Result
The H1 phylogeographic tree showed two large independent clusters within
Iran, suggesting the initiation of two independent effective outbreaks
with different tMRCAs, one in the past years (1997) and the other in
recent years (2005). The first outbreak was initiated from the north
region of the country and most of time was circulated between the north
and northwest region within the country and established some small
introduction in the southwest region in the late years (2008). The
phylogeographic tree showed that the second initiation was introduced in
the north region and had concentrated circulation in the cities within
this region by 2013 and then, circulation of virus expanded to the west
region and continued to 2015, therefore based on our result inferred
cautiously there were small and concentrated epidemics which had
occurred in most parts of the country which came along until recently,
despite the rapid expansion and decline in the region, it has remained
stable (Figure 1). Along with the circulation of virus within the
country, the phylogeographic tree revealed that intercountry
transmission occurred in circulation between southwest region, Turkey
and Afghanistan and again back transmission to the north region in Iran
from 2007 to 2010, in addition, same back transmission was observed in
cities were located in the west region and close to the Turkey border
during 2013 to 2015. So, we could infer cautiously that, the virus has
entered the country many times in the form of multiple introductions and
spread, and according to our data, it did not have a single independent
source and the age of presence of the virus in Iran is older than
neighboring countries. Based on our genome set, lineages from different
parts of the country branched within the lineages from Tehran,
highlighting the role of Tehran as the epicenter of the outbreaks in the
country. After 2010, small and concentrated epidemics in other cities
have their origins in Tehran.
According to the above-mentioned,
generally we concluded that probably the virus has been circulating in
Tehran with low frequency for a little period and has gradually spread
throughout the region and neighboring countries in the following years
in a single and sporadic cases. The small genetic distance between the
different branches of these clusters in this period suggests rapid
spread of the virus in this specific geographical area during the narrow
time (2007-2009) in Iran and neighboring countries. Based on our genome
set the virus was still present in small numbers in some countries, such
as Turkey, where the virus was mainly circulating in comparison to other
neighboring countries.
The H1 phylogeographic tree based on different geographical regions was
constructed separately and showed that two independent clusters same as
the above results were created for our total sequences and
geographically suggested that the virus circulation started from north
and northwest to east and southwest of Iran over time, and then moved to
east and southeast in recent years (Figure 2), and this indicates the
correct goodness of fit of the model.
Results of the N1 phylogeographic tree were in line with the H1 tree,
which two independent clusters among sequences from Iran, Turkey, Iraq,
and Turkmenistan were observed, with the sequences related to Turkey
occupying the root of the tree (Figure 3). The phylogeographic tree
showed that the transmission and circulation pattern was like H1 tree,
and the northern region is the epicenter of this transmission which had
taken place bilaterally and had occurred between the north of the
country and Turkey. During a short time, the circulation of virus
involved the whole country (Figure 4).
It should be noted that, if there were more sequences from other
countries in our genome set, different results could be expected from
the model outputs.
The evolutionary rate for the HA and NA genes of the H1N1 viruses were
estimated at 4.01×10-3 [95% HPD: (2.82-5.40)
×10-3] and 3.30×10-3 [95% HPD:
(1.33-5.43) ×10-3] substitutions/site/year,
respectively. The mean time of the most recent common ancestor of
H1N1 viruses was 1989 (95% HPD: 1980-1994) for HA and NA as well.
The estimation of dN/dS ratio per site for each lineage was conducted.
After excluding the duplicated sequences, 210 sequences with 443 sites
for HA and 85 sequences with 91 sites for NA lineages were analyzed. The
SLAC analysis of HA lineages showed that 17 sites were under
negative/purifying selection while no positive/diversifying selection
were observed. The overall dN/dS for HA lineages was 0.403. For the NA
lineages, 14 negative/purifying selection sites and no
positive/diversifying selection were observed. The overall dN/dS for NA
lineages was 0.286. Based on the FEL analysis of HA lineages, 48 and 2
sites were under negative/purifying and positive/diversifying
selections, respectively. This was 24 and 1 selection sites for the NA
lineages, respectively. The results of Generic Algorithm Recombination
Detection (GARD) showed no evidence of recombination.