Characterization and pathogenicity evaluation of recombinant novel duck reovirus isolated from Southeast China

The novel duck reovirus (NDRV) emerged in southeast China in 2005. The virus causes severe liver and spleen hemorrhage and necrosis in various duck species, bringing serious harm to waterfowl farming. In this study, three strains of NDRV designated as NDRV-ZSS-FJ20, NDRV-LRS-GD20, and NDRV-FJ19 were isolated from diseased Muscovy ducks in Guangdong and Fujian provinces. Pairwise sequence comparisons revealed that the three strains were closely related to NDRV, with nucleotide sequence identities for 10 genomic fragments ranging between 84.8 and 99.8%. In contrast, the nucleotide sequences of the three strains were only 38.9–80.9% similar to the chicken-origin reovirus and only 37.6–98.9% similar to the classical waterfowl-origin reovirus. Similarly, phylogenetic analysis revealed that the three strains clustered together with NDRV and were significantly different from classical waterfowl-origin reovirus and chicken-origin reovirus. In addition, the analyses showed that the L1 segment of the NDRV-FJ19 strain was a recombinant of 03G and J18 strains. Experimental reproduction of the disease showed that the NDRV-FJ19 strain was pathogenic to both ducks and chickens and could lead to symptoms of hemorrhage and necrosis in the liver and spleen. This was somewhat different from previous reports that NDRV is less pathogenic to chickens. In conclusion, we speculated that the NDRV-FJ19 causing duck liver and spleen necrosis is a new variant of a duck orthoreovirus that is significantly different in pathogenicity from any previously reported waterfowl-origin orthoreovirus.

Chicken ARV was first isolated in 1954 from broiler chickens suffering from chronic respiratory diseases (7). Arthritis, tenosynovitis, and intestinal and respiratory diseases were all clinical manifestations of chicken ARV infection, and infection was common in chickens in China, with a positive rate of 27% for antibodies detected in chicken serum samples (8). Classical waterfowl-origin reovirus was designated as Muscovy duck reovirus (MDRV) and was subsequently isolated from Muscovy ducks in France in 1972 and introduced into China in 1997 (9). The primary target of infection was Muscovy ducklings between 10 and 20 days of age, and the white necrosis spots in the liver and spleen were the characteristic pathological changes (10,11). The mortality rate reached 30% (11,12).
In 2002, new viral infections emerged in Muscovy ducks, Peking ducks, and many others in southeast China (13,14). The disease was common in ducklings aged 5-10 days, with an incidence rate of 10-40% and a fatality rate of 15-50%; the younger the age of the impacted ducks, the greater the severity and fatality rate (15,16). Immunosuppression, slow development, severe diarrhea, hemorrhagic patches, and necrosis of the liver and spleen of ducklings were the most common symptoms of this viral infection (15). In addition, the virus causing this disease had more hosts and more significant infection rate than MDRV, and it was pathogenic to a wide range of ducks (12,15,17). Therefore, the virus was named NDRV (13). Phylogenetic analysis of nucleotide sequences based on S1 and S3 segments also showed that NDRV was significantly different from MDRV and chicken ARV (5,13).
Because of its high morbidity and mortality and the susceptibility of many species of ducks, NDRV has become one of the most important infectious diseases in duck breeding in China (17,18). Therefore, continuous surveillance is required to obtain information concerning endemic and emerging wild-type NDRV strains in waterfowl in order to develop effective prevention strategies. In this study, three strains of NDRV designated as NDRV-FJ19, NDRV-ZSS-FJ20, and NDRV-LRS-GD20 were isolated from Guangdong and Fujian provinces between 2019 and 2020. The whole genomes of the three strains were cloned, sequenced, and analyzed, and the pathogenicity of NDRV-FJ19 to ducks and chicks was investigated. The experimental data of these three strains may provide a new theoretical foundation for NDRV monitoring, prevention, and control.

. . Sample collection
Three Muscovy duck farms in China's Fujian and Guangdong provinces had cases of hemorrhagic patches and necrosis of the liver and spleen in Muscovy ducklings. To explore the etiology of the disease, liver and spleen samples from the afflicted ducks were obtained in August 2019 and June and August 2020, mixed with phosphate buffer saline (PBS) (pH 7.2) at a ratio of 1:3, and then freeze-thawed three times.

. . Virus isolation and identification
The supernatant was filtered through a 0.22-µm filter membrane and inoculated on a monolayer of Vero cells after centrifugation at 12 000 r/min for 15 min (17). The viral inoculant was discharged after 1 h of adsorption. The cells were cultured for 5-7 days at 37 • C with 5% CO 2 in DMEM with 1% FBS (Gibco, Shanghai, China). When cytopathic effects (CPEs) of >75% were observed, viral cultures were collected and sub-cultured until a stable CPE could be obtained (19). Then, the virus solution was diluted by gradient and plaque purification technology for virus purification. Finally, the cell cultures were frozen and thawed three times and stored for later use (20).
The viral RNAs of cell cultures were extracted according to the instructions of a virus nucleic acid extraction kit (Magen, Guangzhou, China) and stored at −80 • C. The reverse transcription-polymerase chain reaction (RT-PCR) with a PrimeScript TM One Step RT-PCR Kit (Takara, Dalian, China) was used to identify NDRV, Duck parvovirus virus (DPV), MDRV, and Duck Tembusu virus (DTMUV) viral RNAs. The purified virus was used to measure the TCID 50 , which was computed using the Reed-Muench technique (21). The NDRV-FJ19 strain (MOI = 0.1) was inoculated in three cell lines (Vero cells, BHK-21 cells, and LMH cells), and the viral cultures were harvested at 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h and used to measure the TCID 50 and plot growth curves through data measurement.

. . Experimental reproduction of the disease with the isolated virus
To validate the pathogenicity and infectivity of the NDRV-FJ19 strain, we conducted an experimental reproduction on 30 oneday-old healthy ducklings and 30 one-day-old SPF chicks. Each duckling and chick were injected intramuscularly with 0.5 mL of the NDRV-FJ19 virus (TCID 50 = 10 −4.25 /0.1 mL). As a control, 30 ducklings and 30 chicks were inoculated with PBS in the same manner. Ducklings and chicks were observed daily, and symptoms were recorded. Twelve ducklings (six control and six infected) and 12 chicks (six control and six infected) were .
/fvets. .  killed on the third, sixth, ninth, 12th, and 15th days after the start of infection (22). Liver and spleen tissues were examined to determine if pathological changes had occurred, and then, tissues were collected for pathological examination and virus reisolation. The tissue samples were fixed in 10% formaldehyde solution for 24 h, and embedded in paraffin. Tissue sections were prepared from the paraffin blocks and stained with hematoxylin and eosin (HE) for microscopic observation. Ultimately, the virus was confirmed with RT-PCR analysis and gene sequencing. (All the animal infection experiments were reviewed and approved by Animal Protection and Ethics Committee and Use Committee of Foshan University).

. . Complete genome segment amplification and sequencing
The stored viral RNA samples were collected, and genomes of all segments were cloned using RT-PCR with a PrimeScript TM One Step RT-PCR Kit (Oligonucleotide primers in Additional File 1) (5). The reaction program was as follows: 50 • C for 30 min, 94 • C for 3 min, then 35 cycles at 94 • C for 30 s, 50-60 • C for 30 s, extension at 72 • C for 1 min, and then a further extension for another 10 min at 72 • C. The RT-PCR products were electrophorized on 1% agarose gels, and the results were viewed and recorded using a gel imaging analyzer (Tianneng Technology Co., Ltd., Shanghai, China). A Gel Extraction Kit (Omega Bio-Tek, Beijing, China) was used in the purification of RT-PCR products, and the recovered product was cloned to the pMD18-T vector (TaKaRa Biotechnology Company, Dalian, China) and transformed into E.coli DH5α competent cells (TaKaRa). The positive bacterial solution was sent to Sangon Biotech (Guangzhou, China).

. . Recombination detection
All segment sequences of the three strains were screened for recombination by using the RDP, GENECONV, and BootScan methods in Recombination Detection Program, version 4 (RDP4). Sequence recombination was validated using at least two methods. In addition, the parent strains of the recombinants were visualized by using SimPlot Version 3.5.1, with a window size of 200 bp and a step size of 20 bp.

. . Virus isolation and purification
Three liver and spleen samples were inoculated into Vero cells for five generations, and all were capable of causing visible CPE in Vero cells within 3-5 days, leading to cell fusion and syncytial formation. In the RT-PCR analysis, NDRV was positive, while DPV, MDRV, and DTMUV were negative. Three isolates, NDRV-FJ19, .

. . Experimental reproduction of infection
After the infection with the NDRV-FJ19 strain, the infected group of ducklings experienced a significant peak in mortality at 4-7 days (mortality rate rising to 46.7%). The infected group of chicks experienced a significant peak in mortality at 2-5 days (mortality rate rising to 33.3%). Symptoms of hemorrhage and necrosis in livers and spleens ( Figure 3A) and hemorrhage and swelling of kidneys ( Figure 3A) were observed, similar to symptoms acquired by naturally infected ducks. Histopathological changes in the liver and spleen were observed ( Figure 3B). Finally, the virus was successfully recovered from infected or dead birds and confirmed by RT-PCR and sequencing. The virus was detected from the heart, lungs, liver, spleen, kidneys, small intestine, and bursae of infected animals.

. . Phylogenetic analyses
The ML method in the MEGA V5.0 program was used to create phylogenetic trees based on nucleotide sequences of 10 genome segments to explore the phylogenetic relationships between the three viruses obtained in this experiment and with NDRV, MDRV, and chicken ARV. As illustrated in Figure 4, the reference sequences were clearly split into four monophyletic groups: the NDRV subgroup, the MDRV subgroup, the chicken ARV subgroup, and the MRV subgroup. Meanwhile, all waterfowl-and chicken-origin isolates also formed two separate host-associated clades (except M2 and S2 segments). The three strains of NDRV-ZSS-FJ20, NDRV-FJ19, and NDRV-LRS-GD20 and other NDRV isolates (SH12, DH13, 091, TH11, NP03, J18, ZJ00M, and HN5d) formed a monophyletic branch distinct from MDRV and chicken ARV. Therefore, the three strains in this experiment can be confirmed as NDRV. NDRV-ZSS-FJ20 and NDRV-LRS-GD20 had a close relationship (except S1 segments) and were most closely associated with the DH13 and SH12 strains in the phylogenetic trees based on the L1, L2, M3, and S2 segments; in addition, they were the closest relatives to ZJ00M strains in the phylogenetic trees based on the L3, S1, and S3 segments and were the closest relatives to HN5d strains in the phylogenetic trees based on the M1, M2, .

. . Recombination analyses
The sequences of 10 segments of the three strains were evaluated for the presence of recombination using RDP4 and SimPlot. Using the sequences of J18, 03G, DH13, and 091 as the parental sequences, a recombination event that was supported by the RDP and similarity plot analyses was found in the L1 sequence of the NDRV-FJ19 strain. According to the similarity plot analysis, the recombination breakpoint occurred at position 3,621 of the sequence alignment ( Figure 5A). Therefore, it is reasonable to speculate that the L1 sequence of the NDRV-FJ19 strain was reconstituted by 03G and J18 strains. In addition, another recombination event was found in the L1 sequence of the J18 strain, similar to the NDRV-FJ19 strain, and the recombination breakpoint occurred at position 3,581 of the sequence alignment ( Figure 5B). Apart from the above, the recombination events in the S2 segments of ZJ00M, J18, HN5d, SH12, and DH13 and in the M2 segments of NP03 and D1007 were detected, but these recombination events were not effectively statistically supported by RDP or similarity plot analysis.

. Discussion
Since the outbreak of the first epidemic disease characterized by irregular necrosis of duck liver and spleen in 2005, the industry has suffered massive economic losses (23). As a multi-segment double-stranded RNA virus, NDRV is prone to recombination during transmission, leading to the emergence of new variants (5). Therefore, it is necessary to monitor the sequence of NDRV. In this study, three strains of NDRV were isolated, and the sequence and pathogenicity were preliminarily analyzed.
The sequencing analysis of the three strains revealed that the full genomes were 23,418 bp in length, and the length of each segment was almost identical to that of the reported matching NDRV genome segments (5,13). In terms of genomic structure, three strains of NDRV were more comparable to chicken ARV and were considerably distinct from MDRV S-class segments due to the different locations of the proteins encoded. The S1 segment was polycistronic with three overlapping ORFs, similar to chicken ARV. NDRV S1-S4 segments encoded P10, P18, σC, σA, σB, and σNS proteins, while MDRV S1-S4 segments encoded σA, σB, σNS, P10, and σC proteins (23). The S4 segment of MDRV was a dicistron that did not produce the P18 protein; this was the most significant change in genomic structure between NDRV and MDRV (24).
Many previous studies have shown that the σC protein (16,25,26), as the capsid structural protein of virions, is the most important antigenic protein of viruses, meaning that it primarily determines the pathogenicity of viruses. Therefore, the σC encoding genes can be used as genetic markers for ARV differentiation and classification. The similarity of nucleotide sequence encoding σC between NDRV and MDRV was 37.6-39.5% and that between NDRV and chicken ARV was 38.9-40.5%. The current international classification and naming of NDRV are unclear. As a result, we believe that NDRV should be categorized as a unique waterfowl-origin reovirus distinct from chicken ARV and MDRV. Furthermore, the nucleotide sequences of the L1, L2, and M2 gene segments of a goose-origin (03G) NGRV and a duckorigin NDRV were considerably different. Whether the nucleotide sequences of goose-origin NGRV and duck-origin NDRV differ because of host origin has not been confirmed. This could not be statistically analyzed, since there are few reported cases of gooseorigin NGRV. This perspective should be investigated further.
Looking at the structure of the phylogenetic trees, waterfowl reovirus-origin NDRV and MDRV constituted a substantial clade independent of chicken ARV, suggesting that most chicken ARVs and waterfowl-origin reoviruses (WRVs) have clearly evolved to be host specific. However, NDRV and chicken ARV were on the same branch in the M2 segment, independent of MDRV. This unusual occurrence was not seen in the other segments. We Frontiers in Veterinary Science frontiersin.org . /fvets. . assumed that this was due to recombination between WRVs and chicken ARVs. Recombination events in segments of the genome are very common in ARVs (5). In research reports of recent years, most of the orthoreovirus variants that have emerged are generated by viral recombination, for example, the SDPY-ARV and N-DRV-XT18 strains isolated in Shandong, China (17, 27). These strains were formed by recombination between WRVs or between WRVs and chicken ARVs; this not only resulted in the strains becoming more virulent but also widened the range of hosts. At present, the mechanism and extent of Reoviridae RNA recombination are undefined (1,2,28). Some scholars have found that reovirus accumulates defective gene segments with internal deletions during the passage process, and carries out sequencedirected recombination at different sites (28,29). The titers of these recombinant viruses were significantly increased on the cells8 (29). This may be the reason why the strain NDRV-FJ19 strain was more pathogenic. Therefore, recombination events of ARVs deserve close attention. Strains isolated from different regions of the country were in the same lineage. The isolates from France (D1546, D2044) in the MDRV branch showed significant homology (94.5-98.6%) with the isolates from China, indicating the existence of a regional reovirus difference that should be confirmed further.
From the results of experimental reproduction of infection, we found that NDRV-FJ19 strains cause serious damage to the liver and spleen of ducks and chickens, especially the severe bleeding and necrosis of spleen, necrosis of splenocytes, and lymphocyte depletion. The spleen, as an important immune organ of poultry, plays a major role in the body's immune function (30). However, NDRV infection reduces the number of lymphocytes in the spleen and leads to immunosuppression (27,31). Therefore, the disease should be treated as early as possible; otherwise, it will become more serious. In addition, it has been shown in previous studies that waterfowl-derived orthoreovirus tends to infect only waterfowl or is weakly infectious to chickens (12,15,31), while chicken ARV infects only chickens (32,33). Although NDRV-FJ19 is a waterfowlderived orthoreovirus, it is highly pathogenic to chickens, a phenomenon previously unobserved. Therefore, we speculated that this virus is a new variant of a duck orthoreovirus, and we further verified the tendency of NDRV to adapt to multiple hosts.

. Conclusion
In this investigation, three strains of NDRV were isolated from Muscovy ducks in Guangdong and Fujian provinces, and the whole genome sequences were identified. The three strains were closely related to orthoreovirus isolates derived from ducks and geese, with nucleotide sequence identities for 10 genomic fragments ranging between 37.6 and 99.8%. In contrast, the nucleotide sequences of genomic fragments from the three strains were only 38.9-80.9% similar to a chicken orthoreovirus isolate. Meanwhile, the strains had stronger pathogenic properties than other members in the orthoreovirus genus, suggesting that the waterfowlderived orthoreovirus has mutated in the direction of increased virulence.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethics statement
The animal study was reviewed and approved by Animal Protection and Ethics Committee and Use Committee of Foshan University.