- Short report
- Open Access
First sequence of influenza D virus identified in poultry farm bioaerosols in Sarawak, Malaysia
Tropical Diseases, Travel Medicine and Vaccines volume 6, Article number: 5 (2020)
In 2018, our team collected aerosols samples from five poultry farms in Malaysia. Influenza D virus was detected in 14% of samples. One sample had an 86.3% identity score similar to NCBI accession number MH785020.1. This is the first molecular sequence of influenza D virus detected in Southeast Asia from a bioaerosol sample. Our findings indicate that further study of role of IDV in poultry is necessary.
Newly recognized as a novel species, influenza D virus (IDV) was first isolated in 2011 from a pig exhibiting influenza-like-illness (ILI) . IDV has been detected in various animal species including pigs , cattle , goats and sheep  with the highest prevalence reported in young cattle with symptoms of bovine respiratory disease (BRD) . To date, IDV has not been isolated in poultry . In studies conducted in the United States, serological evidence indicates that IDV has been present in cattle populations since at least 2004  and a cross-sectional study conducted in Florida detected a high prevalence (97%) of neutralizing antibodies in cattle exposed workers compared to non-exposed controls (18%) . IDV transmission has also been noted in comingled cattle herd with 94% seroprevalences of IDV antibodies . More recently in 2015, IDV was isolated in both pigs and cattle during a swine respiratory disease outbreak . The IDV genome associated with the swine respiratory disease was closely related to the viral genome isolated in the United States in 2011. Globally, IDV has been isolated in Morocco, Togo, Benin , China , and Japan , but, prior to this study, has not been detected in Southeast Asia. Despite these initial detections of IDV in swine and cattle, relatively little is known about the potential zoonotic transmission of IDV to humans , and IDV disease has not been described in humans. Currently there is no recommended therapy or vaccine available for IDV, despite active research .
From June 3 to August 3, 2018, our study team collected 28 bioaerosol samples from five poultry farms across the Sibu Division of Sarawak, Malaysia. Bioaerosol sampling was conducted using the National Institute of Occupational Safety and Health’s (NIOSH, Morgantown, West Virginia, USA) model BC 251 two-stage bioaerosol sampler calibrated at a rate of 3.5 L/min [14,15,16]. At the poultry farm, NIOSH samplers were fixed approximately 1 m above the ground on a stationary tripod, set-up near or inside the holding pens of the chickens or ducks for a 1.5-h period. Holding pens were either closed or covered with open-sided enclosures. After collection, samples were immediately transported back to the laboratory on ice and stored at − 80 °C until sample processing could occur.
Viral RNA extracts were analyzed with a real-time polymerase chain reaction (qPCR) for detection of influenza D virus  using Superscript R III Platinum One-Step qRT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Inc., Waltham, MA). Samples positive for influenza D virus were further amplified and sequenced using RT-PCR primers and probes . Partial genome sequencing was performed by Eton Bioscience (Eton Bioscience, Inc., Raleigh, NC, USA). Sequences were then compared to the NCBI sequence database using the BLAST application of BioEdit 7.1.9 (Ibis Biosciences, Carlsband, CA, USA). Sequences were aligned and phylogenetic analysis was performed using the UPGMA method in Geneious Prime 2019.1.1 (Biomatters Inc., San Diego, CA, USA).
IDV was detected in 4 of the 28 (14.29%) samples collected from the poultry farms. One of the four IDV positive samples was successfully sequenced (1200 bp sequence) as influenza D virus. As samples were detected at low concentrations, real time PCR CT values ranged between 34 and 36, no efforts to culture these viruses were attempted.
A phylogenetic tree of five North American influenza D virus isolate sequences currently available in GenBank and the IDV isolated in our study is illustrated in Fig. 1. This tree demonstrates that the genetic distance between strains previously isolated in North America is much less than (nearly zero genetic difference) the strain that we have isolated from our bioaerosol sample collected Southeast Asia. These results suggest that there could be different strains of IDV circulating in animal populations in Asia.
This study of aerosols conducted in Sarawak is the first to successfully sequence influenza D virus in an aerosol sample from a poultry farm. The results of this study as well as our previous work in an airport  suggest that aerosol sampling is a useful technique for respiratory virus surveillance in high traffic and areas of high human-animal interaction. Aerosol sampling has advantages in that it minimally disrupts activities in a commercial setting (abattoir or poultry farm), is simple in setup and operation, and the processing procedures for isolating the viral nucleic acid are relatively simple [16, 18].
A limitation of our study is the inability to link aerosol results with poultry stalls, chickens, or ducks. Also, the inability to link detection of aerosol positivity to presence of virus in poultry hosts, the low concentrations we detected and the lack of viral culture to access viability and infectivity are important limitations. However, the benefits of this environmental sampling approach are the early detection and screening of food animals. Another limitation is that our phylogenetic analysis was primarily focused on the comparison of this bioaerosol sample to North American strains of IDV, this could be expanded to include other reported strains. As other animals present on the farms included in this study might have also influenced the detection of IDV, additional study is needed to determine if poultry can become infected with IDV and/or transmit this virus.
Our finding that IDV is detectable in bioaerosols near poultry farms suggest that commercial food production activities may be generating infectious aerosols. Such industries may, therefore, benefit from aerosol sampling to strengthen surveillance to protect the public from respiratory viruses. This strengthened surveillance may also support public health responses to respiratory virus detection by encouraging the use protective equipment (such as respirators or masks) by at risk workers or customers.
Availability of data and materials
The data collected during the current study are available from the corresponding author on reasonable request.
Basic local alignment search tool
Bovine respiratory disease
Influenza D virus
National center for biotechnology information
National institute of occupational safety and health
Real-time polymerase chain reaction
Unweighted pair group method with arithmetic mean
Hause BM, et al. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses. PLoS Pathog. 2013;9(2):e1003176.
Foni E, Chiapponi C, Baioni L, Zanni I, Merenda M, Rosignoli C, Kyriakis CS, Luini MV, Mandola ML, Bolzoni L, Nigrelli AD, Faccini S. Influenza D in Italy: towards a better understanding of an emerging viral infection in swine. Sci Reports. 2017;7(1):11660. https://doi.org/10.1038/s41598-017-12012-3.
Oliva J, Eichenbaum A, Belin J, Gaudino M, Guillotin J, Alzieu JP, Nicollet P, Brugidou R, Gueneau E, Michel E, Meyer G, Ducatez MF. Serological Evidence of Influenza D Virus Circulation Among Cattle and Small Ruminants in France. Viruses. 2019;11(6):516. https://doi.org/10.3390/v11060516.
Quast M, et al. Serological evidence for the presence of influenza D virus in small ruminants. Vet Microbiol. 2015;180(3–4):281–5.
O'Donovan T, et al. Seroprevalence of influenza D virus in selected sample groups of Irish cattle, sheep and pigs. Ir Vet J. 2019;72(1).
Ferguson L, Olivier AK, Genova S, Epperson WB, Smith DR, Schneider L, Barton K, McCuan K, Webby RJ, Wan XF. Pathogenesis of Influenza D Virus in Cattle. J Virol. 2016;90(12):5636–42. https://doi.org/10.1128/JVI.03122-15.
White SK, et al. Serologic evidence of exposure to influenza D virus among persons with occupational contact with cattle. J Clin Virol : Official Publication Pan Am Soc Clin Virol. 2016;81:31–3.
Ferguson L, et al. Influenza D virus infection in Mississippi beef cattle. Virology. 2015;486:28–34.
Foni E, et al. Influenza D in Italy: towards a better understanding of an emerging viral infection in swine. Sci Rep. 2017;7(1):11660.
Salem E, et al. Serologic evidence for influenza C and D virus among ruminants and Camelids, Africa, 1991-2015. Emerg Infect Dis. 2017;23(9):1556–9.
Zhai SL, et al. Influenza D virus in animal species in Guangdong Province. Southern China Emerg Infect Dis. 2017;23(8):1392–6.
Horimoto T, et al. Nationwide Distribution of Bovine Influenza D Virus Infection in Japan. PLoS One. 2016;11(9).
Hause BM, Huntimer L, Falkenberg S, Henningson J, Lechtenberg K, Halbur T. An inactivated influenza D virus vaccine partially protects cattle from respiratory disease caused by homologous challenge. Vet Microbiol. 2017;199:47–53. https://doi.org/10.1016/j.vetmic.2016.12.024.
Borkenhagen LK, et al. Surveillance for respiratory and diarrheal pathogens at the human-pig interface in Sarawak. Malaysia PLOS ONE. 2018;13(7):e0201295.
Coleman KK, et al. Bioaerosol sampling for respiratory viruses in Singapore’s mass rapid transit network. Sci Rep. 2018;8(1):17476.
Bailey ES, et al. Molecular surveillance of respiratory viruses with bioaerosol sampling in an airport. Trop Dis Travel Med Vaccines. 2018;4:11.
Ducatez MF, Pelletier C, Meyer G. Influenza D Virus in Cattle,. France, 2011–2014. Emerg Infect Dis. 2015;21(2):368–71.
Nguyen TT, et al. Bioaerosol Sampling in Clinical Settings: A Promising, Noninvasive Approach for Detecting Respiratory Viruses. Open Forum Infect Dis. 2017;4(1):ofw259.
We thank William G. Lindsley for his support in providing the NIOSH 2-stage air samplers. We thank the Director General of Health Malaysia for his permission to publish this paper.
This study was supported by Duke University Bass Connections and Duke University discretionary funding (Gray PI).
Ethics approval and consent to participate
The study was exempted from ethical review and approval by the Medical Research & Ethics Committee Ministry of Health Malaysia because all samples were environmental in nature. Permission was sought to sample on farms and near poultry cages.
Consent for publication
The research investigators had complete freedom to conduct this research and report results. There were no other potential conflicts of interest to disclose.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Bailey, E.S., Fieldhouse, J.K., Alarja, N.A. et al. First sequence of influenza D virus identified in poultry farm bioaerosols in Sarawak, Malaysia. Trop Dis Travel Med Vaccines 6, 5 (2020). https://doi.org/10.1186/s40794-020-0105-9
- Influenza D virus