Introduction

Since 2010, a severe infectious disease caused by Tembusu virus (TMUV) has occurred on several duck farms in China, and this virus has spread quickly to most of the duck-producing regions in China [1, 2]. Infection rates are over 90% on some farms, and mortality rates vary from 5% to 30% because of secondary infections [1, 3]. Ducks infected with TMUV mainly show egg drop syndrome and neurological symptoms such as ataxia and paralysis [1, 4]. The ovaries of infected ducks mainly show hyperemia, hemorrhaging, bleeding, and denaturation and other pathological changes include enlargement of the spleen. Neuronophagia has occasionally been found [1, 4]. Since the outbreak began, TMUV has continuously infected ducks and caused huge economic losses to the duck industry.

After it was first isolated from mosquitoes in Malaysia in 1955 [5], TMUV was not reported again until it was isolated from mosquitoes in Thailand in 1992 [6]. Then, in 2000, the Sitiawan strain of TMUV was isolated from domestic chickens in Malaysia [7]. However, at that time, the disease associated with TMUV infections in ducks had not been reported. In 2010, TMUV quickly spread to most of the duck-producing regions in China and caused huge economic losses to the duck industry. Evolutionary analysis of duck TMUV showed that the outbreak of TMUV originated in Malaysia, then spread north to Thailand and northeast to Shandong province of eastern China, and gradually to most of the duck-breading areas of China [8]. Since then, more and more TMUV strains have been isolated and identified from clinical cases. Phylogenetic analysis based on the open reading frame (ORF) and envelope (E) protein sequences of TMUV has shown that TMUV circulating in China can be divided into two lineages: lineage I and lineage II. Lineage II, including lineage IIa, lineage IIb, and lineage IIc, has arisen as the dominant lineage currently circulating in China [9, 10].

Most flaviviruses are transmitted by mosquitoes [11], and since TMUV has been isolated from mosquitoes, it is likely that they are involved in its transmission [12]. However, TMUV can also be spread by vertical transmission, direct contact and aerosol transmission [13, 14]. In addition to many species of ducks, geese [15], chickens [16], pigeons [17] and sparrows [18] have also been found to be infected by TMUV. Furthermore, isolation of TMUV from humans has been reported [19]. The spread of TMUV infections among a wide range of host species is worth paying more attention to due to its possible implications for public health and epidemiology.

Zhejiang province of China is one of the original regions where TMUV outbreaks occurred, and has experienced TMUV infections over the past several years. Therefore, investigation of TMUV epidemiology could provide information about the infection status of TMUV in Zhejiang and in China. In this study, we collected a total of 88 tissue samples and 2,331 serum samples from 2010 to 2016 to carry out a molecular analysis and serological survey of TMUV infection in Zhejiang, and six TMUV strains were isolated and identified. The complete genome sequence of six isolates and the E gene sequences of 19 strains were analyzed, and antibodies to TMUV in ducks from the local areas were detected and analyzed.

Materials and methods

Cells, SPF chick embryos and clinical samples

Tissue samples of suspected TMUV-infected animals were collected by the Yuyao Municipal Institute of Poultry Disease, Zhejiang, China, from 2010 to 2015. Each sample selected for analysis was collected from a different farm. Duck serum samples were provided by Xiaoshan Animal Husbandry and Veterinary Bureau, Zhejiang, China, and were collected from 45 local duck farms from 2013 to 2016.

BHK-21 (Syrian baby hamster kidney) cells and DF-1 (chicken embryo fibroblast) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL Life Technologies, Grand Island, NY) at 37 °C in a 5% CO2 atmosphere. The cell lines were routinely maintained in our laboratory and were used for virus isolation and propagation.

RT-PCR detection

Samples of ovaries from dead ducks were homogenized in sterile phosphate-buffered saline (PBS, pH 7.2) to make a 20% suspension (w/v). After freezing and thawing three times, the suspension was centrifuged at 12,000 g for 10 min, and 500 µl of supernatant was used for RNA extraction and tested by RT-PCR using the primer pair T2F/T2R (T2F, 5’-GTGCCAAGTTTGACTGCA-3’; T2R, 5’-CGTCCAACTGGTGTCAT-3’), to amplify a 716 bp DNA fragment of the E gene of TMUV.

Virus isolation

The rest of the supernatants prepared from the above homogenized duck ovary samples were filtered through a 0.22-µm syringe-driven filter and inoculated into the allantoic cavities of 9-day-old SPF chick embryos (Zhejiang Ceva Ebvac Biotech Co., LTD). The allantoic fluids of the infected embryos and culture medium were collected for further propagation and RNA extraction. Meanwhile, the diluted samples were used to inoculate BHK-21 cells and DF-1 monolayers grown to 80% confluence in 6-well culture plates (Corning, NY, USA), followed by incubation at 37°C for 2 h to allow virus adsorption. After discarding the inoculum, new medium containing 10% FBS was added to the cell cultures, which were incubated at 37°C with the same CO2 conditions for approximately 3 days and then examined daily for evidence of a cytopathic effect (CPE).

Indirect immunofluorescence assay (IFA)

BHK-21 cells were cultured in 96-well plates (Corning, NY, USA) and infected with the isolated virus. The cells were then incubated at 37 °C for 48 h and fixed with 4% paraformaldehyde. The fixed cells were incubated with mAbs to the E protein of TMUV (prepared in our laboratory) [25] for 1 h at 37 °C. They were then washed three times with PBS and incubated with FITC-conjugated anti-mouse IgG secondary antibody (KPL, Gaithersburg, MD) for 1 h at 37 °C. The stained cells were washed five times with PBS and visualized using an inverted fluorescence microscope (Olympus, IX-71, Japan).

Nucleotide sequencing and genetic characterization

RT-PCR-positive clinical samples were selected, and the full-length E gene of TMUV was amplified using specific primers (F, 5’-TTCAGCTGTCTGGGGATGC-3’; R, 5’-GGCATTGACATTTACTGCCAGG-3’). The PCR products were purified using a PCR Purification Kit (Simgen, Hangzhou, China) and cloned into the pMD18-T vector (Takara, Dalian, China) for sequencing. Alignment of the genome sequences of the E gene was performed using DNAMAN and MegAlign software, and analysis of the E genes was performed using the MEGA program (Version 6.0).

For genome sequencing, the total viral RNA of six isolates was extracted from 500 µl of allantoic fluid using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and complementary DNA (cDNA) was synthesized using a PrimeScript first strand cDNA Synthesis Kit (TaKaRa, DaLian, China). Based on a multiple alignment of the complete genome sequence with TMUV sequences obtained from the GenBank database, eleven pairs of primers covering the whole genome were designed using Primer Premier 5 software (Table S1). RT-PCR reactions were performed using EX TaqDNA® Polymerase (TaKaRa, DaLian, China).

Phylogenetic analysis

To investigate the evolution of TMUV nucleotide sequences from a total of 93 strains, including six full genome sequences and 13 E gene sequences from this study and 74 sequences obtained from GenBank were compared (Table S2). Phylogenetic trees were generated based on entire open reading frames (ORF) and the E gene. Multiple alignments and the phylogenetic trees were constructed using the Clustal W method and the neighbor-joining method of the MEGA program (version 6.0) with absolute distances and 1000 bootstrap replicates.

Detection of antibodies in serum samples with indirect EDIII ELISA

Duck sera were examined by ELISA using a purified recombinant EDIII protein. One hundred microliters of the EDIII protein (2 µg/mL in 50 mM sodium carbonate buffer [pH 9.6]) was added to each well of a 96-well microtiter plate and then incubated overnight at 4°C. The plate was then washed twice with PBST (0.25% Tween-20 in PBS) and incubated with the serum sample for 1 h at 37°C. After another washing, the plate was incubated with 100 µL of HRP-conjugated goat anti-duck IgG (KPL, Gaithersburg, MD) (1:5000 dilution) for an additional 1 h at 37°C. The plate was then washed again, and the colorimetric reaction was developed using 3,3’,5,5’-tetramethylbenzidine for 10 minutes at 37°C . The reaction was stopped with 2 M sulfuric acid, and the optical density was read at 450 nm.

Results

Clinical cases of TMUV infection observed in Zhejiang from 2010 to 2016

According to the records of the Yuyao Municipal Institute of Poultry Disease, Yuyao, Zhejiang, China, suspected clinical cases were found during the period 2010-2016 (Fig. 1A). Three epidemic peaks were observed in August of 2010, November of 2012, and July and August of 2015. Only a few cases were observed in 2011, 2013, 2014 and 2016, as well as in most of the months of 2010, 2012 and 2015, except the months experiencing epidemic peaks (Fig. 1A).

Fig. 1
figure 1

Clinical investigation of TMUV infections. (A) Clinical cases of suspected TMUV infections collected from Yuyao Municipal Institute of Poultry Disease, Yuyao, China from 2010 to 2016. (B) Detection of TMUV in clinical samples by RT-PCR. The results for six tissue samples that were used for virus isolation are shown. M, DNA marker; 1, YY1Du; 2, JSGo; 3, WZDu; 4, zjYY150901; 5, zjYY150902; 6, zjYY150903; -, negative control; +, positive control

Full size image

Sick or dead ducks showed neurological symptoms, and a drop in egg production occurred in egg-laying ducks. Pathological changes were observed as oophorrhagia and splenomegaly. A total of 88 samples from clinical cases were collected by the Yuyao Municipal Institute of Poultry Disease from 2010 to 2015 and subjected to further testing. Of the 88 clinical samples examined, 41 were TMUV positive by RT-PCR. Fig. 1B shows positive RT-PCR results for six tissue samples.

TMUV virus isolated and its cell culture

Some of the RT-PCR-positive clinical samples were subjected to virus isolation by inoculation of the homogenized tissue suspension into 10-day-old SPF chick embryos and passed three times. The infected embryo was small, showed hemorrhaging (Fig. 2A), and died 2 days postinoculation with turbid allantoic fluid. The viral RNA extracted from allantoic fluid of infected embryos was confirmed by RT-PCR and sequencing. A total of six TMUV strains, named YY1Du, JSGo, WZDu, zjYY150901, zjYY150902 and zjYY150903, were isolated from different RT-PCR-positive clinical samples.

Fig. 2
figure 2

Characterization of TMUV isolates. (A) SPF chick embryos infected with TMUV isolate zjYY150901. (B) CPE in DF-1 and BHK cells infected with TMUV isolate zjYY150901. (C) YY1Du-infected BHK cells detected by IFA using a monoclonal antibody to the E protein

Full size image

To study the biological characteristics of TMUV, the isolates were adapted to the DF-1 and BHK-21 cell lines. CPE in these cells was characterized by vacuole degeneration and necrocytosis (Fig. 2B). Virus replication in BHK-21 cells was confirmed by IFA using a monoclonal antibody to the E protein of TMUV (Fig. 2C).

Genomic characterization of TMUV isolates

The complete genome sequences of six isolates were obtained by combining the sequences of eleven overlapping cDNA fragments, using DNAMAN software. The genomic sequences of YY1Du, JSGo, WZDu, zjYY150901, zjYY150902 and zjYY150903 were submitted to the GenBank database under the accession numbers AB917088, AB917090, AB917089, MF522174, MF522175, and MF522176, respectively.

All of the isolates had a full-length genome that was 10,990 nucleotides (nt) in length with a single ORF extending from nt 95 to nt 10,372 encoding a 3,425-amino-acid polyprotein. The full-length genomic sequences of the six isolates were 97.3% to 99.9% identical to each other (Table S3) and 87.1% to 99.6% identical to references strains MM-1775, Sitiawan, BYD and YY5 (Table S3).

Phylogenetic analysis

To investigate the genetic evolution of TMUV circulating in Zhejiang, phylogenetic trees were constructed based on TMUV E gene sequences and full sequences encoding the polyprotein. A total of 93 E gene sequences, including 19 sequences obtained in the present study and 74 sequences from GenBank (Table S2), were used to construct a phylogenetic tree based on the E gene (Fig. 3A). A total of 80 genomic sequences, including six sequences obtained in present study and 74 genome sequences from GenBank (Table S2) were used to construct a phylogenetic tree based on the ORF of the TMUV genome (Fig. 3B). Based on the E gene and full genomic sequences, all selected strains were divided into two major lineages. One lineage included isolate MM1775 from Malaysia and isolate Sitiawan from Thailand, both of which were obtained before 2010. Another lineage included one sublineage with D1921/1/3/MY and D1977/1/MY from Malaysia as representatives and another sublineage that was further divided into several subclades that included the strains from China since 2010 (Fig. 3A and B).

Fig. 3
figure 3figure 3

Phylogenetic analysis based on the E gene (A) and ORF (B) of TMUV

Full size image

In the phylogenetic tree based on the E gene, the sequences obtained in this study were distributed in different subclades, but were more concentrated in certain clades (Fig. 3A). Nine of the sequences obtained in 2015 formed an independent sub-branch and clustered together mainly with isolates from the southern part of China, such as GX2013C (GenBank accession no. KP861859). zj-zhashan-2001 and JSGo had a close genetic relationship to AH2011 (GenBank accession no. KJ958533) and the JS-L strain (GenBank accession no. KY626659). YY1Du, zj-04-2015, zjYY150902, zjYY150903 were clustered together with the YY5 strain (GenBank accession no. JF270480). WZDu and zj-02-2011 had a close genetic relationship to XHZD/2010 (GenBank accession no. JQ596407). zj-01-2011 was clustered separately with the Duan strain (GenBank accession no. JQ9260421).

In the phylogenetic analysis based on the entire ORF, the genomes of six strains isolated in this study were also distributed into different subclades (Fig. 3B). The zjYY150901 sequence showed the closest genetic relationship to the HD2-2013 strain (GenBank accession no. KX686575); the genome sequence of the WZDu isolate had a close genetic relationship to the XHZD strain (GenBank accession no. JQ595407); and three strains, YY1Du, zjYY150902, and zjYY150903, clustered together and formed a small group (Fig. 3B).

Genetic characteristics of the E proteins of TMUV isolates from Zhejiang

As the envelope (E) protein of flavivirus is the major surface protein and plays an important role in virus invasion, replication and immunology, the E protein of TMUV was selected for analysis of genetic variation and antigenic properties of TMUV isolates from Zhejiang. The E protein of flavivirus consists of three domains (I, II, and III) and is responsible for induction of neutralizing antibodies, especially against domain III [10].

A total of 19 E gene sequences amplified from clinical samples were obtained in this study and found to be 96.8% to 100.0% identical to each other (Table S4). Like most flaviviruses, TMUV is predicted to have N-linked glycosylation sites at E protein positions 103, 154 and 314 [10, 20]. We observed a total of 19 mutation sites in the 19 sequences that were different from the frequently mutated sites in the E gene that have been reported previously (Table S5). However, none of the 19 mutations were the same as those of the MM-1775 and Sitiawan strains at E protein positions 103, 154 and 314 (Table S5). Among the E sequences of 19 TMUV strains, zj-08-2015 had two mutations, S99G and S150G, near the major glycosylation site motif.

It has been reported that protonation of histidines at acidic pH plays an important role in the flavivirus life cycle, especially during the structural transition that leads to membrane fusion [21]. Some histidines are completely conserved among E proteins of flaviviruses. We found that there are ten histidines present in the E proteins of the 19 strains at positions 144, 153, 163, 219, 246, 263, 285, 320, 398 and 443, as is also seen in those of the MM-1775 and Sitiawan strains, and no mutations were observed in these sites.

Epitopes 87YAEYI91 and 220DLD/NLPWT226, which are located in domain DII, and 374EVE/ DPPFG380, which is located in domain DIII of the TMUV E protein, have been identified as cross-reactive epitopes in most flaviviruses, including West Nile virus, yellow fever virus, and Japanese encephalitis virus [20, 22]. No mutations in the above three epitopes were found in the 19 strains, indicating that the epitope sequences are conserved among many strains of mosquito-borne flaviviruses.

TMUV-specific antibodies in ducks from 2013 to 2016

To further investigate the epidemiology of TMUV infections, 2,331 serum samples of unvaccinated ducks were collected from local duck farms in Zhejiang from 2013 to 2016 and tested for TMUV-specific antibodies by ELISA. The results showed that the antibody-positive rates were 17.4% (105/604), 22.0% (177/805), 26.7% (123/461) and 17.8% (82/461) for the sera collected in 2013, 2014, 2015, 2016, respectively (Fig. 4A), indicating that TMUV infection was persistently present through the years. In each year from April to December, samples were tested for antibodies every two months, and the antibody titers are shown as optical density (OD) measurement at a wavelength of values 450 nm. In 2013, the antibody-positive rate ranged from 10% (2/20, August) to 22.7% (25/110, October), with OD values of less than 0.5. In 2014, the positive rates ranged from 2.5% (1/40, October) to 33.9% (65/192, December). In 2015, the positive rates ranged from 11.2% (25/224, October) to 77.8% (35/45, April) with some OD values reaching 1.0. In 2016, the positive rates ranged from 2.8% (2/73, October) to 31.4% (11/35, April), with higher OD values observed in the sera collected in April and June.

Fig. 4
figure 4

Detection of serum antibody to TMUV in clinical serum samples by EDIII-ELISA. (A)  Ratios of serum samples from 2013-2016 positive for antibody to TMUV. (B) Antibody detection in different years. Duck serum samples were collected from the Xiaoshan area of Zhejiang every two months during 2013-2016. The left y-axis shows the OD450 value of serum samples (scatters). The right y-axis shows the percentage of antibody-positive samples (columns). A dotted line indicates the cutoff value of the ELISA

Full size image

Discussion

Since the outbreak of TMUV in China in 2010, duck farmers have been suffering economic losses, although the epidemic of TMUV has declined slightly in the southeast of China. With the continuous expansion of the infected host and transmission route, we should pay more attention to the prevalence of the virus. In our investigation of TMUV infection in Zhejiang from 2010 to 2016, we found that TMUV outbreaks in recent years have not been as serious as the first outbreak in 2010, although two epidemic peaks were observed in 2012 and 2015. Wave-like infections of TMUV in these years suggest that TMUV infection might be persistent, and we do not know when another epidemic peak will occur. Therefore, continuous investigation of the epidemiology of TMUV infections is necessary.

In this study, we also investigated the epidemiology of TMUV infections by testing for the antibodies to TMUV on local duck farms. The data showed that antibodies to TMUV were frequently present in ducks on local farms, indicating that these ducks were infected with TMUV, since they had not been vaccinated. The highest positive rate for antibody detection was observed in 2015. This result was consistent with the clinical observations.

Since the TMUV outbreaks in 2010, more and more TMUV strains have been isolated. The TMUV strains isolated in this study all caused severe hemorrhage and death of duck and SPF chicken embryos and caused CPE in DF-1 and BHK-21 cells. The sequences obtained in this study all showed a high level of sequence similarity (87.1%-88.8% identity) to the corresponding sequences of the MM-1775 and Sitiawan strains. The sequences of the E genes of the isolates were 96.8%-100.0% identical to each other, and the six genomic sequences were 97.3%-99.9% identical. Therefore, the TMUV strains circulating in Zhejiang were highly conserved in sequence from 2010 to 2015. However, 19 sites in the E protein that were previously reported to be highly conserved were found to be mutated in the current strains in this study, indicating that the TMUV strains in Zhejiang have special variations. Whether these variations contribute to virus pathogenicity, immunogenicity or other biological characteristics still needs to be evaluated.

Generally, N-linked glycosylation sites of viral surface proteins are important for entry into host cells. N-linked glycosylation sites on the E protein of flaviviruses affect viral infectivity and binding to cellular receptors [23, 24]. We did not observe any mutations in N-linked glycosylation sites in the E gene of any of the nineteen strains, suggesting that the glycosylation sites in the E protein have been conserved among the TMUV isolates in different areas since the TMUV infection outbreak. However, we found that zj-08-2015 has mutations around the glycosylation sites (position 103 and 154) at position 99 (Gly to Arg) and position 150 (Gly to Ser). Whether these mutations affect glycosylation requires further study.

We also examined the positions of histidine residues and E protein epitopes in the sequences obtained in this study and found no variation compared to other TMUV isolates, indicating that histidines and epitopes in the E protein are conserved in the TMUV isolates in Zhejiang.

Phylogenetic analysis based on the nucleotide sequence of the entire ORF or the E gene showed that all of the sequences obtained in this study clustered together in the same sublineage, separately from TMUV D1997/1/MY, which was isolated from Malaysia in 2012, and were further divided into several subclades. Most of the sequences obtained in this study have similar evolution patterns based on both phylogenetic trees. Different evolution of three genomes (YY1Du, zjYY150902 and zjYY150903) was observed between the two trees. In the phylogenetic tree based on the ORF, these three genomes were the most distant from D1997/1/MY, but in the tree based on the E gene, they were closer to D1997/1/MY than to other isolates, such as WZDu. This evolutionary difference might be due to the sequence difference in the E genes of the isolates. Based on this phylogenetic analysis, sequences obtained from samples collected in 2010-2014 showed a distant relationship to each other, while the strains circulating in 2015 showed a closer genetic relationship (Fig. 3A). Interestingly, YY1Du and YY5, which were isolated in 2010, were clustered together with strains zj-04-2015, zjYY0902, and zjYY0903 isolated in 2015 (Fig. 3A), indicating that TMUV from Zhejiang province may not have undergone any obvious variation in recent years.

The results of this study emphasize the need for a continuous active serological and molecular surveillance system for TMUV and other flaviviruses, which may provide timely epidemiological data for understanding the evolution of these viruses and finding a way to control the spread of disease caused by TMUV.