In a span of few weeks, A(H1N1)pdm09 viruses had diversified sufficiently to form 7 distinct clades, although the epidemiological behavior of these viruses was largely uniform with certain risk groups (such as pregnant women, diabetics, obesity) more prominently vulnerable than others 
. These viruses were detected in Pakistan as early as June 2009. The earlier cases were travel importations during June–August 2009, and increased number of domestic infections was observed from September 2009 to March 2010.
Present study demonstrates that even the earliest imported isolates clearly belonged to clade 7. All the sequenced viruses belonged to clade 7 with signature change S203T, and no clade 5 or 6 that first appeared in Asia in May–September, 2009 
were found in this study. Since the first confirmed cases from Pakistan were reported relatively late (June; week 13), and the limited number of viruses sequenced in this study; this may partly explain the presence of only clade 7 isolates in current analysis which were prominent in Asia from week 9 onwards.
The epidemiological data (not shown here) demonstrated that the early cases in Pakistan were imported through foreign travelers; however soon after that indigenous evolution and transmission resulted in widespread of the virus. The presence of a large number of mild or sub-clinical cases combined with a limited surveillance network for case detection may explain the relatively low number of reported and confirmed A(H1N1)pdm09 cases in Pakistan.
The antigenic profile of all tested 2009–2010 Pakistan isolates showed that these viruses were antigenically close to the A/California/7/2009 vaccine virus. Therefore, the advisability of identifying risk groups through serological studies and developing appropriate vaccination strategies cannot be overstressed.
Diagnostic use of real-time PCR assay has already proven to be highly effective in the detection of seasonal influenza viruses. The rapid development and prompt dissemination of the real time PCR diagnostic assay for detection of swine influenza A (H1N1) virus was instrumental in institution of rapid response and control measures across the Global Influenza Surveillance Network (GISN). The typical period during which the virus may be detected with the use of real-time RT-PCR is 6 days (whether or not fever was present at the time of sample collection). In the absence of a fully robust and proficient surveillance and sample delivery capacity, we believe that many asymptomatic or mildly symptomatic cases may have escaped testing and treatment.
The sequence data of the Pakistan viruses showed a high homology for all eight genes to the A(H1N1)pdm09 viruses from neighboring countries and to A/California/07/2009 (nucleotide identity ranged from 99–100%). The isolates group indistinguishably with other viruses on phylogenetic analysis. There is no evidence of gene reassortment between pandemic strain and co-circulating seasonal influenza H1N1 or H3N2 viruses during this time period 
. One isolateA/154/09 had a prominent long branch on HA tree since the number of analyzed viruses is quite small. As more viruses are sequenced and added to GenBank, the apparent gaps/distances observed will become clear.
Certain amino acid substitutions such as D222G in HA have been reported in connection with severe disease and poor outcome 
. As none of the Pakistan viruses analyzed in this study had this substitution or a fatal outcome, their significance merits further study especially in more serious cases reported from Pakistan. One Pakistan isolate, A/Pak/81/09 showed D222N substitution that has been reported for some H1N1 viruses from Netherlands and Malaysia (). Only one isolate, A/Pakistan/81/2009 retained isoleucine (I) at position 321 in HA, while the rest of the remaining three viruses; A/Pak/1/09, A/Pak/27/09 and A/Pak/154/09 - had I to V change at residue 321 that was seen in certain European viruses 
. In contrast to D222G change in the HA 
, the significance of retaining isoleucine at this position for disease severity has not been clearly demonstrated. More recent substitutions such as E374K and S451N reported in isolates from Iran, Netherlands and India 
were absent in the analyzed Pakistan viruses
In the NA gene, the clade 7 specific substitutions (V106I and N248D) were uniformly seen in the analyzed viruses while the characteristic substitutions reportedly associated with drug resistance were absent. Other substitutions such as S95N and R257K reported previously in some viruses from Finland were not present in Pakistan isolates.
Two amino acid substitutions in the PB2 gene at positions 627-K(Lysine) and 701-N (Aspargine) have been reported as important in adaptation of avian influenza viruses to mammalian hosts. Pandemic 2009 viruses that contain the PB2 gene of avian origin appear to lack these adaptive changes, however their transmission and replication efficiency is comparable to those of human viruses. Recent studies report that a basic amino acid substitution (R or K) at position 591 of PB2 counterbalances for the lack of E627K change and allows the A(H1N1)pdm09 viruses to replicate efficiently in mammalian hosts and in humans 
. There are numerous as yet unexplained factors that made this novel virus highly transmissible as opposed to a highly pathogenic H5N1 that has shown poor human to human transmission capacity despite being around for over 14 years.
One of the impediments in understanding the severity of this epidemic in Pakistan is the paucity of available patient data. Out of the reported 262 positive cases, 52 hospitalized cases and 29 deaths were confirmed to be caused by Pdm H1N1 associated pneumonia and complications (Epidemiological data not shown). The viruses analyzed in the present study came from cases that recovered completely. These viruses were randomly selected for sequencing and isolates from cases with adverse or fatal outcomes are currently being subjected to detailed analysis for any significant mutations associated with high virulence. In the following months since the end of pandemic, no new reassortant events or key changes have been observed in these viruses.
Antiviral susceptibility surveillance conducted by the WHO GISN has shown that resistance for oseltamivir was sporadically detected in these viruses with rare onward transmission. None of the fourteen viruses analyzed in the present study showed the H275Y substitution associated with resistance to neuraminidase inhibitors which is not surprising since majority of cases that were virologically confirmed presented in outpatient clinic as influenza Like Illness(ILI) and most likely were not treated.(Epidemiological data on antiviral treatment is unavailable). Furthermore, data on prophylactic use of antiviral medication in suspected cases from Pakistan prior to diagnostic confirmation is currently limited, and therefore hinders analysis of potential non responders to oseltamivir therapy.
As more epidemiological and sequencing data on Pakistan influenza viruses becomes available, a better understanding of their continuing evolution will be achieved. In particular, it will be established whether any reassortment events with local seasonal influenza viruses may have occured. The continued surveillance of A(H1N1)pdm09 viruses through the coming years can ensure early detection of new antigenic or drug resistant variants. This will facilitate better pandemic planning and response capacity at national as well as global levels.