Influenza D virus in animal species in Guangdong Province, southern China.
In 2016, we collected 607 clinical samples from 4 species of animals with different clinical diseases and 250 nasal swab samples from asymptomatic animals (Table) from 16 farms in 4 cities of Guangdong Province: Guangzhou, Qingyuan, Heyuan, and Jiangmen (Figure 1). In addition, we randomly chose 200 archived Holstein dairy cattle serum samples, 40 per year, from 2011-2015 to investigate possible early RNA distribution of influenza D virus in this region. We used the reverse transcription PCR method and subcloning protocol (online Technical Appendix, https://www.nc.cdc.gov/EID/article/23/8/170059-Techapp.pdf). We performed sequence alignment using ClustalW implemented in DNAStar software (DNAStar, Madison, WI, USA), and we conducted phylogenetic analyses based on our obtained sequences and reference truncated sequences (496-bp) of influenza D viruses from GenBank by using MEGA 5.1 software (http://www.megasoftware.net; online Technical Appendix Table).
After testing by reverse transcription PCR with further sequencing confirmation, we found influenza D virus-positive rates in 230 total nasal swab samples of 12.8% (20/156) for dairy cattle, 7.3% (4/55) for native yellow cattle, and 36.8% (7/19) for pigs. Rates in 324 total serum samples were 7.8% (15/193) for dairy cattle, 5.9% (3/51) for buffalo, and 33.8% (27/80) for goats. The influenza D virus-positive rate was also high (28.9%, 13/45) in swine lung samples. In contrast, we found no or low prevalence ([less than or equal to] 2%) in asymptomatic animals tested (Table). Moreover, all of the archived serum samples were found to be influenza D virus negative. Interestingly, 1 of 8 rectal swabs of goats with severe diarrhea tested positive (Table). Samples from animals with reproductive problems had a positive rate of 4.3% (5/116) (Table).
Sequence alignment analysis showed that the nucleotide sequences of influenza D viruses found in this study shared high similarity (99%-100%) with previously described sequences from China (7) and low similarity (93.8%-98.8%) with sequences originating from the United States, France, Italy, Mexico, and Japan (1, 8-12). Similarly, phylogenetic analysis revealed that all influenza D virus sequences in this study clustered together with previous sequences from China and belonged to the D/OK lineage (Figure 2).
When first discovered, influenza D virus was reported in diseased pigs in the United States (1). Later, it was identified in cattle and swine herds in several other countries, with or without clinical manifestation (7-11). Moreover, antibodies to influenza D virus were detected in goats, sheep, and humans (5-6). Under experimental conditions, influenza D virus replicated and transmitted among ferrets and guinea pigs (13). We confirmed that influenza D virus is widely present in cattle species (dairy cattle, yellow cattle, and buffalo). We also found influenza D virus at a high prevalence (>30%) in pigs and goats (Table), which is in contrast to the low prevalence found in previous investigations (1,5,10). The high prevalence may be caused by poor biosecurity measures and highdensity feeding mode practices in China's animal industry as well as possible cross-species transmission (13). Taken together, our findings expand the host range of influenza D virus and further emphasize the health concern this virus poses to multiple animal species.
Previous studies have shown that influenza D viruses are mainly found in respiratory tract samples (1-4,7,9-12) and that they have played an etiologic role in bovine respiratory diseases (2-4). In this study, we found that influenza D virus RNA was present in cattle and goat serum samples; it was also present in goat rectal swabs, accompanied by peste des petits ruminants virus and caprine kobuvirus (data not shown). The distribution of influenza D virus in our study is not the same as that described under experimental conditions (3).
Influenza viremia, an indicator of disease severity (14), has been detected in 20.9% of severe cases during the acute phase of infection or before host death. Our detection of influenza D virus genome in serum samples from severely diseased animals (Table) implies that the virus could enter transiently into the animal's circulatory system through capillaries lining the respiratory tract, which further contributes to the possibility of detecting virus in other organs. Similar to previous studies (2, 4), we also found that the reverse transcription PCR positive rate was significantly higher (4%-40%) in diseased animals than the rate ([less than or equal to] 2%) observed in asymptomatic animals (p<0.05), which suggests a potential correlation between the disease severity and presence of influenza D virus. For influenza D virus found in rectal swabs, it might be that animals have swallowed the virus. Another possibility is that, similar to influenza A and B viruses, influenza D virus can replicate within the intestinal tract (15).
We detected influenza D virus in cattle with reproductive disorders. However, we could not determine whether influenza D virus is associated with reproductive problems. Future studies can be designed to investigate these scientific issues.
To date, 2 lineages of influenza D virus (D/OK and D/660) co-circulate in North America and Europe (8-10,12). However, only the D/OK lineage has been found in China, and a potential third lineage was found in Japan (7,11). Our study confirms and further extends the previous observation that D/OK lineage circulates in East Asia. The viral, host, and ecologic factors that shape the observed contrasting phylodynamics of influenza D viruses among different geographic regions warrant further investigation.
In addition, we found different minor genetic variants circulating on the same farm (Figure 2), indicating the ongoing evolution of influenza D viruses in their hosts (7,8,11). In comparing our sequences to the reference sequences from different animal species, we found 4 frequent nucleotide mutations (at positions 136, 231, 263, and 486) (online Technical Appendix Figure 1), which caused 2 amino acid mutations at positions 77 and 88 (online Technical Appendix Figure 2). Interestingly, among 4 nucleotide mutations, 1 unique nucleotide (T at position 486) was originally from the D/660 lineage. Moreover, we found several consistent sequences co-circulating in multiple animal species (online Technical Appendix Figure 1). Our speculation is that homologous recombination among different influenza D viruses and potential cross-species transmission under field conditions are possible, but further study is needed.
In summary, our study investigating the infection status of influenza D virus in different farmed animal species in Guangdong Province provides novel insights into the epidemiology and evolution of this virus. In particular, we document the molecular evidence for influenza D virus infection in goats and buffalo.
We are very grateful to Ben M. Hause, Milton Thomas, and Hunter Nedland for their suggestions and English editing when we revised this manuscript.
This work was supported by Guangdong Provincial Department of Science and Technology (Grant no. 2016A040403083), Guangdong Provincial Agricultural Department (Grant no. 2016LM3177), and Ministry of Science and Technology of the People's Republic of China (Grant no. 2015GA780010). The work was also supported in part by SDSU AES Fund 3AH-477 to F.L. and D.W. S.-L.Z. is sponsored by Guangdong Academy of Agricultural Sciences, Guangzhou, China.
Dr. Zhai is an associate professor at Animal Disease Diagnostic Center, Institute of Animal Health, Guangdong Academy of Agricultural Sciences. His research interests focus on surveillance and rapid response research of emerging or reemerging animal pathogens. In 2016-17, he is a visiting scholar at the Department of Biology and Microbiology, South Dakota State University.
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Address for correspondence: Shao-Lun Zhai, Guangdong Key Laboratory of Animal Disease Prevention, Animal Disease Diagnostic Center, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, No. 21 Baishigang St, Tianhe District, Guangzhou, 510640, China; email: email@example.com; Feng Li, Department of Biology and Microbiology & Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD 57007, USA; email: firstname.lastname@example.org
Shao-Lun Zhai,  He Zhang,  Sheng-Nan Chen,  Xia Zhou, Tao Lin, Runxia Liu, Dian-Hong Lv, Xiao-Hui Wen, Wen-Kang Wei,  Dan Wang, Feng Li
Author affiliations: Guangdong Academy of Agricultural Sciences, Guangzhou, China (S.-L. Zhai, D.-H. Lv, X.-H. Wen, W.-K. Wei); South Dakota State University, Brookings, South Dakota, USA (S.-L. Zhai, S.-N. Chen, T Lin, R. Liu, D. Wang, F. Li); South China Agricultural University, Guangzhou (H. Zhang, X. Zhou)
DOI: https://doi.org/ 10.3201/eid2308.170059
 These authors contributed equally to this article.
Caption: Figure 1. Farm locations for study of influenza D viruses in cattle, goats, buffalo, and pigs, Guangdong Province, China.
Caption: Figure 2. Phylogenetic analysis of viruses from study of influenza D viruses in cattle, goats, buffalo, and pigs in Guangdong Province, China, compared with reference viruses. Partial hemagglutininesterase-fusion gene sequences (496 bp) were aligned by using ClustalW implemented in DNAStar software (DNAStar, Madison, WI, USA), and the phylogenetic tree was obtained using neighbor-joining method within MEGA 5.1 software (http://www.megasoftware. net). Numbers at nodes are percentages of bootstrap values obtained by repeated analyses (1,000 times) to generate majority consensus tree. Only bootstrap scores of at least 50 were retained. Scale bar indicates 0.5% nucleotide sequence divergence. Gray shading indicates viruses from this study; reference viruses obtained from the United States are marked with * ; from China, **; from Italy, ***; from Mexico, ****; from France, *****; and from Japan ******. Note that D/ swine/Guangdong/YS1/2016 and D/swine/Guangdong/YS2/2016 are from the same farm; D/swine/ Guangdong/P8/2016 and D/swine/ Guangdong/P14/2016 are from the same farm; D/swine/Guangdong/ U1/2016 and D/swine/Guangdong/ U16/2016 are from the same farm; D/bovine/Guangdong/LG2/2016, D/ bovine/Guangdong/LG5/2016 and D/bovine/Guangdong/LG9/2016 are from the same farm; D/bovine/ Guangdong/QQ1/2016, D/bovine/ Guangdong/QQ4/2016, D/bovine/ Guangdong/QQ7/2016 and D/ bovine/Guangdong/QQ12/2016 are from the same farm; D/bovine/ Guangdong/RS1/2016 and D/ bovine/Guangdong/RS4/2016 are from the same farm.
Table. Animal species, location, sample data, and detection rate of influenza D virus, Guangdong Province, China * Animal species Farm typet Farm location No. and farm animals Holstein dairy cattle A Not all-in-all-out Guangzhou: Tianhe 2,000 A Not all-in-all-out Guangzhou: Tianhe 2,000 B Not all-in-all-out Guangzhou: 800 Luogang B Not all-in-all-out Guangzhou: 800 Luogang C Not all-in-all-out Guangzhou: Tianhe 175 American Landrace pig D Not all-in-all-out Guangzhou: Huadu 200 E All-in-all-out Heyuan: 1,000 Yuancheng E All-in-all-out Heyuan: 1,000 Yuancheng F All-in-all-out Jiangmen: Kaiping 800 F All-in-all-out Jiangmen: Kaiping 800 G All-in-all-out Heyuan: Dongyuan 600 Native hybrid white goat H Not all-in-all-out Guangzhou: 200 Zengcheng I Not all-in-all-out Guangzhou: 300 Luogang Native hybrid black goat J Not all-in-all-out Qingyuan: Jiangkou 150 K Not all-in-all-out Jiangmen: Enping 500 Asian buffalo L Not all-in-all-out Guangzhou: 150 Nansha M Not all-in-all-out Guangzhou: Panyu 180 N Not all-in-all-out Qingyuan: Yingde 400 Native yellow cattle O Not all-in-all-out Qingyuan: Qingxin 200 P Not all-in-all-out Qingyuan: Fogang 230 Animal species Age range Sample type No. positive/ and farm of animals no. samples Holstein dairy cattle A 3-5 y Nasal swab 14/86 ([double dagger]) A 3-5 y Serum 10/94 ([double dagger]) B 3-6 y Nasal swab 6/70 ([double dagger]) B 3-6 y Serum 5/99 ([double dagger]) C 2-5 y Nasal swab 1/50 ([section]) American Landrace pig D 10-15 wks Lung 4/10 ([double dagger]) E 5-5 wks Nasal swab 4/10 ([double dagger]) E 3-5 wks Lung 1/8 ([double dagger]) F 8-20 wks Nasal swab 3/9 ([double dagger]) F 8-20 wks Lung 8/27 ([double dagger]) G 9-15 wks Nasal swab 1/50 ([section]) Native hybrid white goat H 0.5-5 y Serum 7/25 ([double dagger]) I 2-4 y Serum 20/55 ([paragraph]) Native hybrid black goat J 1-3 y Rectal swab 1/8 (#) K 1 -4 y Nasal swab 0/50 ([section]) Asian buffalo L 3-5 y Serum 2/26 ([paragraph]) M 3-6 y Serum 1/25 ([paragraph]) N 1 -4 y Nasal swab 0/50 ([section]) Native yellow cattle O 2-5 y Nasal swab 4/55 ([double dagger]) P 1 -3 y Nasal swab 0/50 ([section]) Animal species Detection and farm rate, % Holstein dairy cattle A 16.3 A 10.6 B 8.57 B 5.05 C 2 American Landrace pig D 40 E 40 E 12.5 F 30 F 29.6 G 2 Native hybrid white goat H 28 I 36.4 Native hybrid black goat J 12.5 K 0 Asian buffalo L 7.7 M 4 N 0 Native yellow cattle O 7.3 P 0 * Feeding type of farms A-G was in captivity (poor biosecurity and high density). Feeding type of farms H-K and N-P was free grazing on the hills in the daytime and in captivity (poor biosecurity and high density) in the nighttime. Feeding type of farms L and M was free grazing in wetland in the daytime and in captivity (poor biosecurity and high density) in the nighttime. ([dagger]) All-in-all-out is a strategy for the control of infectious disease. The barn is emptied of all animals and the accommodation is cleaned and disinfected and then refilled, all on 1 day. ([double dagger]) These animals had severe respiratory diseases with a 10%-30% mortality rate, mainly characterized by expiratory dyspnea and abdominal respiration. ([section]) These animals were asymptomatic. ([paragraph]) These animals had severe reproductive disorders with a 60%-70% abortion rate. (#) These animals had severe diarrheal disease, characterized by watery diarrhea, limb weakness, and nearly dying.
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|Author:||Zhai, Shao-Lun; Zhang, He; Chen, Sheng-Nan; Zhou, Xia; Lin, Tao; Liu, Runxia; Lv, Dian-Hong; Wen, Xi|
|Publication:||Emerging Infectious Diseases|
|Date:||Aug 1, 2017|
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