Sunday, July 08, 2018

BMC Infect Dis.: Key Amino Acid Substitutions In Egyptian H5N1 Isolates

Credit BMC Infectious Diseases


Despite ambitious International Health Regulations adopted by WHO member states in 2005 - which required all nations to develop mandated surveillance and testing systems, and to report certain disease outbreaks and public health events to WHO - there remain large swaths of the globe where surveillance and reporting are either weak, suppressed, or virtually nonexistent. 
The implementation of these standards is a big job, of course (see Ten things you need to do to implement the IHR), and requires a major commitment of money and resources by all member states.
While many of the delays in implementing the IHR over the past decade can be attributed to a lack of resources, or there are many countries where compliance just isn't at the top of their to-do list  - or worse - where reporting of diseases is thwarted for political or economic reasons.
One of those places from where - for a number of years - we've gotten precious little information is Egypt (see Why No News Isn't Necessarily Good News), which holds the world's record for the most human H5N1 infections (n=359). 
While impressive, the official number of 359 human infections is believed to be a major underestimate.  This passage from the 2016 EID Journal: H5N1 In Egypt explains why:

In Egypt, the number of reported human cases of avian influenza infection appears to be underestimated. An underestimation might result in an overestimation of the case-fatality rate, but it would certainly underestimate the extent of human infection with avian influenza viruses.
Results from a controlled, serologic cohort study of persons in Egypt exposed and not exposed to poultry estimated the seroprevalence of antibodies against H5N1 (titers >80) at 2% (19). If this seroprevalence were to be extrapolated to the entire poultry-exposed population in Egypt, the true number of infections would amount to several hundred thousand.
These figures are even more striking when it comes to human infection with H9N2 viruses. The seroprevalence of H9N2 antibodies detected in the same cohort study (19) ranged from 5.6% to 7.5%, whereas just 1 case of H9N2 infection was reported.
Although the government remains tight lipped about H5N1 (and other avian viruses), we do see occasional studies published, which provide us with a sense of what may be going on with HPAI viruses in Egypt.

A few recent examples include:
Sci. Reports: Efficacy Of AI Vaccines Against The H5N8 Virus in Egypt
EID Journal: Multiple Introductions Of HPAI H5N8 In Egypt

Virology Journal: Evolution of H5N1 Clade 2.2.1 In Egypt
We've a new study to look at today - from BMC Infectious Diseases -  that examines the prevalence and spatial patterns of key amino acid substitutions (KS) found in Egyptian HPAI H5N1 isolates deposited in Genbank between 2005-2015.
These Key Substitutions (KS) are those mutations that have been previously identified as potentially aiding the virus in becoming better adapted to mammalian hosts, or that increase its transmissibility, or virulence.
Given past studies suggesting that the right 3 to 5 key substitutions occurring simultaneously might greatly increase H5N1's pandemic potential (see The Fouchier Ferret Study and the The Kawaoka H5N1 Study), tracking the actual progression and prevalence of these changes in the wild might give us a head's up on H5N1's threat level.

Unfortunately, as the following passage from today's study indicates, the quantity and quality of raw data out of Egypt has declined significantly since 2011 (emphasis mine).
Samples with governorate location information uploaded to GenBank varied from year to year, with a low of 39 in 2005–6 and a high in 2009 with 155 isolates, however the sample sizes dropped after 2011.
It is not entirely clear why fewer isolates from Egypt are being uploaded to GenBank than previously, although political turmoil and social unrest in the wake of the 2011 revolution are likely responsible.
Detection rates of KS also varied, trending down during the study period from a high of 0.38 in 2007 to a low of 0.18 in 2014 (see Fig. 3). This decline is likely driven a smaller number of protein sequences per sample being uploaded in later years of the study period, with the HA gene being the most commonly uploaded.
HA sequences tend to have lower detection rates due to the larger number of known HA KS being tested for. The problem of outdated or incomplete reporting of avian influenza is not new in the country, and several studies suggest the true burden of disease in Egypt is severely underestimated [3, 61].

Despite these limitations, what follows is a fascinating survey of 39 known key amino acid substitutions identified in Egyptian H5N1 isolates over a 10 year span.  
Whether the `right' combination needed to turn H5N1 into a pandemic strain will ever become fixed in the wild remains a matter of conjecture. But the potential certainly exists.
The full open-access study is available at the link below, so I've only excerpted the abstract.  Follow the link to read it in its entirety.
Unlocking pandemic potential: prevalence and spatial patterns of key substitutions in avian influenza H5N1 in Egyptian isolates

Sean G. YoungEmail authorView ORCID ID profile,  Andrew Kitchen,
Ghazi Kayali and  Margaret Carrel
BMC Infectious Diseases201818:314

© The Author(s). 2018

Published: 6 July 2018



Avian influenza H5N1 has a high human case fatality rate, but is not yet well-adapted to human hosts. Amino acid substitutions currently circulating in avian populations may enhance viral fitness in, and thus viral adaptation to, human hosts. Substitutions which could increase the risk of a human pandemic (through changes to host specificity, virulence, replication ability, transmissibility, or drug susceptibility) are termed key substitutions (KS). Egypt represents the epicenter of human H5N1 infections, with more confirmed cases than any other country. To date, however, there have not been any spatial analyses of KS in Egypt.


Using 925 viral samples of H5N1 from Egypt, we aligned protein sequences and scanned for KS. We geocoded isolates using dasymetric mapping, then carried out geospatial hot spot analyses to identify spatial clusters of high KS detection rates. KS prevalence and spatial clusters were evaluated for all detected KS, as well as when stratified by phenotypic consequence.


A total of 39 distinct KS were detected in the wild, including 17 not previously reported in Egypt. KS were detected in 874 samples (94.5%). Detection rates varied by viral protein with most KS observed in the surface hemagglutinin (HA) and neuraminidase (NA) proteins, as well as the interior non-structural 1 (NS1) protein. The most frequently detected KS were associated with increased viral binding to mammalian cells and virulence. Samples with high overall detection rates of KS exhibited statistically significant spatial clustering in two governorates in the northwestern Nile delta, Alexandria and Beheira.


KS provide a possible mechanism by which avian influenza H5N1 could evolve into a pandemic candidate. With numerous KS circulating in Egypt, and non-random spatial clustering of KS detection rates, these findings suggest the need for increased surveillance in these areas.
       (Continue . . . . )

Given 12 years of intensive H5N1 activity in poultry and in humans, and the recent introduction of both H5N8 and H9N2 - along with a reasonably modern medical infrastructure and the presence of U.S. Naval Medical Research Unit-No. 3 (NAMRU-3) in Cairo - Egypt ought to be the ideal place to study and monitor the evolution of HPAI H5 viruses.
Sadly, for the past 5 or 6 years, those opportunities have been largely squandered, and avian flu research is much poorer for it. 
For now, what happens with avian flu in Egypt, pretty much stays in Egypt.  Unfortunately, should H5N1 acquire the right key substitutions, there isn't much hope of it remaining just an Egyptian problem.