New Avian Viruses Discovered with the Help of Sanger Sequencing

Written by Caroline McMahon and Cleta D’Sa

Iryna Goraichuk’s arrival in the United States to work for the U.S. Department of Agriculture, Agriculture Research Service (USDA-ARS) Exotic and Emerging Avian Viral Disease Unit in 2015 could not have come at a more opportune time—an unknown virus originally isolated from a goose had not yet been characterized. That goose and Goraichuk just happened to share a homeland: Ukraine. This instant connection strongly compelled Goraichuk to take on the difficult task of identifying a completely new avian virus, paramyxovirus 13 (APMV-13).

Iryna Goraichuk received her PhD in Biology at the National Scientific Center, Institute of Clinical and Veterinary Medicine (NSC IECVM), in Kharkiv, Ukraine, and is currently a visiting scientist at the USDA-ARS, where she studies Newcastle disease and avian influenza viruses. She developed expertise in the study of avian viruses when the NSC IECVM conducted avian influenza virus and paramyxovirus wild bird surveillance in the Azov and Black Sea regions of Ukraine.^1,2,3 Also, Ukraine was one of 119 countries that conducted avian influenza virus surveillance in wild birds between 2008 and 2013, and responded to aWorld Organisation for Animal Health-administeredsurvey during The Global Avian Influenza Surveillance in Wild Birds.⁴ Since birds come in from all directions during the winter, the Azov and Black Sea regions of Ukraine presented a perfect opportunity for researchers to study many birds at one time.

What are the benefits of using Sanger sequencing to identify new avian viruses?

Next-generation sequencing (NGS) is a relatively new technology that did not exist when the almost-complete genome of avian paramyxovirus 10 (APMV-10), which was found in penguins from the Falkland Islands during routine surveillance and which has yet to be found in any other birds, was shotgun-sequenced using Sanger sequencing.⁵ The small fragments (100-1000 bp) of the complete genome were randomly amplified, sequenced and assembled like a puzzle. Specific design primers were used to close any gaps, a labor-intensive but very reliable process. In 2010, Sanger sequencing was the only technique that enabled scientists to collaborate to obtain the whole genome of an unknown virus at the USDA-ARS Southeast Poultry Research Laboratory (SEPRL). Years later, Goraichuk arrived there and was able to confirm the accuracy of Sanger sequencing and to obtain the last 231 bp using NGS.⁶

The APMV-13 case was completely the opposite. Following up on the research associated with the influenza surveillance in the Azov and Black Sea regions of Ukraine, Goraichuk sequenced the almost-complete genome of a previously unknown serotype of avian paramyxovirus.⁷ But even though NGS has high sensitivity, the team faced a common issue in viral NGS sequencing: missing data at the genome termini. And only Sanger sequencing, with its high sensitivity and ability to sequence specific regions of the genome, enabled the team to close the gaps and obtain the first complete genome of the new serotype 13.

According to Goraichuk, “Even though you can split molecular biology [techniques] into ‘old’ and ‘new’ by their dates of invention, it does not mean that older techniques are not applicable anymore. Today, the NGS whole-genome sequencing supersedes the Sanger shotgun sequencing due to the time-consuming protocol of the latter. However, the high-precision and sensitivity of Sanger sequencing make it irreplaceable in confirming point mutations, determining the position of cloned DNA, sequencing short internal gaps, or missing data at the genome termini.” ^{8, 9, 10}

Goraichuk notes that the primary benefit of using Sanger sequencing is that short pieces can be sequenced, and you can sequence exactly what you want. “If you’re not interested in a whole genome and want to stick on just a particular gene, you can do this. Plus, it’s very affordable. There are a lot of lesser economically developed countries that cannot afford NGS, but they can always do PCR, cut pieces of gels, and send them to labs who will do Sanger sequencing for them.”

Research and global impact

Between 1926 and 1978, nine different serotypes of avian paramyxovirus were discovered. The serotype 10 (APMV-10) found in the penguins from the Falkland Islands was the first new serotype isolated after 30 years of “silence.” Since then, 10 more new serotypes have been discovered, proving how Sanger sequencing and NGS techniques can lead to successful discovery of new viruses.

Viruses, especially RNA viruses, tend to have high rates of mutation. Avian viruses in particular are very easy to spread around the globe due to bird migration and interactions between wild and domestic birds. Goraichuk is very passionate about the One Health Initiative, which highlights the relevance of zoonotic diseases and their global impact, and believes it is important to conduct surveillance to identify any new viruses due to potential risks to wildlife, poultry and human health. For example, the bird flu, caused by the avian influenza virus, can also infect humans. To date, avian influenza viruses have crossed the species barrier multiple times, causing human infections and leading to pandemics in the past. The original APMV-10⁵ and the APMV-13^{7, 11} research was presented at international conferences and is leading to the research and development of vaccines against avian influenza and Newcastle disease viruses.

Goraichuk enjoys other passions as well, and hones her analytical skills outside of work. As a painter herself, she enjoys attending “downtown painting facilities,” where she meets new people who all offer different thoughts, perspectives and interpretations of a single painting. She combines her research work with her hobby as she can often be found working in her home office, surrounded by her paint supplies, plants, and her many, many research papers.

Explore Sanger sequencing and fragment analysis applications, instrumentation, reagents, consumables, and software designed to respond to the unlimited potential of scientific inquiry.

References:

1. Muzyka, D., Pantin-Jackwood, M. et al. (2016) “Isolation and genetic characterization of avian influenza viruses isolated from wild birds in the Azov-Black Sea region of Ukraine (2001-2012),” Avian Diseases, 60(1s), pp. 365–377. doi:10.1637/11114-050115-Reg
2. Muzyka, D., Pantin-Jackwood, M. et al. (2014) “Wild bird surveillance for avian paramyxoviruses in the Azov-black sea region of Ukraine (2006 to 2011) reveals epidemiological connections with Europe and Africa,” Appl Environ Microbiol, 80(17), pp. 5427-5438. doi: 10.1128/AEM.00733-14
3. Muzyka, D., Pantin-Jackwood, M. et al. (2012) “Avian influenza virus wild bird surveillance in the Azov and Black Sea regions of Ukraine (2010-2011),” Avian Diseases, 56(4Suppl), pp. 1010-1016. doi: 10.1637/10157-040912-ResNote.1
4. Machalaba, C., Elwood, S. et al. (2015) “Global avian influenza surveillance in wild birds: a strategy to capture viral diversity,” Emerg Infect Dis. 21(4), pp. e1–7. doi: 10.3201/eid2104.141415
5. Miller, P.J., Afonso, C.L. et al. (2010) “Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands,” J Virol, 84(21), pp. 11496-11504. doi:10.1128/JVI.008220-10
6. Goraichuk, I.V., Dimitrov, K.M. et al. (2017) “Complete genome sequences of four avian paramyxoviruses of serotype 10 isolated from rockhopper penguins on the Falkland Islands,” Genome Announc, 5(22), pp. e00472-17. doi:10.1128/genomeA.00472-17
7. Goraichuk, I., Sharma, P., Stegniy, B. et al. (2016) “Complete genome sequence of an avian paramyxovirus representative of putative new serotype 13,” Genome Announc, 4(4),pp.e00729-16. doi:10.1128/genomeA.00729-16.
8. Goraichuk, I.V., Msoffe, P.L.M., Chiwanga, G.H. et al. (2019) “First complete genome sequence of a subgenotype Vd Newcastle disease virus isolate,” Microbiol Resour Announc, 8(27), pp. e00436-19. doi: 10.1128/MRA.00436-19
9. Sabra, M., Dimitrov, K.M., Goraichuk, I.V. et al. (2017) “Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe, Asia, and Africa,” BMC Veterinary Research, 13(1), p. 291. doi: 10.1186/s12917-017-1211-4.
10. Ramey, A.M., Goraichuk, I.V., Hicks, J.T. et al. (2017) “Assessment of contemporary genetic diversity and inter-taxa/inter-region exchange of avian paramyxovirus serotype 1 in wild birds sampled in North America,” Virology Journal, 14(1), p. 43. doi: 10.1186/s12985-017-0714-8
11. Goraichuk, I., Poonam, S. et al. (2016) “Phylogenetic analysis of the complete genome of the APMV-13 isolate from Ukraine,” International Journal of Infectious Diseases, Poster Presentation, 45(Supplement 1), p. 459. doi: 10.1016/j.ijid.2016.02.972