Page 29 - Avian Virology: Current Research and Future Trends

22 | Perez et al.

The HAI assay cannot detect all anti-HA antibodies, only well-trained personnel, ample knowledge in bioinformatics, and
those that target regions close to the receptor binding site. Alter- expensive equipment.
native methods detection of antibodies against influenza are
the traditional agar gel immunodiffusion (AGID) assay and the Prevention and control of avian influenza
enzyme-linked immunosorbent assay (ELISA). AGID is consid- Close contact resultant from domestication and captivity of many
ered the ‘gold standard’ by the OIE for detection of anti-influenza bird species has led to exposure of humans to the potential bio-
antibodies. AGID is a low-cost assay that uses an agar matrix to hazards carried and/or transmitted by these animals, such as AI.
support the precipitation of the antigen–antibody complex. This Live bird markets have played a major role in the emergence of
assay is particularly sensitive for detection of anti-influenza NP HPAI, with many being the originators of very important HPAI
or M1 antibodies in chicken and turkey sera, but it is less reli- outbreaks (e.g. markets in the east coast of the USA, in Hong
able for other avian species (Spackman et al., 2009). The ELISA Kong, and Italy). Without proper biosecurity measures, the virus
is a plate-based assay in which the antigen–antibody complex is can be easily spread from flock to flock. Excretions from infected
detected by a secondary antibody conjugated to an enzyme that birds are the major source of contamination, and movement of
in presence of its substrate produces a colorimetric reaction. infected birds, contaminated equipment, clothing, hands, shoes,
Commercially available, species-independent, kits are available egg flats, feed trucks, water, and food are all major contributors
that detect the anti-NP antibodies. Suggested by the OIE, AGID in the spread of the virus. Studies have shown that vehicles and
may be used to confirm the ELISA results. With the advent of equipment are important for transmission of HPAI viruses from
protein microarrays and other diagnostic technologies, several farm to farm (Capua et al., 2003a; Biswas et al., 2008; Chaudhry
groups have devised alternative multiplex diagnostic assays for et al., 2015). Airborne transmission may occur, but it is rather
simultaneous detection of viral antigen of (or antibodies against) limited to birds that are in close proximity.
different subtypes in simplified formats. Implementation of biosecurity measures is the first line of
To assess infection, screening tests that allow high-throughput defence against AI, and essential to secure the production sector
detection can complement and/or replace VI. The Real time and food security, as well as to limit the risk of human infection
reverse transcription polymerase chain reaction (RRT-PCR) (OIE, 2017). Thus, strict biosecurity measures and good hygiene
is widely used for its high sensitivity, specificity, and speed of are key for preventing and controlling the spread of AI and are
detection. The most common RRT-PCR methods are the SYBR related to controlling the contamination of equipment and
Green-based or TaqMan-based methods. The SYBR Green personnel. Since wild birds are considered the major source of
method is based on the binding of a fluorescent dye into the PCR infection for domestic poultry, it is essential to reduce the con-
product. The TaqMan methods uses a dual-labelled probe to the tact between these two groups. Flocks and production facilities
PCR product developing a signal by loss of fluorescence quench- should maintain strict control over access by vehicles, people and
ing as PCR degrades the probe. The TaqMan is the method of equipment, and guarantee proper cleaning and disinfecting of
choice and probes to detect the highly conserved matrix gene seg- the facility and equipment. Appropriate education programs are
ment or that are subtype specific have been developed are widely essential to guarantee that the population in contact with poultry
used (Spackman et al., 2002; Das et al., 2006). species understands about AI and its risks, monitoring, report-
Next generation sequencing (NGS) is quickly replacing not ing and response initiatives. This level of awareness assures that
only the traditional Sanger-based sequencing but also shows producers and workers know how to identify clinical signs of the
several advantages over traditional diagnostic methods NGS disease and report any AI-like illness and deaths to the authorities
approaches are also more powerful since the output is the entire and Veterinary Services.
virus genome which helps in understanding and defining poten- Another essential step for avian influenza prevention is the
tial virulent markers, zoonotic potential and epizootiological epizootiological surveillance and early detection followed by a
significance (Grad and Lipsitch, 2014). Different platforms are rapid response. Surveillance efforts during the 1983–1984 out-
available but currently, the Illumina is the most widely used. breaks in Pennsylvania were able to track the origin of the virus to
For IAV Illumina-based NGS sequencing, a multisegment live bird markets. Likewise, the constant monitoring of influenza
RT-PCR (MS-RTPCR) amplification is carried out to enrich activity in the live bird markets of Hong Kong helped to quickly
the sample. The MS-RTPCR is performed by using primers characterize the 1997 H5N1 virus that transmitted to humans
targeting the conserved 12–13 nucleotides at the end of all from chickens (Sims et al., 2003b). Through data obtained by
8 genomic segments (Zhou and Wentworth, 2012; Mena et global surveillance, it was possible to trace back the origin of
al., 2016). Once the full genome is amplified, the sample can the reassortant H5N2 virus that caused the recent outbreak in
be processed for NGS that produces massive high-throughput the USA, which resulted in culling of more than 50 million tur-
producing millions of sequencing reactions at the same time, keys and chickens. It seems that this virus emerged in China in
is highly sensitive and specific. One of the most valuable char- 2013, spread through Asia and reached the USA via migratory
acteristics of this technology is that it allows sequencing from birds (Lee et al., 2015). State and federal authorities are respon-
the original sample avoiding the selective bottleneck imposed sible for developing surveillance, quarantine, stamping out, and
by VI since this selection may distort the characteristics of indemnification programs and policies, following the guidelines
the virus present in the natural hosts. NGS is replacing first of the OIE Terrestrial Animal Health Code, particularly in states
generation sanger sequencing. However, this technique requires where poultry represents an economically important commodity.