Nagai, M., Fukushima, R., Shibutani, M., Yamaguchi, T., Uematsu, Y., Shirota, K., Mizutani, T., 2014. Existence of feline morbillivirus infection in Japanese cat populations. Arch. Virol. 159, 371–373. Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2000. Sister-Scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16, 573–582. Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2001. Recombination in the hemagglutinin gene of the 1918 “Spanish flu“. Science 293, 1842–1845. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98. Han, G.Z., He, C.Q., Ding, N.Z., Ma, L.Y., 2008a. Identification of a natural multirecombinant of Newcastle disease virus. Virology 371, 54–60. Han, G.Z., Liu, X.P., Li, S.S., 2008b. Cross-species recombination in the haemagglutinin gene of canine distemper virus. Virus Res. 136, 198–201. Han, G.Z., Worobey, M., 2011. Homologous recombination in negative sense RNA viruses. Viruses 3, 1358–1373. He, C.Q., Han, G.Z., Wang, D., Liu, W., Li, G.R., Liu, X.P., Ding, N.Z., 2008. Homologous recombination evidence in human and swine influenza A viruses. Virology 380, 12–20. He, C.Q., Xie, Z.X., Han, G.Z., Dong, J.B., Wang, D., Liu, J.B., Ma, L.Y., Tang, X.F., Liu, X.P., Pang, Y.S., Li, G.R., 2009. Homologous recombination as an evolutionary force in the avian influenza A virus. Mol. Biol. Evol. 26, 177–187. Holmes, E.C., Worobey, M., Rambaut, A., 1999. Phylogenetic evidence for recombination in dengue virus. Mol. Biol. Evol. 16, 405–409. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Lemey, P., Lott, M., Martin, D.P., Moulton, V., 2009. Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning. BMC Bioinform. 10, 126.
531
Lo, M.K., Coughlin, M.M., Rota, P.A., 2011. Hendra and Nipah viruses. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 211–234. Lole, K.S., Bollinger, R.C., Paranjape, R.S., Gadkari, D., Kulkarni, S.S., Novak, N.G., Ingersoll, R., Sheppard, H.W., Ray, S.C., 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73, 152–160. Lukashev, A.N., 2005. Evidence for recombination in Crimean-Congo hemorrhagic fever virus. J. Gen. Virol. 86, 2333–2338. Martin, D., Rybicki, E., 2000. RDP: detection of recombination amongst aligned sequences. Bioinformatics 16, 562–563. Martin, D.P., Posada, D., Crandall, K.A., Williamson, C., 2005. A modified BOOTSCAN algorithm for automated identification of recombinant sequences and recombination breakpoints. AIDS Res. Hum. Retrovir. 21, 98–102. Mazzariol, S., Peletto, S., Mondin, A., Centelleghe, C., Di, Guardo, G., DiFrancesco, C.E., Casalone, C., Acutis, P.L., 2013. Dolphin morbillivirus infection in a captive harbor seal (Phoca vitulina). J. Clin. Microbiol. 51, 708–711. McGuire, G., Wright, F., 2000. TOPAL 2.0: improved detection of mosaic sequences within multiple alignments. Bioinformatics 16, 130–134. Padidam, M., Sawyer, S., Fauquet, C.M., 1999. Possible emergence of new geminiviruses by frequent recombination. Virology 265, 218–225. Posada, D., Crandall, K.A., 2001. Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc. Natl. Acad. Sci. USA 98, 13757–13762. Roelke-Parker, M.E., Munson, L., Packer, C., Kock, R., Cleaveland, S., Carpenter, M., O'Brien, S.J., Pospischil, A., Hofmann-Lehmann, R., Lutz, H., Mwamengele, G.L., Mgasa, M.N., Machange, G.A., Summers, B.A., Appel, M.J., 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379, 441–445. Rubio-Guerri, C., Melero, M., Rivera-Arroyo, B., Bellière, E.N., Crespo, J.L., GarcíaPárraga, D., Esperón, F., Sánchez-Vizcaíno, J.M., 2013. Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay. Vet. Microbiol. 165, 109–114. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sakaguchi, S., Nakagawa, S., Yoshikawa, R., Kuwahara, C., Hagiwara, H., Asai, K., Kawakami, K., Yamamoto, Y., Ogawa, M., Miyazawa, T., 2014. Genetic diversity of feline morbilliviruses isolated in Japan. J. Gen. Virol. , http://dx.doi.org/10.1099/ vir.0.065029-0. Sakai, K., Nagata, N., Ami, Y., Seki, F., Suzaki, Y., Iwata-Yoshikawa, N., Suzuki, T., Fukushi, S., Mizutani, T., Yoshikawa, T., Otsuki, N., Kurane, I., Komase, K., Yamaguchi, R., Hasegawa, H., Saijo, M., Takeda, M., Morikawa, S., 2013a. Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008. J. Virol. 87, 1105–1114. Sakai, K., Yoshikawa, T., Seki, F., Fukushi, S., Tahara, M., Nagata, N., Ami, Y., Mizutani, T., Kurane, I., Yamaguchi, R., Hasegawa, H., Saijo, M., Komase, K., Morikawa, S., Takeda, M., 2013b. Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors. J. Virol. 87, 7170–7175. Saliki, J.T., Cooper, E.J., Gustavson, J.P., 2002. Emerging morbillivirus infections of marine mammals: development of two diagnostic approaches. Ann. NY Acad. Sci. 969, 51–59. Schneider-Schaulies, S., Paul, Duprex, W., 2011. Measles virus. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 245–266. Smith, J.M., 1992. Analyzing the mosaic structure of genes. J. Mol. Evol. 34, 126–129. Takeda, M., Tahara, M., Nagata, N., Seki, F., 2012. Wild-type measles virus is intrinsically dual-tropic. Front Microbiol., http://dx.doi.org/10.3389/fmicb.2011.00279, eCollection 2011. Tamura, K., Nei, M., Kumar, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101, 11030–11035. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Weiller, G.F., 1998. Phylogenetic profiles: a graphical method for detecting genetic recombinations in homologous sequences. Mol. Biol. Evol. 15, 326–335. Wittmann, T.J., Biek, R., Hassanin, A., Rouquet, P., Reed, P., Yaba, P., Pourrut, X., Real, L.A., Gonzalez, J.P., Leroy, E.M., 2007. Isolates of Zaire ebolavirus from wild apes reveal genetic lineage and recombinants. Proc. Natl. Acad. Sci. USA 104, 17123–17127. Woo, P.C., Lau, S.K., Wong, B.H., Fan, R.Y., Wong, A.Y., Zhang, A.J., Wu, Y., Choi, G.K., Li, K.S., Hui, J., Wang, M., Zheng, B.J., Chan, K.H., Yuen, K.Y., 2012. Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats. Proc. Natl. Acad. Sci. USA 109, 5435–5440. Yoshikawa, Y., Ochikubo, F., Matsubara, Y., Tsuruoka, H., Ishii, M., Shirota, K., Nomura, Y., Sugiyama, M., Yamanouchi, K., 1989. Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata). Vet. Microbiol. 20, 193–205. Zhang, R., Wang, X., Su, J., Zhao, J., Zhang, G., 2010. Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China. Virus Res. 151, 45–53.
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
Brief Communication
Identification of a natural recombination in the F and H genes of feline morbillivirus Eun-Sil Park a, Michio Suzuki a, Masanobu Kimura a, Keiji Maruyama b, Hiroshi Mizutani b, Ryuichi Saito b, Nami Kubota b, Tetsuya Furuya c, Tetsuya Mizutani c, Koichi Imaoka a, Shigeru Morikawa a,n a
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan Tokyo Metropolitan Animal Care and Consultation Center Jounanjima Branch Office, Tokyo 143-0002, Japan c Research and education center for prevention of global infectious diseases of animals, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Returned to author for revisions 31 July 2014 Accepted 4 September 2014
Feline morbillivirus (FmoPV) has recently been identified in Hong Kong and Japan. FmoPV is considered to belong to the genus Morbillivirus, in the family Paramyxoviridae. In this study, the complete nucleotide sequences of three strains of FmoPV detected in cats in Japan were determined. Among the six genes in FmoPV; N, P/V/C, M, F, H and L, the P gene showed the highest polymorphism in the nucleotide and putative amino acid sequences among the FmoPV strains. There was no geographical association in terms of the FmoPV phylogeny; however, from extensive phylogenetic and recombination analyses, we found that one Japanese FmoPV strain, MiJP003, was a probable recombinant between two virus strains in the independent lineages found in Japan and Hong Kong, respectively. The recombination was considered to have occurred within the F and H genes. Such recombination is thought to be involved in the evolution of FmoPV. & 2014 Elsevier Inc. All rights reserved.
Keywords: Feline morbillivirus Fusion protein Hemagglutinin protein Recombination
Introduction Morbilliviruses belong to the family Paramyxoviridae and possess a non-segment single-stranded negative RNA genome. Morbilliviruses include the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RV) and peste des petits ruminants virus (PPRV), and these viruses cause similar diseases in their respective host species. They use the SLAM (CD150) and nectin4 of their hosts as common receptors (Baron, 2011; Bevan et al., 2001; Birch et al., 2013; Schneider-Schaulies et al., 2011; Takeda et al., 2012). Recently, feline morbillivirus (FmoPV) has been newly isolated and reported in Hong Kong and Japan (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). FmoPV is phylogenetically related to other morbilliviruses, and thus is considered to belong to the genus Morbillivirus. Similar to other morbilliviruses, the genome of FmoPV encodes eight non-structural and structural proteins, which are the N, P/V/C, M, F, H and L proteins. The complete genome size of FmoPV is 16,050 bases, which is the largest among the morbilliviruses. FmoPV may be associated with
n
Corresponding author. E-mail address: [email protected] (S. Morikawa).
http://dx.doi.org/10.1016/j.virol.2014.09.003 0042-6822/& 2014 Elsevier Inc. All rights reserved.
feline tubulointerstitial nephritis, although more studies are needed to understand the virological and molecular basis of the disease (Furuya et al., 2014; Woo et al., 2012). Recently, newly emerging viruses in the genus Morbillivirus, such as porcine distemper virus (PDV) and cetacean morbillivirus (CMV), were identified (Mazzariol et al., 2013; Rubio-Guerri et al., 2013; Saliki et al., 2002). The CDV has a wide host range, including the felidae (except domestic cats), marine mammals and primates (Roelke-Parker et al., 1996, Sakai et al., 2013a, Sakai et al., 2013b; Yoshikawa et al., 1989). Indeed, PDV, CMV and CDV are reported to be genetically and antigenically related, and they have also been shown to cross-react in serological tests (Saliki et al., 2002). FmoPV is a novel morbillivirus that infects domestic cats, which have not been a natural host of the previously known morbilliviruses. The cluster of this virus is genetically different from that of CDV, but is close in the phylogenetic tree (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). It is uncertain whether FmoPV is a newly emerged virus or whether it has existed, undetected, and it is also uncertain whether it causes diseases in the other felidae. Re-assortment and non-homologous or homologous recombination have been reported to contribute to the evolution and the virulence of negative-strand RNA viruses, although the rates of these processes are controversial (Archer
E.-S. Park et al. / Virology 468-470 (2014) 524–531
and Rico-Hesse, 2002; Chare et al., 2003; Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; Han and Worobey, 2011; He et al., 2008; He et al., 2009; Lukashev, 2005; Wittmann et al., 2007; Zhang et al., 2010). To elucidate the evolution of FmoPV, it is necessary to understand its genome on the genetic and molecular bases. Therefore, in this study, we determined the whole genome sequences of three strains of FmoPV that were detected in Japan, and found a possible recombination event between two FmoPV strains from independent lineages initially isolated in Japan and Hong Kong.
Results We collected cat urine samples in Tokyo and detected FmoPV by RT-PCR (Supplementary Table 1). FmoPV was detected in 13.7% of the cat urine specimens. Among these FmoPV-positive specimens, we amplified the whole genome region by overlapping RTPCR amplicons and sequenced the full genomes of three FmoPV strains, OtJP001 (Accession no. AB924120), MiJP003 (Accession no. AB924121) and ChJP073 (Accession no. AB924122) to compare them with those reported in Hong Kong and Japan (Sakaguchi et al., 2014; Woo et al., 2012). The genome organization was also consistent with that of the previously reported strains. The genome of all three FmoPV strains possessed six genes, N, P/V/C, M, F, H and L. The degree of nucleotide and deduced amino acid identity was obtained by pairwise comparisons among the three Japanese FmoPV strains and four previously reported strains (Table 1). The P gene and the F gene showed the highest rates of nucleotide polymorphism having 88.6% to 99.3% and 88.6% to 99.2% nucleotide identities, respectively. The P (V) gene also showed the highest rate of amino acid polymorphism having 85.5–98.9% amino acid identity. Phylogenetic trees were constructed using the complete genomic and amino acid sequences of FmoPV and other paramyxoviruses deposited in the GenBank (Supplementary Table 2). The three Japanese FmoPV strains were clustered with the four reported strains, and grouped against other viruses, indicating that FmoPVs form a distinct group in the phylogenetic tree (Fig. 1). Interestingly, the phylogenetic grouping of the intragenomic region, F and H genes, of the MiJP003 strain was different from those observed for other genes (Fig. 1). Thus, the complete genome sequences of the three Japanese strains, OtJP001, MiJP003 and ChJP073, the three Hong Kong strains, M252A, 761U and 776U, and the one Japanese strain, SS1, were aligned using the MEGA6 program, and then were screened for any possible recombination events using the SimPlot and Recombination Detection Program (RDP) software platforms. When the standard similarity plots were constructed using all sequences as queries with SimPlot, the sequence of MiJP003 showed high similarity with that of ChJP073 except in the F and H genes which had high similarity with that of 776U, suggesting the presence of recombination (Fig. 2A and B). To obtain a more accurate overview of the recombination, a subsequent bootscanning analysis was carried out with a SimPlot subprogram. Bootstrap values of over 80% are considered to be significant. When the sequences of MiJP003, ChJP073 and 776U were studied as queries, reliable bootstrap values in the bootscanning analysis were produced (Fig. 2B). These results indicated the putative recombination among FmoPV strains. To obtain detailed information about such putative recombination, all sequences were studied using several methods included in the RDP. The results are shown in Fig. 3 and Supplementary Table 3. Similar to the results of the SimPlot analyses, one possible recombination signal was detected. When we further limited the analyses to the three strains, MiJP003, ChJP073 and 776U, MiJP003, a Japanese strain, was identified as the putative recombinant by
525
the RDP (Fig. 3A C). The major parental-like strain was ChJP073, another Japanese strain, and the minor parental-like strain was 776U, a Hong Kong strain, with nucleotide identities of 99.2% and 98.2%, respectively. The break-points were identified using the RDP and MaxChi methods, which are two of the methods in the RDP (Fig. 3D). The beginning break-point was located from position 5600 to 5703 in the alignment (exactly at position 5670, corresponding to the F protein, Po 0.05) and the ending breakpoint was located from position 7951 to 8030 (exactly at position 7983, corresponding to the H protein, P o0.05). The results of the RDP supported those of SimPlot, indicating that there was recombination among the FmoPV strains. To provide strong evidence of the putative recombination event, a further phylogenetic analysis was performed. The phylogenetic trees were constructed from the nucleotide sequences of the seven FmoPV strains from Japan and Hong Kong and two currently available sequences of CDV, 50Con and 011C (as outgroups), with the MEGA6 program. To avoid topological errors resulting from the putative recombination, separate phylogenetic trees were constructed from the sequences on either side of the break-point and the putative recombination region. The phylogenetic trees were delimitated by the break-points (Fig. 3E). Whereas both sides of the break-points in MiJP003, the putative recombinant, were most closely related to ChJP073, it was most closely related to 776U in the putative recombination region (Po 0.01, Shimodaira–Hasegawa test). Conflicting phylogenetic trees for different regions of the sequence supported the results of the SimPlot analysis and RDP, and provided strong evidence of recombination.
Discussion In this study, we investigated the whole genome sequences of FmoPV from cats in Japan, and detected one possible recombination event between strains from Japan and Hong Kong. To date, FmoPV has only been found in Hong Kong and Japan, and the whole genome sequences of these FmoPV strains have been reported (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). At present, it has not been confirmed whether FmoPV belongs to the genus Morbillivirus, although it was closest to the morbillivirus groups in the phylogenetic analysis (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012). It was first considered that the Hendra virus belonged to genus Morbillivirus when it had been isolated (Lo et al., 2011). Now, the Hendra virus is included in a novel genus, Henipavirus, with the Nipah virus. Thus, it is necessary to obtain more information on FmoPV to determine whether the virus belongs to the genus Morbillivirus. In this regard, we determined the complete nucleotide sequences of three Japanese FmoPV strains. The FmoPV strains detected in Japan showed high nucleotide and amino acid identities to those from Hong Kong. In the phylogenetic analysis, the FmoPV from Japan and Hong Kong formed the same group apart from other morbilliviruses. Our study and previous studies (Furuya et al., 2014; Sakaguchi et al., 2014; Woo et al., 2012) showed that FmoPV is distributed in at least Japan and Hong Kong. In addition, when we aligned the nucleotide sequences of FmoPV, we recognized that the phylogenetic grouping of the intragenomic region, the F and H genes, of a Japanese FmoPV strain, MiJP003, was different from that observed for other genes. We could detect one possible recombination event between the F and H gene regions by further recombination detection analyses. The phylogenetic analyses and statistical tests strongly supported these results. The rate of recombination in negative-strand RNA viruses is controversial, due to the possible presence of laboratory artifacts or contamination (Chare et al., 2003; Han, Worobey,
526
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Table 1 Similarities in the FmoPV strains for each protein. N Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 776U 761U M252A
OtJP001
MiJP003
ChJP073
SS1
776U
761U
M252A
0.947
0.949 0.998
0.992 0.951 0.953
0.965 0.965 0.967 0.965
0.965 0.963 0.965 0.965 0.992
0.949 0.984 0.986 0.953 0.961 0.959
0.901 0.901 0.992 0.9 0.9 0.896
0.996 0.9 0.921 0.926 0.981
0.9 0.922 0.927 0.981
0.9 0.901 0.895
0.985 0.917
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.859
0.859 0.989
0.987 0.863 0.863
0.859 0.971 0.973 0.863
0.855 0.896 0.896 0.861 0.885
0.859 0.902 0.902 0.865 0.892 0.973
0.923
P Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.901 0.9 0.993 0.901 0.886 0.888
0.993 0.901 0.984 0.924 0.931
0.901 0.984 0.924 0.931
0.901 0.886 0.888
0.921 0.928
0.982
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.943
0.94 0.997
0.997 0.94 0.937
0.94 0.997 0.994 0.937
0.961 0.967 0.964 0.958 0.964
0.952 0.961 0.958 0.949 0.958 0.988
M Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.895 0.897 0.989 0.898 0.905 0.896
0.993 0.894 0.994 0.923 0.922
0.896 0.993 0.921 0.92
0.897 0.902 0.893
0.922 0.921
0.987
OtJP001
MiJP003
ChJP073
SS1
M252A
776U
761U
0.926
0.926 0.974
0.994 0.928 0.928
0.922 0.968 0.985 0.924
0.926 0.966 0.963 0.928 0.959
0.93 0.97 0.966 0.931 0.963 0.988
F Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
0.888 0.888 0.992 0.887 0.886 0.89
0.958 0.89 0.955 0.956 0.957
0.89 0.991 0.931 0.936
0.889 0.887 0.891
0.928 0.933
0.979
H Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 SS2 SS3 M252A 776U 761U
OtJP001
0.898 0.907 0.988 0.906 0.908 0.908 0.898 0.902
MiJP003
ChJP073
SS1
SS2
SS3
M252A
776U
761U
0.932
0.937 0.964
0.986 0.932 0.937
0.936 0.957 0.964 0.936
0.941 0.968 0.993 0.941 0.968
0.941 0.968 0.986 0.941 0.968 0.989
0.932 0.971 0.952 0.932 0.949 0.952 0.952
0.942 0.981 0.963 0.942 0.959 0.963 0.963 0.989
0.952 0.897 0.939 0.954 0.95 0.96 0.965
0.906 0.952 0.994 0.985 0.927 0.93
0.906 0.907 0.906 0.898 0.902
0.954 0.952 0.925 0.927
0.987 0.928 0.932
0.927 0.929
0.988
L Amino acid Nucleotide OtJP001 MiJP003 ChJP073 SS1 M252A 776U 761U
OtJP001
0.9 0.901 0.993 0.903 0.896 0.896
MiJP003
ChJP073
SS1
M252A
776U
761U
0.96
0.962 0.992
0.997 0.961 0.962
0.963 0.988 0.991 0.963
0.962 0.972 0.974 0.961 0.973
0.96 0.969 0.971 0.959 0.97 0.993
0.993 0.9 0.983 0.932 0.932
0.901 0.984 0.933 0.932
0.903 0.896 0.896
0.933 0.932
0.985
E.-S. Park et al. / Virology 468-470 (2014) 524–531
527
Fig. 1. Phylogenetic trees based on the N, P, M, F, H and L amino acid sequences of FmoPV. Phylogenetic trees were inferred using the maximum likelihood method in the MEGA6 package. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The trees are drawn to scale, and the scale bars indicate the branch length corresponding to 0.2 substitutions per site. The three strains of FmoPV from Japan were named OtJP001, MiJP003 and ChJP073 (red). The strains from Hong Kong and other morbilliviruses are represented by blue and green, respectively. Information about the strain names and accession numbers is listed in Supplementary Table 2.
528
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Fig. 2. The results of the SimPlot analyses. (A) The genomic organization of FmoPV. The putative recombination region was found within the F and H genes. The break-points are shown with arrowheads. (B) The standard similarity plot was constructed using MiJP003 as a query for comparison with other strains, with a window size of 200 bp and a step size of 20 bp. (C) The bootscan analysis was performed using MiJP003 as a query for comparison with other strains, with a window size of 200 bp and a step size of 20 bp. The bootstrapping tests were performed with 1000 replicates.
2011). We designed new primers and re-sequenced the region including the putative recombination area, and confirmed the identical recombinant sequences (data not shown). Therefore, contamination and artifacts should not have been present. It is thought that the recombination or reassortment of the virus genome affects the evolution and virulence of the virus (Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; He et al., 2008; He et al., 2009; Wittmann et al., 2007; Zhang et al., 2010). In particular, the recombination of envelope genes has been reported in
negative-strand RNA viruses, such as CDV, influenza A virus and Newcastle disease virus (Chare et al., 2003; Gibbs et al., 2001; Han et al., 2008a; Han et al., 2008b; Zhang et al., 2010). The recombination of genes related to the entry of viruses may affect the host ranges. In the case of CDV, a putative recombination in the H gene region was found in a CDV strain isolated from a giant panda, and the recombination event was thought to confer the ability for CDV to infect different host species (Han et al., 2008b). Therefore, the recombination of FmoPV might result in changes in the host range.
E.-S. Park et al. / Virology 468-470 (2014) 524–531
Genetic typing using partial sequences of viruses has been widely performed as a means of tracing the spread of viruses, such as the Newcastle disease virus, influenza A virus and CDV (Gibbs et
529
al., 2001; Han et al., 2008a; Han et al., 2008b; He et al., 2008; He et al., 2009; Zhang et al., 2010). It is also helpful for epidemiologists to survey the origin and epidemics of the viruses. From
530
E.-S. Park et al. / Virology 468-470 (2014) 524–531
these reports, it has been emphasized that ignoring recombination might severely distort the results of the phylogenetic analysis (Han et al., 2008a; Han et al., 2008b). The present study indicates that this is also the case for FmoPV. Therefore, recombination should be considered when a phylogenetic analysis of FmoPV is performed using partial sequences. In the present study, we showed that the MiJP003 strain was a putative recombinant strain, with the parents being viruses closely related to ChJP073 and 776U. The recombination is considered to have occurred in a cat infected with two different virus strains, and this suggests that an FmoPV strain genetically closely related to the 776U strain is also distributed in Japan. A previous report indicated that FmoPV is associated with tubulointerstitial nephritis, which could be an autoimmune response caused by the infection (Furuya et al., 2014; Woo et al., 2012). However, the association of FmoPV with tubulointerstitial nephritis is not observed (Sakaguchi et al., 2014). A cat infected with MiJP003, a putative recombinant, had mild pathological lesions and clinical characterstics in this study (Supplementary Table 1). This disease could be acute, chronic or persistent. The chronic or persistent infection conditions would be favorable for the recombination of FmoPV. Given the remarkable development of the global pet business, the FmoPV strains may expand their distribution in association with this development. However, further studies are needed to better understand the virulence of FmoPV, including the identification of receptors and a survey of host animal species for FmoPV.
Materials and methods Sample collection Cat urine samples (Supplementary Table 1) were collected from Tokyo Metropolitan Animal Care and Consultation Center and were used under the approval of the Bureau of Social Welfare and Public Health of Tokyo Metropolitan Government in Japan (24-DouSou-4557). The maintenance and handling of cats were performed at the Tokyo Metropolitan Animal Care and Consultation Center (Jounanjima Branch Office) in accordance with the Act on the Welfare and Management of Animals (Article 40) of Japan. Virus RNA was extracted from the urine samples using ISOGEN (Wako, Osaka, Japan) and a precipitation carrier (Ethachin mate, Wako) and was used as the template for RT-PCR. RT-PCR of the L gene of FmoPV and DNA sequencing FmoPV was detected by amplifying a 401 bp fragment of the L gene of FmoPV. Reverse transcription (RT) was performed using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad CA, USA) with random hexamer primers, according to the manufacturer's manual. Then, two-step nested PCR was performed with sets of primers (Furuya et al., 2014). The PCR products were purified from agarose gels using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE healthcare, Buckinghamshire, UK).
Both strands of the extracted PCR products were sequenced with an ABI 3130 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). The sequences were compared with known sequences of the L genes of FmoPV in the GenBank database. Complete genome sequencing and analysis Three complete genomes, OtJP001, MiJP003 and ChJP073, determined in this study, were amplified and sequenced using virus cDNA converted from RNA using random hexamer primers as templates. The primers used to amplify separated fragments of the FmoPV whole genome were designed with reference to the conserved regions of the three FmoPV sequences (Woo et al., 2012) available in the GenBank database. The primers are listed in Supplementary Table 4. The PCR products were purified from agarose gels using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE healthcare). Both strands of the extracted PCR products were sequenced with an ABI 3130 Genetic Analyzer (Applied Biosystems). The sequences were assembled and edited with the MEGA version 6 software package (Tamura et al., 2004; Tamura et al., 2013). The sequences were compared with the sequences of FmoPV (M252A, 776U and 761U, Hong Kong) in the GenBank database, and were aligned by the MEGA6 program. The nucleotide and amino acid sequence identities were determined with the Bioedit version 7.1.11 software program (Hall, 1999). For the phylogenetic analysis, the nucleotide and amino acid sequences of paramyxoviruses were aligned by the clustalW and clustalW (codon) programs included in the MEGA6 software package (Felsenstein, 1985; Tamura et al., 2004; Tamura et al., 2013; Saitou and Nei, 1987). Information about the sequences used in this analysis is shown in Supplementary Table 2. A phylogenetic tree was constructed using the maximum likelihood method with the Kimura two-parameter model included in the MEGA6 package. Analysis of recombination Six complete genomes from the strains isolated in Japan and Hong Kong were aligned with the clustalW program using the MEGA6 package. To detect the probable recombination event(s), the standard similarity plot and bootscanning analysis were performed using all six sequences as queries with the SimPlot v.3.5.1 software program (Lole et al., 1999). To identify the putative recombinant sequence and the parent sequences, further analyses were implemented in the Recombination Detection Program (RDP) v.4.27 using the putative recombinants as queries (Boni et al., 2007; Gibbs et al., 2000; Holmes et al., 1999; Lemey et al., 2009; Martin and Rybicki, 2000; Martin et al., 2005; McGuire and Wright, 2000; Padidam et al., 1999; Posada, Crandall, 2001; Smith, 1992; Weiller, 1998). The sliding window size was 200 base pairs, and the step size was 20 nucleotides. For this purpose, bootstrapping tests were performed with 1000 replicates. To analyze the break-points, the maximization of ϰ2 using the MaxChi method in RDP was performed (Smith, 1992). Phylogenetic trees using the sequences on either side of the break-point and the putative recombination region were constructed with the max-
Fig. 3. The results of the RDP analyses. (A) The genomic organization of FmoPV. The putative recombination region was found within the F and H genes. The break-points are shown with arrowheads. (B) The pairwise identity was determined using MiJP003, the putative recombinant, ChJP073, the major parent, and 776U, the minor parent. The number in the upper region of the graph (black) indicates the average P value (*) calculated by the RDP. The numbers on the bottom of the graph (red) were the positions of the break-points identified by the RDP. The χ2 value of each break-point is shown in the middle part of the graph. (C) A bootscan analysis was performed using MiJP003, ChJP073 and 776U. The black dashed line indicates the cut-off of the bootstrapping test ( 4 80%). (D) The break-point distribution test was performed using the RDP. The beginning and ending break-points on either side of the break-point (red) were identified with the MaxChi program in the RDP, and are shown at the bottom of the graph, with the 99% CI. (E) Phylogenetic trees were inferred from the sequences on either side of the break-point and the putative recombinant region using six strains of FmoPV (red indicates strains from Japan and blue indicates those from Hong Kong), and two strains of CDV were used as outgroups. Phylogenetic trees were constructed by using the maximum likelihood method based on the Kimura 2-parameter model. The trees with the highest log likelihood ( 19850.9468 left, 8010.0924 middle, 25071.1108 right) are shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The trees are drawn to scale, and the scale bars indicate the branch length corresponding to 0.1 substitutions per site.
E.-S. Park et al. / Virology 468-470 (2014) 524–531
imum likelihood method based on the Kimura 2-parameter model with the MEGA6 package (Kimura, 1980). The sequences of two CDV strains were used as outgroups in the phylogenetic analysis. These trees were tested by bootstrapping with 1000 replicates. All branches supported by 480% bootstrap values were considered to be in the same group in the trees. The Shimodaira–Hasegawa test was implemented to prove whether the phylogenetic trees inferred from the different regions were significantly different using the PHYLIP 3.6a2.1 software program (Felsenstein, 1989). Acknowledgments This work was supported by a grant-in-aid from the Ministry of Health, Labor and Welfare of Japan (grants H25-shinkou-ippan008). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.09.003. References Archer, A.M., Rico-Hesse, R., 2002. High genetic divergence and recombination in arenaviruses from the Americas. Virology 304, 274–281. Baron, M.D., 2011. Rinderpest and Peste des Petits Ruminants viruses. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 293–327. Bevan, S., Sebastien, D., Veronika, von M., 2001. Canine distemper virus. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 275–284. Birch, J., Juleff, N., Heaton, M.P., Kalbfleisch, T., Kijas, J., Bailey, D., 2013. Characterization of ovine Nectin-4, a novel peste des petits ruminants virus receptor. J. Virol. 87, 4756–4761. Boni, M.F., Posada, D., Feldman, M.W., 2007. An exact nonparametric method for inferring mosaic structure in sequence triplets. Genetics 176, 1035–1047. Chare, E.R., Gould, E.A., Holmes, E.C., 2003. Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J. Gen. Virol. 84, 2691–2703. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Felsenstein, J., 1989. PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166. Furuya, T., Sassa, Y., Omatsu, T., Nagai, M., Fukushima, R., Shibutani, M., Yamaguchi, T., Uematsu, Y., Shirota, K., Mizutani, T., 2014. Existence of feline morbillivirus infection in Japanese cat populations. Arch. Virol. 159, 371–373. Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2000. Sister-Scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16, 573–582. Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2001. Recombination in the hemagglutinin gene of the 1918 “Spanish flu“. Science 293, 1842–1845. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98. Han, G.Z., He, C.Q., Ding, N.Z., Ma, L.Y., 2008a. Identification of a natural multirecombinant of Newcastle disease virus. Virology 371, 54–60. Han, G.Z., Liu, X.P., Li, S.S., 2008b. Cross-species recombination in the haemagglutinin gene of canine distemper virus. Virus Res. 136, 198–201. Han, G.Z., Worobey, M., 2011. Homologous recombination in negative sense RNA viruses. Viruses 3, 1358–1373. He, C.Q., Han, G.Z., Wang, D., Liu, W., Li, G.R., Liu, X.P., Ding, N.Z., 2008. Homologous recombination evidence in human and swine influenza A viruses. Virology 380, 12–20. He, C.Q., Xie, Z.X., Han, G.Z., Dong, J.B., Wang, D., Liu, J.B., Ma, L.Y., Tang, X.F., Liu, X.P., Pang, Y.S., Li, G.R., 2009. Homologous recombination as an evolutionary force in the avian influenza A virus. Mol. Biol. Evol. 26, 177–187. Holmes, E.C., Worobey, M., Rambaut, A., 1999. Phylogenetic evidence for recombination in dengue virus. Mol. Biol. Evol. 16, 405–409. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Lemey, P., Lott, M., Martin, D.P., Moulton, V., 2009. Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning. BMC Bioinform. 10, 126.
531
Lo, M.K., Coughlin, M.M., Rota, P.A., 2011. Hendra and Nipah viruses. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 211–234. Lole, K.S., Bollinger, R.C., Paranjape, R.S., Gadkari, D., Kulkarni, S.S., Novak, N.G., Ingersoll, R., Sheppard, H.W., Ray, S.C., 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73, 152–160. Lukashev, A.N., 2005. Evidence for recombination in Crimean-Congo hemorrhagic fever virus. J. Gen. Virol. 86, 2333–2338. Martin, D., Rybicki, E., 2000. RDP: detection of recombination amongst aligned sequences. Bioinformatics 16, 562–563. Martin, D.P., Posada, D., Crandall, K.A., Williamson, C., 2005. A modified BOOTSCAN algorithm for automated identification of recombinant sequences and recombination breakpoints. AIDS Res. Hum. Retrovir. 21, 98–102. Mazzariol, S., Peletto, S., Mondin, A., Centelleghe, C., Di, Guardo, G., DiFrancesco, C.E., Casalone, C., Acutis, P.L., 2013. Dolphin morbillivirus infection in a captive harbor seal (Phoca vitulina). J. Clin. Microbiol. 51, 708–711. McGuire, G., Wright, F., 2000. TOPAL 2.0: improved detection of mosaic sequences within multiple alignments. Bioinformatics 16, 130–134. Padidam, M., Sawyer, S., Fauquet, C.M., 1999. Possible emergence of new geminiviruses by frequent recombination. Virology 265, 218–225. Posada, D., Crandall, K.A., 2001. Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc. Natl. Acad. Sci. USA 98, 13757–13762. Roelke-Parker, M.E., Munson, L., Packer, C., Kock, R., Cleaveland, S., Carpenter, M., O'Brien, S.J., Pospischil, A., Hofmann-Lehmann, R., Lutz, H., Mwamengele, G.L., Mgasa, M.N., Machange, G.A., Summers, B.A., Appel, M.J., 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379, 441–445. Rubio-Guerri, C., Melero, M., Rivera-Arroyo, B., Bellière, E.N., Crespo, J.L., GarcíaPárraga, D., Esperón, F., Sánchez-Vizcaíno, J.M., 2013. Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay. Vet. Microbiol. 165, 109–114. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sakaguchi, S., Nakagawa, S., Yoshikawa, R., Kuwahara, C., Hagiwara, H., Asai, K., Kawakami, K., Yamamoto, Y., Ogawa, M., Miyazawa, T., 2014. Genetic diversity of feline morbilliviruses isolated in Japan. J. Gen. Virol. , http://dx.doi.org/10.1099/ vir.0.065029-0. Sakai, K., Nagata, N., Ami, Y., Seki, F., Suzaki, Y., Iwata-Yoshikawa, N., Suzuki, T., Fukushi, S., Mizutani, T., Yoshikawa, T., Otsuki, N., Kurane, I., Komase, K., Yamaguchi, R., Hasegawa, H., Saijo, M., Takeda, M., Morikawa, S., 2013a. Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008. J. Virol. 87, 1105–1114. Sakai, K., Yoshikawa, T., Seki, F., Fukushi, S., Tahara, M., Nagata, N., Ami, Y., Mizutani, T., Kurane, I., Yamaguchi, R., Hasegawa, H., Saijo, M., Komase, K., Morikawa, S., Takeda, M., 2013b. Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors. J. Virol. 87, 7170–7175. Saliki, J.T., Cooper, E.J., Gustavson, J.P., 2002. Emerging morbillivirus infections of marine mammals: development of two diagnostic approaches. Ann. NY Acad. Sci. 969, 51–59. Schneider-Schaulies, S., Paul, Duprex, W., 2011. Measles virus. In: Samal, SK (Ed.), The Biology of Paramyxoviruses. Caister Academic Press, Norfolk, UK, pp. 245–266. Smith, J.M., 1992. Analyzing the mosaic structure of genes. J. Mol. Evol. 34, 126–129. Takeda, M., Tahara, M., Nagata, N., Seki, F., 2012. Wild-type measles virus is intrinsically dual-tropic. Front Microbiol., http://dx.doi.org/10.3389/fmicb.2011.00279, eCollection 2011. Tamura, K., Nei, M., Kumar, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101, 11030–11035. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Weiller, G.F., 1998. Phylogenetic profiles: a graphical method for detecting genetic recombinations in homologous sequences. Mol. Biol. Evol. 15, 326–335. Wittmann, T.J., Biek, R., Hassanin, A., Rouquet, P., Reed, P., Yaba, P., Pourrut, X., Real, L.A., Gonzalez, J.P., Leroy, E.M., 2007. Isolates of Zaire ebolavirus from wild apes reveal genetic lineage and recombinants. Proc. Natl. Acad. Sci. USA 104, 17123–17127. Woo, P.C., Lau, S.K., Wong, B.H., Fan, R.Y., Wong, A.Y., Zhang, A.J., Wu, Y., Choi, G.K., Li, K.S., Hui, J., Wang, M., Zheng, B.J., Chan, K.H., Yuen, K.Y., 2012. Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats. Proc. Natl. Acad. Sci. USA 109, 5435–5440. Yoshikawa, Y., Ochikubo, F., Matsubara, Y., Tsuruoka, H., Ishii, M., Shirota, K., Nomura, Y., Sugiyama, M., Yamanouchi, K., 1989. Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata). Vet. Microbiol. 20, 193–205. Zhang, R., Wang, X., Su, J., Zhao, J., Zhang, G., 2010. Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China. Virus Res. 151, 45–53.
