Geographically, Pakistan is located (33°40′N and 73°10′E) at the crossroads of the strategically important regions of South, Central and Western Asia (Figure 1). From 2009 to mid 2012, a number of outbreaks of Newcastle disease have been reported to World Organization for Animal Health (OIE-WAHID interface, available at: http://www.oie.int/wahis/public.php) from Pakistan as well as neighboring countries. Most of the outbreaks have been reported from Iran and India that shares border with Pakistan. Despite some of the reports from selective regions only and emergence of novel NDV (5i) from Pakistan , it is of the essence to screen and characterize the NDV throughout the country.
The F gene sequence data of all the analyzed samples clustered virulent NDVs among the lineage 5 (sub-lineage 5b) except KPK province, where the ND strains were clustered together in lineage 4 (sub-lineage 4c). Throughout the world, the NDVs belonging to lineage 5 are considered to be the one involved in outbreaks in Far East [29–31] South Africa  and Europe . Among many Asian countries and particularly those that shares border with Pakistan, there have been found and characterized number of velogenic NDVs within class II that belongs to various lineage and sub-lineages [7, 33–35]; however, the dominant one is found to be lineage 5. Likewise, NDVs belonging to various sub-lineage like 5a, 5b, and novel 5i has been reported from Pakistan from different type of birds recently [9, 11, 16]. Contrary to recent study analysis of genetic nature of circulating NDVs except those of KPK province, where sub-lineage 5b was found circulating among rural, commercial and wild birds, we have seen a clear distinction between the viruses isolated from various types of birds, wild and commercial in the recent past. The isolates from wild birds were clustered within sub-lineage 5a and closely related to Indonesian isolate (AY562985) [15, 17] while the ones from commercial and rural poultry were found within sub-lineage 5b, novel 5i and closely related to Swedish (GU585905) and Russian isolates (AY865652) [17, 19, 20].
Since 2005, lineage 4 (sub-lineage 4c) has been found now and from a different geographical region, Abbottabad and Mansehra districts of KPK province. Previously, the lineage 4 was isolated and characterized from areas in and around coastal border of Pakistan . The re-emergence of lineage 4 (sub-lineage 4c) from KPK province might be attributed either to the fact that viruses have not been characterized from this particular region before or either to the movement of migratory/caged birds across the country or from north to south involving Europe, Asia and Middle East. Historically, lineage 4 has been isolated and characterized among domestic fowls, ostrich, falcons and pigeons from middle east countries (United Arab Emirates and Saudi Arabia), Asian (Japan and China) and European countries (UK, Italy, Peru and Belgium) [1, 34]; however, most of NDVs belonging to sub-lineage 4c have been reported from wild captive and domestic fowls from United Arab Emirates . Isolation and characterization of NDV (JP/Chiba-pa/97) from parakeets exported to Japan in 1997  gives an evidence that lineage 4 is present in Pakistan since 1997. Since it is the only report from past, it could be hypothesized that lineage 4 is present even before and is still circulating in the environment. Further, it is imperative to describe the fact that samples were collected during a designated period of six month throughout the country and all of the clinical outbreaks are not necessarily included in the study. This means that there may exist more diversity in NDV strains in Pakistan and therefore, presence of lineage 4 (sub-lineage 4c) in already reported area (Karachi) or others cannot be ignored. The isolation and subsequent characterization of isolates from turkey and domestic fowls in UK (Q-GB506/97) , exported parakeets from Pakistan to Japan (JP/Chiba-pa/97) , and from pigeons in china (C/98-1)  which clustered together with Italian exotic isolate (IT-148/94)  in lineage 4 provides a possible epidemiological transmission through migratory birds from north to south. Considering migratory/caged birds as reservoir/carrier of NDVs in an inapparent form and the potential of viruses to infect multiple avian species without prior adaptation [1, 2, 26, 36], raises the concerns in worldwide distribution of velogenic pathotypes through trading and migration of birds across regional and international boundaries.
Recent outbreaks of ND in Pakistan are supposed to be due to a breach in the biosecurity measures. However, the role of vaccines in providing protection against the field challenge also needs to be evaluated. Inability of live vaccines to elicit protective immune response might be due to several reasons like improper cold chain supply system, inappropriate route of vaccination, or uneven vaccination schedules. Administration of both live and inactivated ND vaccine could be practiced to protect the birds from virulent NDVs . Presently, lentogenic (LaSota) or mesogenic strains (Muktesewar) of NDVs are being used to vaccinate the birds in Pakistan. However, it is still a matter of question whether these vaccinal strains are able to elicit a protective immune response against the prevailing field strains with high genetic gap in relation to vaccinal strains as evidenced and reported previously . It has been reported that the currently practiced NDV vaccines give better protection against the velogenic NDVs isolated in 1930 to 70s (Herts33/56, California 71) than the ones, which have been isolated in past few years [5, 9]. Hence parameters for selection of a vaccinal strain are needed to be reconsidered. Furthermore, monitoring the immune response of birds to NDV vaccines along with strict biosecurity measures should be employed to keep the flocks free of vNDVs infection.