Isolation of buffalo poxvirus from clinical case and variations in the genetics of the B5R gene over fifty passages

Outbreaks of buffalopox affect udder and teats, which may ultimately lead to mastitis in dairy buffalo and can significantly compromise the production. In this study, we report isolation of buffalo poxvirus and sequence analysis of the B5R gene collected from the buffalo clinically suspected to be poxvirus infected. The virus was isolated on BHK-21 cell line and was passaged for 50 times, B5R gene was amplified and sequenced using gene-specific primers, and analyzed at both nucleotide and amino acid levels. Phylogenetically, the isolate can be classified close to the previously reported Pakistani and Indian isolates with certain level of differential clustering patterns. Three significant putative mutations (I2K, N64D, and K111E) were observed in the B5R protein. The K111E was common with previous human isolate from Karachi, Pakistan in 2005. These mutations differed from poxviruses reported from the neighboring countries. Some deletion mutations were observed which were recovered in upcoming passages. The K111E mutation suggests potential to cause zoonotic infection in human all over the country

Isolation and characterization of low pathogenic H9N2 avian influenza A viruses from a healthy flock and its comparison to other H9N2 isolates

Several outbreaks of avian influenza (AI) caused by H9N2 subtype, have been reported in the poultry industry during 1990 around the globe. Currently, H9N2 are endemic in the large area of Middle and Far East, including Pakistan. Since H9N2 AI viruses are sporadically reported from humans, extensive incidence of H9N2 in poultry imposes a great risk for human health. In this context, continuous monitoring of the poultry and determining the genetic nature of these viruses are fundamental to predict any future threat. Thus gene sequences of one isolate of H9N2, isolated from commercial poultry flocks, were analyzed. The results of this investigation, based on hemagglutinin (HA), neuraminidase (NA) and nonstructural genes, showed that Pakistani H9N2 isolates are closely related to each other and to other H9N2 isolates from the Middle East. However, several unusual substitutions with unknown functional consequences were observed in HA and NA proteins and thus warrant further investigations for their possible role in viral biology. In conclusion, these findings provide information regarding the genetic nature of H9N2 avian influenza viruses in Pakistani poultry and necessitate the sequencing of more H9N2 viruses from both naturally infected and vaccinated flocks

Biological characterization and phylogenetic analysis of a novel genetic group of Newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in Pakistan

Newcastle disease (ND) is a contagious viral disease of many avian species particularly domestic poultry, and is responsible for devastating outbreaks in the poultry industries around the globe. In spite of its importance and endemicity in Southern Asia, data on the genetic nature of the viruses and epizootiological information of the disease is scarce. In this study, six isolates from an emerging wave of ND outbreaks in the north of Pakistan and two isolates from healthy poultry flocks were biologically and genetically characterized. Based on pathogenicity indices such as intracerebral pathogenicity index (ICPI), mean death time (MDT) and cleavage motifs in the fusion protein, all these isolates were classified as virulent. Phylogenetic analysis of the fusion (F), hemagglutinin-neuraminidase (HN) and matrix (M) genes indicated the emergence of a novel genetic group within lineage 5, distinct from isolates previously reported in the region. Several mutations in the neutralizing epitopes and functionally important motifs of the F and HN genes pose a need for re-evaluation of the currently used vaccine and vaccination practices. The characteristics of Newcastle disease virus (NDV) as virulent (F protein cleavage site, ICPI and MDT) in apparently healthy backyard poultry (BYP) explain that BYP can play crucial role in the epizootiology and spread of the disease. The present investigation provides essential information on the genetic nature of NDV circulating in Pakistan and its implication on disease diagnosis and control. Furthermore, these investigations emphasize the importance of continuous surveillance of ND in developing countries