Page 28 - Avian Virology: Current Research and Future Trends

Avian Influenza Virus | 21

single-stranded, uncapped vRNAs is usually driven by a host’s detection of IAV by either classical methods of virus isolation, or
RNA polymerase I (pol I) promoter (Engelhardt, 2013). In detection of a subcomponent of the viral particle such as nucleic
eukaryotic cells, the RNA pol I complex produces uncapped acids or proteins. Post-exposure evaluation is usually performed
ribosomal RNA. RNA pol I-mediated transcription of vRNAs is by the presence of antibodies against some of the viral proteins.
initiated and terminated on defined sites. The most common pol-I With technologies ever evolving, development of novel aims for
driven RG systems for influenza viruses use the murine RNA more specific, more sensitive and more cost-effective assays. The
polymerase I terminator (T-1) sequence, also known as Sal I box. ‘gold standard’ for avian-origin IAV identification is viral isolation
An alternative method to produce vRNAs is through the use of a (VI) in specific pathogen free (SPF) embryonated chicken eggs
T7 RNA polymerase promoter directly upstream of viral cDNA (Hirst, 1941; OIE, 2017). VI is highly sensitive and widely used
cloned in the negative sense (de Wit et al., 2007). The 3′ end of (Cross et al., 2012). The procedure is performed by inoculating
the vRNA is formed by the hepatitis delta ribozyme cleavage the swab containing media into the allantoic cavity of 9- to 11-day-
sequenced cloned immediately downstream. Transcription of the old chicken embryonated eggs in the presence of high antibiotic
eight vRNAs, together with the four protein expression plasmids concentration to avoid bacterial growth. After 48 hours of incuba-
responsible for viral replication and transcription (PB2, PB1, PA tion at 35–37°C, IAV presence in the allantoic fluid is assessed by
and NP), allows for the generation of influenza in HEK293T and a secondary detection method (Spackman and Killian, 2014).
a variety of other human derived cell types (Fig. 1.5) (Fodor et al., The most commonly used method is the haemagglutination assay
1999; Neumann et al., 1999). Different protocols have been devel- (see below). If the allantoic fluid of the first passage is negative,
oped (Chen et al., 2012, 2014). However, the most widely used a second (and even a third) blind passage is performed using the
method is based on the eight-plasmid system with bidirectional undiluted allantoic fluid from the previous passage. Once isolated,
promoters (Hoffmann et al., 2000a,b). Bi-directional vectors have this method allows further laboratory analysis of the specimen.
been made that contain pol II and pol I promoters in opposite Despite its historical relevance, this method is not suited for high-
directions to drive expression of both viral mRNAs and vRNAs throughput detection, it is time consuming and likely imposes a
from the same plasmid, thus consolidating the viral rescue system bottleneck on strains that are poorly fit to replicate in eggs.
into eight plasmids (Hoffmann et al., 2000a). Alternative RG The principle of the haemagglutination assays (HA assay) is
systems have been developed since recovery of the virus depends based on the capacity of the HA protein to agglutinate with red
on the capacity of the cell line to be transfected and the capacity blood cells (RBC) forming a lattice and preventing their sedimen-
of the virus to replicate in the given cell line (Fig. 1.5). Since pol I tation (Killian, 2008). The RBCs can be of multiple species and
promoter activity is species specific, RG systems require a species different IAV strains have different capacities to agglutinate them.
match between the promoter and the cell type. For instance, the Chicken and turkey RBCs are commonly used in the detection
swine polymerase I promoter is used in swine cell lines whereas of IAVs of avian-origin. The RBC suspension is incubated in the
the chicken (avian) polymerase I promoter is used in avian cell presence of serial two-fold dilutions of the virus in a 96 V-bottom
lines (Perez et al., 2017). From studies that allowed a better well plastic plate. Negative samples form a red dot at the bottom
understanding of the host range and the transmission of zoonotic of the well due to sedimentation of the non-agglutinated RBC.
strains to the unravelling of the mechanisms of pathogenesis of In order to produce haemagglutination, the assay requires a rela-
the once extinct 1918 Spanish pandemic influenza virus, RG tively high load of virus making it a good complement of VI. The
has revolutionized IAV research. Furthermore, novel attenuating result is expressed as haemagglutination units (HAU). Although
mutations and large genomic modifications have been intro- not all the HA subtypes show the same capacity to agglutinate
duced for the design and production of live attenuated vaccines, RBCs and other infectious agents show this property as well, the
improving live vaccines safety and with the potential to produce assay is relatively simple and does not require specialized equip-
universal vaccines (Finch et al., 2015). Fluorescent proteins are ment.
coupled to the virus segments to track different aspects of the Several serological tests are available for detection of
viral infectious cycle (Nogales et al., 2015). Chemiluminescent antibodies against IAV. A modification of the HA assay, the
proteins are used as markers for anti-viral selection (Sutton et al., haemagglutination inhibition assay (HAI), allows diagnosis of
2014). The diverse applications of RG highlight the robustness of previous exposures to IAV by detecting antibodies that prevent
an always adaptable tool. haemagglutination (Pedersen, 2014). The HAI is used to perform
subtyping of IAV isolates and to distinguish them from other
RBC agglutinating agents, such as NDV, using a panel of hyperim-
Diagnosis mune sera prepared against the different HA subtypes and NDV.
Definitive diagnosis of avian influenza must be made by serologi- Similarly, NA subtyping can be performed with neuraminidase
cal and virological methods to differentiate from other diseases inhibition assay (NI) using antisera prepared against the nine NA
that can cause similar disease signs such as Newcastle disease virus, subtypes. Newer sequencing technologies allow for much faster
avian pneumovirus, infectious laryngotracheitis virus, infectious and easier subtyping than the aforementioned traditional meth-
bronchitis virus, chlamydia, mycoplasma, fowl cholera (Pasteurella ods. The HAI assay, however, remains as the method of choice to
multocida), E. coli, and other bacteria. Secondary and concurrent determine antigenically distinct variants within a single subtype,
infections with avian influenza are common in poultry. Cloacal, which is not yet possible using the current methods of sequence
faecal or tracheal swab samples obtained from birds are used for analysis.