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Abstract Eleven strains of duck Tembusu virus （TMUV） were isolated from diseased ducks at different duck farms in South China during 2011–2015， and their whole genomes were sequenced. The 11 isolated strains shared high sequence identity from 95.9% to 99.5%. Phylogenetic analysis revealed that all TMUV strains isolated after 2010 are clustered into two distinct branches， one branch comprising 2 Malaysian strains， and the other branch comprising all Chinese and Thai strains forming Chinese cluster 1 and Chinese cluster 2. Five of the 11 isolated strains were closely related to classical Chinese strains （BYD） belonging to Chinese cluster 1， and the remaining 6 isolated strains were closely related to strains newly isolated in South China and Thailand， belonging to Chinese cluster 2 （a mixed China-Thailand cluster）. Phylogenetic analysis of the partial E and NS5 gene of TMUVs also showed the similar results. The results collectively showed a new trend of TMUV disease that two Chinese clusters of TMUV have been concurrently circulating in South China. This study provides practical guidance for preparing vaccines against TMUV in South China.
Key words Duck; Tembusu virus; Phylogenetic analysis
Duck Tembusu virus （TMUV） is a mosquitoborne Ntaya group flavivirus in the family Flaviviridae， infecting ducks[1]， geese[2]， chickens[3]， pigeon[4]， sparrows[5]and mosquitos[6]. The main clinical signs include anorexia， depression， retarded growth， greenish diarrhea， neurological symptoms， ataxia， reluctance to walk， progressive paralysis with absent gross pathological lesions[7]. Infected egglaying ducks typically exhibit a remarkable drop in egg production， with the main pathologic changes of ovaritis， ovarian hemorrhage， ovarian atrophy and rupture[8]. The infection of TMUV caused up to 90% morbidity and mortality rate varied from 5% to 30%[8-9]. TMUV has spread fast to the major duckfarming region from eastern China to southern China since April2010[9-10]. After a few years of spreading in the field， the TMUV occurrence in layer ducks became frequent and the morbidity and mortality rate of infected broiler ducks and geese increased due to secondary bacterial infections[11-12]. In Malaysia and Thailand， TMUV rapidly spread through duck farms during the rainy season （July-December） since 2012[13]and 2013[11]， respectively. To date， though commercial vaccines have been developed， the outbreaks of this severe contagious disease among domestic fowl have resulted in greater economic losses to the poultry industry in China and Southeast Asia[11，13-14]（ Fig. 1）. TMUVs are spherical and enveloped viruses of approximately 40-60 nm in diameter. The genome consists of singlestranded， positivesense RNA of approximately 11 kb in length with a long open reading frame （ORF） that encodes a large polyprotein[15]. The polyprotein is cleaved by viral and cellular proteases into three structural proteins （C， prM/M， and E） and seven nonstructural proteins （NS1， NS2A， NS2B， NS3， NS4A， NS4B， and NS5）[15]. More than 60 genome sequences of TMUV strains have been submitted to Genbank from 2010 so far， most of them were duck origin strains， five （JS804， ZJ407， lq1， GSPT7， goose） were goose origin strains， two （CJD05， CKSD11） were chicken origin strains， each 1 were pigeon （pigeon） origin strain， house sparrow （SDHS） origin strain， Anas platyrlynels （ZJ6） origin strain and Culex （SDMS） origin strain， respectively （https：//www.ncbi.nlm.nih.gov/）. The isolated TMUV strains shared high sequence similarity and clustered tightly together regardless of isolation host， isolation year and isolation area before 2012[7]. Thai TMUV strains showed high nucleotide identity with Chinese TMUVs （97.9%）[11]. However， Malaysian TMUV strains were grouped into a subcluster apart from Thai and Chinese TMUVs[13]. In 2013， GX2013G and CQW1， isolated in South China[16-18]， was reported formed a new clade genetically divergent apart from other Chinese TMUVs， but most closely related to Thai strains on phylogenetic tree[11].
JM and FS strain were isolated in 2011 in our lab[19]， and 11 duck TMUV strains （Table 1） were isolated in South China from clinical outbreaks occurred during 2011-2015. The isolated TMUV strains were molecularly characterized by sequencing the whole genome. Sequence alignment and phylogenetic analysis of the isolated TMUV strains together with other published flavivirus strains provided useful information as to the nature of the circulating TMUV strains， and the implications of this study in the strategies for future protection of TMUV infections are also discussed.
The provinces， autonomous regions， or municipalities regions of isolated TMUVs of Chinese cluster 1 are indicated in blue; the provinces of China and Thailand regions of isolated TMUVs of Chinese cluster 2 are indicated in green; the Guangdong Province having both Chinese cluster 1 and Chinesecluster 2 are indicated in bluegreen （the merge of blue and green）; and regions of TMUVs infection outbreaks in Malaysia are indicated in gray. Materials and Methods
Virus isolation
TMUVs were isolated from diseased ducks at different duck farms located in Guangdong， China， during 2011-2015. The diseased ducks showed clinical signs including severe dropping in egg production， declining of feed uptake and pathological changes in ovary tissues of layer ducks， and greenish diarrhea， ataxia， reluctance to walk， and progressive paralysis of broiler ducks.
The heart， liver， spleen， and/or ovary tissues from the diseased ducks were homogenized in sterile phosphatebuffered saline （PBS， pH 7.2）， and then centrifuged at 3 000 g for 10 min. After filtered through a 0.22 μm syringedriven filter， the supernatants were then inoculated into allantoic cavities of 10dayold sheldrake embryonated eggs， cultured in 37 ℃ incubator. Embryos were checked daily， and the allantoic fluids were collected after 3-4 d postinoculation. Viruses were propagated by more than three passages. And the harvested allantoic fluids were frozen at -80 ℃ for RNA extraction and genome amplification. PCR detections were processed using specific primers of TMUV.
Primers for virus detection and genome sequence
Specific primers for detection of TMUVs were designed using Primer Premier 5.0 software according to the published complete genome sequence of Baiyangdian virus BYD1 from GenBank （accession number： JF312912）.
Nine pairs of overlapping primers were designed using Primer Premier 5.0 software according to Tembusu virus strain JS （GenBank. No JQ920422） to amplify the whole genome sequence of TMUVs. The expected amplification segments are about 400-2 000bp （Table 2）.
RNA extraction and RTPCR
Viral RNAs were extracted using TRIzol reagent （TaKaRa， Japan） according to the manufacturers instructions. The firststrand cDNAs were synthesized using PrimeScriptTM1stStrand cDNA Synthesis Kit （TaKaRa， Japan）. According to the manufacturers instructions， reverse transcription reaction mixture was set up as follows： 1 μl of Random 6 mers （50 mM）， 1 μl of 10 mmol/L dNTPmix， 2 μl of RNA extract， 6 μl RNase freed H2O. The reaction mixture was incubated at 65 ℃ for 5 min， and then quickly frozen on ice. After cooling， reagents were added in the same tube as follows： 4 μl 5×PrimeScript Buffer， 0.5 μl of 40 U/μl RNase inhibitor， 1 μl of 200 U/μl PrimeScript Rtase， 4.5 μl Rnase free dH2O. The total volume of reverse transcription reaction mixture was 20 μl; the reaction was carried out at 30 ℃ for 10 min， 42 ℃ for 30 min， 70 ℃ for 15 min. About 1 μl of the cDNA was used for amplification in PCR reactions. The PCR reactions （total volume of 50 μl） contained 5 μl of 10×PCR buffer， 4 μl of 2.5mmol/L dNTP， 2 μl of 10 mmol/L of each of the two primers， and 0.25 μl of 2 U/μl TaqDNA polymerase. The PCR conditions for amplification were 94 ℃ for 3 min， 30 cycles of 94 ℃ for 30 s， 52 ℃ for 30 s， and 72 ℃ for 2 min， followed by 72 ℃ for 10 min. The products were analyzed on 1.0% agarose gel. DNA cloning
PCR products of each RTPCR were purified using Axy PrepTM DNA Gel Extraction Kit （AXYGEN）. Purified PCR products ligated with a TA cloning vector， pMD18T （TaKaRa， Japan）， were transformed into DH5α E. coli competent cells. Cells carrying recombinant plasmid were screened on Luriabertani （LB） agar plates containing Ampicillin （50 μg/ml）. Positive clones were initially screened from three different PCRs through blue and white colonies; then the inserts were amplified by PCR which had the same conditions as that for the abovementioned PCR amplification. The verified recombinant plasmid DNAs were used for sequencing.
Gene sequencing and analysis
The purified recombinant plasmids were sequenced by Invitrogen Trading （Shanghai） Co.， Ltd. Translation of the nucleotide sequence and sequence alignments were performed by the DNAStar software （DNASTAR Inc.， USA）. Multiplesequence alignments were performed and analyzed using ClustalX 1.83 software. Phylogenetic analysis was performed with the neighborjoining method using MEGA （version 5.2） software. The bootstrap values were determined from 1 000 replicates of the original data.
Nucleotide sequence accession number
The whole genome sequences of the 11 isolated TMUV strains described in this study were submitted to the GenBank database with the accession numbers KX686570KX686580， respectively.
Linlin LI et al. Phylogenetic Analysis Revealed Concurrent Circulation of Two Clusters of Duck Tembusu Virus in South China
Results
Eleven strains of duck tembusu virus were isolated from diseased ducks from South China
Diseased ducks showing ovaritis， ovarian hemorrhage and regression， even ovarian follicles rupture in layer ducks and greenish diarrhea， neurological symptoms， ataxia， reluctance to walk， and progressive paralysis in broiler ducks were collected from different duck farms from South China in the period of 2011-2015. Their heart， liver， spleen， and/or ovary tissues were processed for virus isolation using sheldrake embryonated eggs. By RTPCR detection， 11 strains of TMUV were successfully isolated， eight from layer ducks and 3 from broiler ducks （Table 1）. The whole genome sequences of 11 isolated strains were amplified by 9 different pairs of overlapping primers of TMUV.
Eleven isolated strains share high sequence similarity
The whole genome sequences of the 11 isolated strains were sequenced， 3 strains comprising 10 990 nucleotides， 6 strains comprising 10 991 nucleotides， 2 strains comprising 10 992 nucleotides. All 11 strains have an ORF encoding 3 425 amino acids （aa）. The 5′terminal and 3′terminal noncoding regions were 93-95 and 618-619 nucleotides， respectively. Sequence similarities among these 11 isolated strains were 95.9%-99.5%. Phylogenetic analyses show that 11 isolated strains belong to two Chinese clusters of TMUVs
Phylogenetic analyses based on the whole genome sequences were performed consisting of 11 isolated strains and 68 reference strains of TMUVs and Ntaya group Flavivirus. All TMUV strains isolated after 2010 were clustered into two distinct branchs， 2 Malaysian strains were formed a small branch， and all Chinese and Thai strains were formed a big branch above the phylogenetic tree （Fig. 2A）， in which， 5 isolated strains were closely clustered to classical Chinese strains （BYD， JS804） as Chinese cluster 1 （a clustercontaining duck， goose， chicken， pigeon， house sparrow， anas platyrlynels， mosquito origin TMUVs from China）， and 6 isolated strains were closely related to Chinese strains isolated in Guangxi （GX2013C， GX2013E， GX2013G， GX2013H）， Chongqing（CQW1） and Thai strains（DK/TH/CU1， KPS54A61/THA） as Chinese cluster 2 （a mixed China-Thailand cluster）. TMUV strain MM1775 and sitiawan， isolated before 2010， were clustered into a small branch which distinct far from the big branch of TMUVs isolated after 2010. Overall， the phylogenic relationships among isolates did not depend on the isolated host， area and year.
Phylogenetic analysis based on the nucleotide sequences of the partial E and NS5 gene were performed comparing with representative flavivirus obtained from genBank.
The nucleotide sequences of the partial E and NS5 gene of the 11 isolated strains were tightly clustered into a big branch on the phylogenetic tree （Fig. 2B and Fig. 2C） which divided into Chinese cluster 1 and Chinese cluster 2.4 Malaysian strains isolated in 2012 were clustered into a branch distinct far from Chinese genotypes， the rest TMUV strains isolated in Thailand and Malaysian before 2010 were clustered closely to a branch below the phylogenetic tree （Fig. 2B and Fig. 2C）.
Alignment analysis of deduced amino acid sequence of 11 strains between two clusters
Alignment analysis of deduced amino acid sequences of the entire coding sequence of these 11 isolated strains were performed， showing amino acid similarities of 98.4%-99.8%. More high variable regions were found located among NS protein （NS1NS5）， especially NS1， only 3 high variable sites were found amongstructure protein. Detailed amino acid changes in complete genomic sequence among 11 isolated strains between Chinese cluster 1 and Chinese cluster 2 were showed in table 3.
The 11 isolates were marked with anteblack triangle， and 2 isolates preserved in our lab （JM and FS） were marked with anteblack circle. The nucleotide sequences were aligned by using clustalx l.83. The phylogenetic trees were constructed in MEGA version 5.2 by using the neighborjoining algorithm with the Kimura2 parameter model applied to 1 000 replications of bootstrap.
Fig. 2 Phylogenetic tree of the whole genome sequences constructed by neighborjoining method （Mega 5.2） （A）， partial envelope gene （941 bp） （B）， and partial nonstructural 5 gene （900 bp） （C） of Tembusu viruses isolated in China， Southeast Asia and selected reference strains of flaviviruse
Discussion
TMUV belongs to the Ntaya group of mosquitoborne Flaviviruses. As a member of the flavivirus genus， TMUV has a high potential to become a zoonotic pathogen that threatens public health[20]. The history of TMUV can be traced back to 1955. TMUV strain MM1775 was originally isolated from mosquitoes of the genus Culex in Malaysia[21]. Then， Sitiawan virus， isolated from sick chickens in Malaysia， was one of the first Tembusu virus strains reported to cause disease in domestic poultry[22]. After ten years of latent， duck TMUVs has spread fast to the major duckfarming region in China[23]and strain BYD1 becomes classical as the firstly reported duckorigin Tembusu virus[10]. Nevertheless， It was reported that duck TMUVs was isolated in Thailand in 2007[24]. To date， the diseases have been reported in China[14，25]， Thailand[11]and Malaysia[13]. The report of detection of both antibodies and viral RNA to TUMV in duck industry workers in China indicates possible infection in human[20]， thus it is urgent to track and understand the nature of the circulating TMUV strains.
Most of the reports showed that TMUV strains isolated in Chinawere clustered tightly forming one genotype[3，19]， However， the CQW1 isolate was reported most closely related to GX2013H[18]， and these two isolates formed a separate clade distinct from other TMUV isolates. Furthermore， it was reported that 38 TMUV China isolates between 2010 and 2014 were clustered into two lineages， with lineage II being further divided into three sublineages， IIa， b， and c， and that sublineage IIc became the dominant lineage currently circulating[4].
In this paper， phylogenetic analysis of the whole genome sequences， partial E and NS5 gene revealed that all Chinese TMUV were clustered into two clusters， Chinese cluster 1 （a classical duck TMUV cluster） and Chinese cluster 2 （a mixed ChinaThailand TMUV cluster）. Besides strains CQW1 and GX2013H， the previously reported genome sequence of Chinese TMUV strains all belonged to Chinese cluster 1， while Chinese cluster 2 contained Chinese TMUV strains only isolated in South China. Surprisingly， our phylogenetic analysis reveals that two clusters of TMUVs are concurrently circulating in South China. This finding has two important implications. First， whether South China with two clusters of strains circulating at the same time will serve as a breeding ground for new TMUV clusters. This will be investigated in continuityin the future. Second， with great practical significance， the development of a TMUV vaccine to be used in the South China has to consider of including two strains from these two clusters respectively. Acknowledgement
The funders had no role in study design， data collection and analysis， decision to publish， or preparation of the manuscript. We thank to Dr. George D. Liu and Dr. Yun Zhang for critical review and revision of the manuscript.
References
[1] SHEN HQ， LIN WC， WANG ZX， et al. Pathogenicity and genetic characterization of a duck Tembusu virus associated with eggdropping in Muscovy ducks[J]. Virus Res， 2016， 223： 52-56.
[2] TI J， ZHANG L， LI Z， et al. Effect of age and inoculation route on the infection of duck Tembusu virus in Goslings[J]. Vet Microbiol， 2015， 181 （3-4）： 190-197.
[3] CHEN S， WANG S， LI Z， et al. Isolation and characterization of a Chinese strain of Tembusu virus from Hyline Brown layers with acute eggdrop syndrome in Fujian， China[J]. Arch Virol， 2014， 159 （5）： 1099-1107.
[4] DAI L， LI Z， TAO P. Evolutionary analysis of Tembusu virus： Evidence for the emergence of a dominant genotype[J]. Infect Genet Evol， 2015， 32： 124-129.
[5] TANG Y， DIAO Y， YU C， et al. Characterization of a Tembusu virus isolated from naturally infected house sparrows （Passer domesticus） in Northern China[J]. Transbound Emerg Dis， 2013， 60 （2）： 152-158.
[6] TANG Y， DIAO Y， CHEN H， et al. Isolation and genetic characterization of a Tembusu virus strain isolated from mosquitoes in Shandong， China[J]. Transbound Emerg Dis， 2015， 62（2）： 209-216.
[7] YUN T， YE W， NI Z， et al. Identification and molecular characterization of a novel flavivirus isolated from Pekin ducklings in China[J]. Vet Microbiol， 2012，157 （3-4）： 311-319.
[8] WANG J， LIU W， MENG G， et al. Isolation and genome characterization of a novel duck Tembusu virus with a 74 nucleotide insertion in the 3′ nontranslated region[J]. Avian Pathol， 2015， 44 （2）： 92-102.
[9] YAN P， ZHAO Y， ZHANG X， et al. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China[J]. Virology， 2011， 417 （1）： 1-8.
[10] SU J， LI S， HU X， et al. Duck eggdrop syndrome caused by BYD virus， a new Tembusurelated flavivirus[J]. PLoS One， 2011， 6 （3）： e18106.
[11] THONTIRAVONG A， NINVILAI P， TUNTERAK W， et al. Tembusurelated Flavivirus in Ducks， Thailand[J]. Emerg Infect Dis， 2015， 21 （12）： 2164-2167.
[12] ZHAO D， HUANG X， HAN K， et al. Protective immune response against newly emerging goose tembusu virus infection induced by immunization with a recombinant envelope protein[J]. Lett Appl Microbiol， 2015， 61 （4）： 318-324. [13] HOMONNAY ZG， KOVACS EW， BANYAI K， et al. Tembusulike Flavivirus （Perak virus） as the cause of neurological disease outbreaks in young Pekin ducks[J]. Avian Pathol， 2014， 43 （6）： 552-560.
[14] LU Y， DOU Y， TI J， et al. The effect of Tembusu virus infection in different weekold Cherry Valley breeding ducks[J]. Vet Microbiol， 2016， 192： 167-174.
[15] LIU P， LU H， LI S， et al. Duck egg drop syndrome virus： an emerging Tembusurelated flavivirus in China[J]. Sci China Life Sci， 2013， 56 （8）： 701-710.
[16] XIE Z， ZENG T， XIE L， et al. Genome Analysis of a Tembusu Virus， GX2013H， Isolated from a Cheery Valley Duck in Guangxi， China[J]. Genome Announc， 2014， 2（4）： e00466-14.
[17] ZENG T， XIE Z， XIE L， et al. Identification and wholegenome sequence analysis of Tembusu virus GX2013G， isolated from a cherry valley duckling in southern China[J]. Genome Announc， 2015， 3（1）： e00007-15.
[18] ZHU K， HUANG J， JIA R， et al. Identification and molecular characterization of a novel duck Tembusu virus isolate from Southwest China[J]. Arch Virol， 2015，160 （11）： 2781-2790.
[19] LI L， AN H， SUN M， et al. Identification and genomic analysis of two duckorigin Tembusu virus strains in southern China[J]. Virus Genes， 2012， 45 （1）： 105-112.
[20]. TANG Y， GAO X， DIAO Y， et al. Tembusu virus in human， China[J]. Transbound Emerg Dis， 2013， 60 （3）： 193-196.
[21] KUNO G， CHANG GJ， TSUCHIYA KR， et al. Phylogeny of the genus Flavivirus[J]. J Virol， 1998， 72 （1）： 73-83.
[22] KONO Y， TSUKAMOTO K， ABD HAMID M， et al. Encephalitis and retarded growth of chicks caused by Sitiawan virus， a new isolate belonging to the genus Flavivirus[J]. Am J Trop Med Hyg， 2000， 63 （1-2）： 94-101.
[23] ZHANG Y， LI X， CHEN H， et al. Evidence of possible vertical transmission of Tembusu virus in ducks[J]. Vet Microbiol， 2015，179 （3-4）： 149-154.
[24] NINVILAI P， NONTHABENJAWAN N， LIMCHAROEN B， et al. The presence of duck Tembusu virus in Thailand since 2007： A retrospective study[J].Transbound Emerg Dis， 2018， 65（5）： 1208-1216.
[25] YAN Z， SHEN H， WANG Z， et al. Isolation and characterization of a novel Tembusu virus circulating in muscovy ducks in South China[J]. Transbound Emerg Dis， 2017， 64（5）： e15-e17.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words Duck; Tembusu virus; Phylogenetic analysis
Duck Tembusu virus （TMUV） is a mosquitoborne Ntaya group flavivirus in the family Flaviviridae， infecting ducks[1]， geese[2]， chickens[3]， pigeon[4]， sparrows[5]and mosquitos[6]. The main clinical signs include anorexia， depression， retarded growth， greenish diarrhea， neurological symptoms， ataxia， reluctance to walk， progressive paralysis with absent gross pathological lesions[7]. Infected egglaying ducks typically exhibit a remarkable drop in egg production， with the main pathologic changes of ovaritis， ovarian hemorrhage， ovarian atrophy and rupture[8]. The infection of TMUV caused up to 90% morbidity and mortality rate varied from 5% to 30%[8-9]. TMUV has spread fast to the major duckfarming region from eastern China to southern China since April2010[9-10]. After a few years of spreading in the field， the TMUV occurrence in layer ducks became frequent and the morbidity and mortality rate of infected broiler ducks and geese increased due to secondary bacterial infections[11-12]. In Malaysia and Thailand， TMUV rapidly spread through duck farms during the rainy season （July-December） since 2012[13]and 2013[11]， respectively. To date， though commercial vaccines have been developed， the outbreaks of this severe contagious disease among domestic fowl have resulted in greater economic losses to the poultry industry in China and Southeast Asia[11，13-14]（ Fig. 1）. TMUVs are spherical and enveloped viruses of approximately 40-60 nm in diameter. The genome consists of singlestranded， positivesense RNA of approximately 11 kb in length with a long open reading frame （ORF） that encodes a large polyprotein[15]. The polyprotein is cleaved by viral and cellular proteases into three structural proteins （C， prM/M， and E） and seven nonstructural proteins （NS1， NS2A， NS2B， NS3， NS4A， NS4B， and NS5）[15]. More than 60 genome sequences of TMUV strains have been submitted to Genbank from 2010 so far， most of them were duck origin strains， five （JS804， ZJ407， lq1， GSPT7， goose） were goose origin strains， two （CJD05， CKSD11） were chicken origin strains， each 1 were pigeon （pigeon） origin strain， house sparrow （SDHS） origin strain， Anas platyrlynels （ZJ6） origin strain and Culex （SDMS） origin strain， respectively （https：//www.ncbi.nlm.nih.gov/）. The isolated TMUV strains shared high sequence similarity and clustered tightly together regardless of isolation host， isolation year and isolation area before 2012[7]. Thai TMUV strains showed high nucleotide identity with Chinese TMUVs （97.9%）[11]. However， Malaysian TMUV strains were grouped into a subcluster apart from Thai and Chinese TMUVs[13]. In 2013， GX2013G and CQW1， isolated in South China[16-18]， was reported formed a new clade genetically divergent apart from other Chinese TMUVs， but most closely related to Thai strains on phylogenetic tree[11].
JM and FS strain were isolated in 2011 in our lab[19]， and 11 duck TMUV strains （Table 1） were isolated in South China from clinical outbreaks occurred during 2011-2015. The isolated TMUV strains were molecularly characterized by sequencing the whole genome. Sequence alignment and phylogenetic analysis of the isolated TMUV strains together with other published flavivirus strains provided useful information as to the nature of the circulating TMUV strains， and the implications of this study in the strategies for future protection of TMUV infections are also discussed.
The provinces， autonomous regions， or municipalities regions of isolated TMUVs of Chinese cluster 1 are indicated in blue; the provinces of China and Thailand regions of isolated TMUVs of Chinese cluster 2 are indicated in green; the Guangdong Province having both Chinese cluster 1 and Chinesecluster 2 are indicated in bluegreen （the merge of blue and green）; and regions of TMUVs infection outbreaks in Malaysia are indicated in gray. Materials and Methods
Virus isolation
TMUVs were isolated from diseased ducks at different duck farms located in Guangdong， China， during 2011-2015. The diseased ducks showed clinical signs including severe dropping in egg production， declining of feed uptake and pathological changes in ovary tissues of layer ducks， and greenish diarrhea， ataxia， reluctance to walk， and progressive paralysis of broiler ducks.
The heart， liver， spleen， and/or ovary tissues from the diseased ducks were homogenized in sterile phosphatebuffered saline （PBS， pH 7.2）， and then centrifuged at 3 000 g for 10 min. After filtered through a 0.22 μm syringedriven filter， the supernatants were then inoculated into allantoic cavities of 10dayold sheldrake embryonated eggs， cultured in 37 ℃ incubator. Embryos were checked daily， and the allantoic fluids were collected after 3-4 d postinoculation. Viruses were propagated by more than three passages. And the harvested allantoic fluids were frozen at -80 ℃ for RNA extraction and genome amplification. PCR detections were processed using specific primers of TMUV.
Primers for virus detection and genome sequence
Specific primers for detection of TMUVs were designed using Primer Premier 5.0 software according to the published complete genome sequence of Baiyangdian virus BYD1 from GenBank （accession number： JF312912）.
Nine pairs of overlapping primers were designed using Primer Premier 5.0 software according to Tembusu virus strain JS （GenBank. No JQ920422） to amplify the whole genome sequence of TMUVs. The expected amplification segments are about 400-2 000bp （Table 2）.
RNA extraction and RTPCR
Viral RNAs were extracted using TRIzol reagent （TaKaRa， Japan） according to the manufacturers instructions. The firststrand cDNAs were synthesized using PrimeScriptTM1stStrand cDNA Synthesis Kit （TaKaRa， Japan）. According to the manufacturers instructions， reverse transcription reaction mixture was set up as follows： 1 μl of Random 6 mers （50 mM）， 1 μl of 10 mmol/L dNTPmix， 2 μl of RNA extract， 6 μl RNase freed H2O. The reaction mixture was incubated at 65 ℃ for 5 min， and then quickly frozen on ice. After cooling， reagents were added in the same tube as follows： 4 μl 5×PrimeScript Buffer， 0.5 μl of 40 U/μl RNase inhibitor， 1 μl of 200 U/μl PrimeScript Rtase， 4.5 μl Rnase free dH2O. The total volume of reverse transcription reaction mixture was 20 μl; the reaction was carried out at 30 ℃ for 10 min， 42 ℃ for 30 min， 70 ℃ for 15 min. About 1 μl of the cDNA was used for amplification in PCR reactions. The PCR reactions （total volume of 50 μl） contained 5 μl of 10×PCR buffer， 4 μl of 2.5mmol/L dNTP， 2 μl of 10 mmol/L of each of the two primers， and 0.25 μl of 2 U/μl TaqDNA polymerase. The PCR conditions for amplification were 94 ℃ for 3 min， 30 cycles of 94 ℃ for 30 s， 52 ℃ for 30 s， and 72 ℃ for 2 min， followed by 72 ℃ for 10 min. The products were analyzed on 1.0% agarose gel. DNA cloning
PCR products of each RTPCR were purified using Axy PrepTM DNA Gel Extraction Kit （AXYGEN）. Purified PCR products ligated with a TA cloning vector， pMD18T （TaKaRa， Japan）， were transformed into DH5α E. coli competent cells. Cells carrying recombinant plasmid were screened on Luriabertani （LB） agar plates containing Ampicillin （50 μg/ml）. Positive clones were initially screened from three different PCRs through blue and white colonies; then the inserts were amplified by PCR which had the same conditions as that for the abovementioned PCR amplification. The verified recombinant plasmid DNAs were used for sequencing.
Gene sequencing and analysis
The purified recombinant plasmids were sequenced by Invitrogen Trading （Shanghai） Co.， Ltd. Translation of the nucleotide sequence and sequence alignments were performed by the DNAStar software （DNASTAR Inc.， USA）. Multiplesequence alignments were performed and analyzed using ClustalX 1.83 software. Phylogenetic analysis was performed with the neighborjoining method using MEGA （version 5.2） software. The bootstrap values were determined from 1 000 replicates of the original data.
Nucleotide sequence accession number
The whole genome sequences of the 11 isolated TMUV strains described in this study were submitted to the GenBank database with the accession numbers KX686570KX686580， respectively.
Linlin LI et al. Phylogenetic Analysis Revealed Concurrent Circulation of Two Clusters of Duck Tembusu Virus in South China
Results
Eleven strains of duck tembusu virus were isolated from diseased ducks from South China
Diseased ducks showing ovaritis， ovarian hemorrhage and regression， even ovarian follicles rupture in layer ducks and greenish diarrhea， neurological symptoms， ataxia， reluctance to walk， and progressive paralysis in broiler ducks were collected from different duck farms from South China in the period of 2011-2015. Their heart， liver， spleen， and/or ovary tissues were processed for virus isolation using sheldrake embryonated eggs. By RTPCR detection， 11 strains of TMUV were successfully isolated， eight from layer ducks and 3 from broiler ducks （Table 1）. The whole genome sequences of 11 isolated strains were amplified by 9 different pairs of overlapping primers of TMUV.
Eleven isolated strains share high sequence similarity
The whole genome sequences of the 11 isolated strains were sequenced， 3 strains comprising 10 990 nucleotides， 6 strains comprising 10 991 nucleotides， 2 strains comprising 10 992 nucleotides. All 11 strains have an ORF encoding 3 425 amino acids （aa）. The 5′terminal and 3′terminal noncoding regions were 93-95 and 618-619 nucleotides， respectively. Sequence similarities among these 11 isolated strains were 95.9%-99.5%. Phylogenetic analyses show that 11 isolated strains belong to two Chinese clusters of TMUVs
Phylogenetic analyses based on the whole genome sequences were performed consisting of 11 isolated strains and 68 reference strains of TMUVs and Ntaya group Flavivirus. All TMUV strains isolated after 2010 were clustered into two distinct branchs， 2 Malaysian strains were formed a small branch， and all Chinese and Thai strains were formed a big branch above the phylogenetic tree （Fig. 2A）， in which， 5 isolated strains were closely clustered to classical Chinese strains （BYD， JS804） as Chinese cluster 1 （a clustercontaining duck， goose， chicken， pigeon， house sparrow， anas platyrlynels， mosquito origin TMUVs from China）， and 6 isolated strains were closely related to Chinese strains isolated in Guangxi （GX2013C， GX2013E， GX2013G， GX2013H）， Chongqing（CQW1） and Thai strains（DK/TH/CU1， KPS54A61/THA） as Chinese cluster 2 （a mixed China-Thailand cluster）. TMUV strain MM1775 and sitiawan， isolated before 2010， were clustered into a small branch which distinct far from the big branch of TMUVs isolated after 2010. Overall， the phylogenic relationships among isolates did not depend on the isolated host， area and year.
Phylogenetic analysis based on the nucleotide sequences of the partial E and NS5 gene were performed comparing with representative flavivirus obtained from genBank.
The nucleotide sequences of the partial E and NS5 gene of the 11 isolated strains were tightly clustered into a big branch on the phylogenetic tree （Fig. 2B and Fig. 2C） which divided into Chinese cluster 1 and Chinese cluster 2.4 Malaysian strains isolated in 2012 were clustered into a branch distinct far from Chinese genotypes， the rest TMUV strains isolated in Thailand and Malaysian before 2010 were clustered closely to a branch below the phylogenetic tree （Fig. 2B and Fig. 2C）.
Alignment analysis of deduced amino acid sequence of 11 strains between two clusters
Alignment analysis of deduced amino acid sequences of the entire coding sequence of these 11 isolated strains were performed， showing amino acid similarities of 98.4%-99.8%. More high variable regions were found located among NS protein （NS1NS5）， especially NS1， only 3 high variable sites were found amongstructure protein. Detailed amino acid changes in complete genomic sequence among 11 isolated strains between Chinese cluster 1 and Chinese cluster 2 were showed in table 3.
The 11 isolates were marked with anteblack triangle， and 2 isolates preserved in our lab （JM and FS） were marked with anteblack circle. The nucleotide sequences were aligned by using clustalx l.83. The phylogenetic trees were constructed in MEGA version 5.2 by using the neighborjoining algorithm with the Kimura2 parameter model applied to 1 000 replications of bootstrap.
Fig. 2 Phylogenetic tree of the whole genome sequences constructed by neighborjoining method （Mega 5.2） （A）， partial envelope gene （941 bp） （B）， and partial nonstructural 5 gene （900 bp） （C） of Tembusu viruses isolated in China， Southeast Asia and selected reference strains of flaviviruse
Discussion
TMUV belongs to the Ntaya group of mosquitoborne Flaviviruses. As a member of the flavivirus genus， TMUV has a high potential to become a zoonotic pathogen that threatens public health[20]. The history of TMUV can be traced back to 1955. TMUV strain MM1775 was originally isolated from mosquitoes of the genus Culex in Malaysia[21]. Then， Sitiawan virus， isolated from sick chickens in Malaysia， was one of the first Tembusu virus strains reported to cause disease in domestic poultry[22]. After ten years of latent， duck TMUVs has spread fast to the major duckfarming region in China[23]and strain BYD1 becomes classical as the firstly reported duckorigin Tembusu virus[10]. Nevertheless， It was reported that duck TMUVs was isolated in Thailand in 2007[24]. To date， the diseases have been reported in China[14，25]， Thailand[11]and Malaysia[13]. The report of detection of both antibodies and viral RNA to TUMV in duck industry workers in China indicates possible infection in human[20]， thus it is urgent to track and understand the nature of the circulating TMUV strains.
Most of the reports showed that TMUV strains isolated in Chinawere clustered tightly forming one genotype[3，19]， However， the CQW1 isolate was reported most closely related to GX2013H[18]， and these two isolates formed a separate clade distinct from other TMUV isolates. Furthermore， it was reported that 38 TMUV China isolates between 2010 and 2014 were clustered into two lineages， with lineage II being further divided into three sublineages， IIa， b， and c， and that sublineage IIc became the dominant lineage currently circulating[4].
In this paper， phylogenetic analysis of the whole genome sequences， partial E and NS5 gene revealed that all Chinese TMUV were clustered into two clusters， Chinese cluster 1 （a classical duck TMUV cluster） and Chinese cluster 2 （a mixed ChinaThailand TMUV cluster）. Besides strains CQW1 and GX2013H， the previously reported genome sequence of Chinese TMUV strains all belonged to Chinese cluster 1， while Chinese cluster 2 contained Chinese TMUV strains only isolated in South China. Surprisingly， our phylogenetic analysis reveals that two clusters of TMUVs are concurrently circulating in South China. This finding has two important implications. First， whether South China with two clusters of strains circulating at the same time will serve as a breeding ground for new TMUV clusters. This will be investigated in continuityin the future. Second， with great practical significance， the development of a TMUV vaccine to be used in the South China has to consider of including two strains from these two clusters respectively. Acknowledgement
The funders had no role in study design， data collection and analysis， decision to publish， or preparation of the manuscript. We thank to Dr. George D. Liu and Dr. Yun Zhang for critical review and revision of the manuscript.
References
[1] SHEN HQ， LIN WC， WANG ZX， et al. Pathogenicity and genetic characterization of a duck Tembusu virus associated with eggdropping in Muscovy ducks[J]. Virus Res， 2016， 223： 52-56.
[2] TI J， ZHANG L， LI Z， et al. Effect of age and inoculation route on the infection of duck Tembusu virus in Goslings[J]. Vet Microbiol， 2015， 181 （3-4）： 190-197.
[3] CHEN S， WANG S， LI Z， et al. Isolation and characterization of a Chinese strain of Tembusu virus from Hyline Brown layers with acute eggdrop syndrome in Fujian， China[J]. Arch Virol， 2014， 159 （5）： 1099-1107.
[4] DAI L， LI Z， TAO P. Evolutionary analysis of Tembusu virus： Evidence for the emergence of a dominant genotype[J]. Infect Genet Evol， 2015， 32： 124-129.
[5] TANG Y， DIAO Y， YU C， et al. Characterization of a Tembusu virus isolated from naturally infected house sparrows （Passer domesticus） in Northern China[J]. Transbound Emerg Dis， 2013， 60 （2）： 152-158.
[6] TANG Y， DIAO Y， CHEN H， et al. Isolation and genetic characterization of a Tembusu virus strain isolated from mosquitoes in Shandong， China[J]. Transbound Emerg Dis， 2015， 62（2）： 209-216.
[7] YUN T， YE W， NI Z， et al. Identification and molecular characterization of a novel flavivirus isolated from Pekin ducklings in China[J]. Vet Microbiol， 2012，157 （3-4）： 311-319.
[8] WANG J， LIU W， MENG G， et al. Isolation and genome characterization of a novel duck Tembusu virus with a 74 nucleotide insertion in the 3′ nontranslated region[J]. Avian Pathol， 2015， 44 （2）： 92-102.
[9] YAN P， ZHAO Y， ZHANG X， et al. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China[J]. Virology， 2011， 417 （1）： 1-8.
[10] SU J， LI S， HU X， et al. Duck eggdrop syndrome caused by BYD virus， a new Tembusurelated flavivirus[J]. PLoS One， 2011， 6 （3）： e18106.
[11] THONTIRAVONG A， NINVILAI P， TUNTERAK W， et al. Tembusurelated Flavivirus in Ducks， Thailand[J]. Emerg Infect Dis， 2015， 21 （12）： 2164-2167.
[12] ZHAO D， HUANG X， HAN K， et al. Protective immune response against newly emerging goose tembusu virus infection induced by immunization with a recombinant envelope protein[J]. Lett Appl Microbiol， 2015， 61 （4）： 318-324. [13] HOMONNAY ZG， KOVACS EW， BANYAI K， et al. Tembusulike Flavivirus （Perak virus） as the cause of neurological disease outbreaks in young Pekin ducks[J]. Avian Pathol， 2014， 43 （6）： 552-560.
[14] LU Y， DOU Y， TI J， et al. The effect of Tembusu virus infection in different weekold Cherry Valley breeding ducks[J]. Vet Microbiol， 2016， 192： 167-174.
[15] LIU P， LU H， LI S， et al. Duck egg drop syndrome virus： an emerging Tembusurelated flavivirus in China[J]. Sci China Life Sci， 2013， 56 （8）： 701-710.
[16] XIE Z， ZENG T， XIE L， et al. Genome Analysis of a Tembusu Virus， GX2013H， Isolated from a Cheery Valley Duck in Guangxi， China[J]. Genome Announc， 2014， 2（4）： e00466-14.
[17] ZENG T， XIE Z， XIE L， et al. Identification and wholegenome sequence analysis of Tembusu virus GX2013G， isolated from a cherry valley duckling in southern China[J]. Genome Announc， 2015， 3（1）： e00007-15.
[18] ZHU K， HUANG J， JIA R， et al. Identification and molecular characterization of a novel duck Tembusu virus isolate from Southwest China[J]. Arch Virol， 2015，160 （11）： 2781-2790.
[19] LI L， AN H， SUN M， et al. Identification and genomic analysis of two duckorigin Tembusu virus strains in southern China[J]. Virus Genes， 2012， 45 （1）： 105-112.
[20]. TANG Y， GAO X， DIAO Y， et al. Tembusu virus in human， China[J]. Transbound Emerg Dis， 2013， 60 （3）： 193-196.
[21] KUNO G， CHANG GJ， TSUCHIYA KR， et al. Phylogeny of the genus Flavivirus[J]. J Virol， 1998， 72 （1）： 73-83.
[22] KONO Y， TSUKAMOTO K， ABD HAMID M， et al. Encephalitis and retarded growth of chicks caused by Sitiawan virus， a new isolate belonging to the genus Flavivirus[J]. Am J Trop Med Hyg， 2000， 63 （1-2）： 94-101.
[23] ZHANG Y， LI X， CHEN H， et al. Evidence of possible vertical transmission of Tembusu virus in ducks[J]. Vet Microbiol， 2015，179 （3-4）： 149-154.
[24] NINVILAI P， NONTHABENJAWAN N， LIMCHAROEN B， et al. The presence of duck Tembusu virus in Thailand since 2007： A retrospective study[J].Transbound Emerg Dis， 2018， 65（5）： 1208-1216.
[25] YAN Z， SHEN H， WANG Z， et al. Isolation and characterization of a novel Tembusu virus circulating in muscovy ducks in South China[J]. Transbound Emerg Dis， 2017， 64（5）： e15-e17.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU