Page 27 - Avian Virology: Current Research and Future Trends

20 | Perez et al.

of multiple stochastic instances of reassortment among these example, clade 2 has expanded into five second order clades, with
lineages in many parts of the world (Makarova et al., 1999; some of them undergoing further expansion and being assigned
Hansbro et al., 2010; Gonzalez-Reiche and Perez, 2012; Barriga into third and fourth order clades (WHO et al., 2015). Recently, a
et al., 2016; Hurt et al., 2016; Nelson et al., 2016; Gonzalez- novel approach called Lineage Assignment By Extended Learning
Reiche et al., 2016, 2017). The endemic nature of LPAIVs and (LABEL) was developed to quickly identify the clade assignment
HPAIVs in poultry in Southeast Asia, the spill over of these information of H5 and H9 HA genes without the need for time-
viruses to wild birds, climate change that may alter the migra- consuming sequence alignment, phylogenetic tree construction,
tory patterns of birds, and perhaps unique features of particular or manual annotation (Shepard et al., 2014).
strains can result in the emergence of strains with a mixture of The term ‘reverse genetics’ (RG) is an approach to unravel the
genetic lineages, such as the HPAIV virus responsible for the function of a gene by establishing and analysing the phenotypic
outbreak in turkeys and layers in the Midwest of the US in 2014 effects of (artificially) engineered gene sequences (Fig. 1.5). In
(Spackman et al., 2016; Santos et al., 2017), or the emergence case of RNA viruses, reverse genetics invariably requires the de
in wild birds of IAVs carrying the Eurasian-lineage H14 HA novo reconstitution of the virus from a cDNA copy (Perez, 2017).
and PA gene segments in Central America (Gonzalez-Reiche Using molecular biology, cDNA copies of RNA viruses are cloned
et al., 2017). into a variety of vectors, most typically and in order of prefer-
Evolution of IAVs in poultry in Asia is very complex and ence, plasmids, bacterial artificial chromosomes or bacmids, or
affected by spatiotemporal factors (Sims et al., 2005; Skeik and recombinant viral vectors (Fig. 1.5). The generation of viral
Jabr, 2008; Lvov et al., 2010; Lei and Shi, 2011; Su et al., 2015; Lee particles in vitro through RG is one of the most powerful tools
et al., 2017). Thus, once adapted to poultry, the viruses may follow in modern virology. Different RG systems for RNA viruses have
alternative evolutionary pathways that increase their divergence, been available for many years now (Fig. 1.5) allowing a deeper
exemplified by the evolution observed in the H9N2 LPAIVs in understanding of replication, virulence, pathogenicity, transmis-
Asia and the Middle East and the H5N1 HPAIVs in Eurasia and sibility and host range (Stobart and Moore, 2014; Perez, 2017).
Africa (Perez et al., 2003a; Choi et al., 2004; Xu et al., 2007a,b; The principle is based on the capacity of cells to be transfected
Cattoli et al., 2009; Guan et al., 2009; Lvov et al., 2010; Chu et al., by a vector (e.g. plasmids) containing the necessary functional
2011; Tombari et al., 2011; Lee et al., 2012; Shahsavandi et al., elements to produce the viral particle.
2012; Guan and Smith, 2013; Sonnberg et al., 2013; Dalby and RG systems for negative-sense RNA viruses are engineered
Iqbal, 2014; Davidson et al., 2014; Kandeil et al., 2014; Abdel- to produce not only the negative-sense vRNA, but also the posi-
whab et al., 2016). In the case of the Asian-origin H5N1 HPAIVs, tive sense viral mRNAs in order to initiate de novo virus progeny.
such divergence led the WHO, OIE, and FAO to adopt a unified For reverse genetics of influenza viruses, transcription of the
nomenclature to classify them into specific 10 clades (clade 0 viral mRNA proteins is typically under the control of a cellular
through 9) based on the HA sequence (WHO et al., 2009, 2012, RNA polymerase II (pol II) promoter element upstream of the
2014, 2015). Depending on the clade and whether it continues to cloned viral cDNA and expressed in a manner similar to the host’s
circulate, subclade assignments are adopted and/or updated. For mRNAs (Engelhardt, 2013). Synthesis of the negative-sense

Figure 1.5 Reverse genetic systems for engineering influenza A viruses.