Siamak Zohari

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.

Genetic characterization of the NS gene indicates co-circulation of two sub-lineages of highly pathogenic avian influenza virus of H5N1 subtype in Northern Europe in 2006

The non-structural (NS) gene of highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI-H5N1) isolated in Baltic Sea area of Sweden in 2006 was studied. The phylogenetic analysis data demonstrated that two distinct sub-lineages of HPAI-H5N1 were circulating during the outbreak in Northern Europe in Spring 2006. Sub-lineage I viruses fell into the same clade as viruses found in Denmark and Germany and formed a sub-clade which also included viruses isolated in the Russian Federation in late 2005. Sub-lineage II viruses formed a sub-clade closely related to European, Middle Eastern and African isolates reported in 2006. Analysis of the inferred amino acid sequences of the NS1 protein showed a deletion of five amino acids at positions 80–84. No viruses represented in this study contained Glu92 in the NS1 and all isolates contained the avian-like ESKV amino acid sequences at the NS1 C-terminal end. Sub-lineage I isolates contained unique substitutions V194I in NS1 and G63E in Nuclear export protein (NEP).

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

BackgroundThe analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized.ResultsThe presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described.ConclusionThe comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.

Pathology of natural highly pathogenic avian influenza H5N1 infection in wild tufted ducks (Aythya fuligula)

Highly pathogenic avian influenza (HPAI) subtype H5N1 is an infectious systemic viral disease that results in high morbidity and mortality in poultry, and has been reported in a wide range of wild bird species during the last few years. An outbreak of HPAI H5N1 occurred in wild birds in Sweden in 2006 that affected several duck species, geese, swans, gulls, and raptors. Tufted ducks (Aythya fuligula) accounted for the largest number of positive cases and, therefore, were selected for more in-depth histologic and immunohistochemical evaluations. The main histologic lesions associated with the presence of avian influenza antigen were found in the brain, pancreas, and upper respiratory tract. Other tissues in which influenza antigen was variably found included liver, lung, adrenal glands, kidneys, and peripheral nerve ganglia. The current study describes the pathology and viral tissue targeting of H5N1 by using histology, polymerase chain reaction, and immunohistochemistry, and highlights the range and variation in the presentation of the natural disease in tufted ducks.

Emergence of novel strains of avian infectious bronchitis virus in Sweden

Abstract Infectious bronchitis virus (IBV) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. Recent IBV infections in Sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. The complete spike gene of selected isolates from IBV cases was amplified and sequenced using conventional RT-PCR. Nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the QX-like genotype. The phylogenetic analysis revealed that strains predominant in the nineties, which were of the Massachusetts type, have been replaced by D388/QX-like strains, however the evolutionary link could not be established. The homology between the two genotypes was 79 and 81%. Remarkably, a strong positive selection pressure was determined, mostly involving the S1 subunit of the S gene. This strong selective pressure resulted in recombination events, insertions and deletions in the S gene. Two new isolates generated from recombination were found with nucleotide sequence diverging 1.7–2.4% from the D388/QX-like branch, indicating the emergence of a new lineage. The study demonstrates a constant evolution of IBV that might be in relation to increased poultry farming, trade and vaccine pressure. The findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario.

Detection and Phylogenetic Analysis of Peste des Petits Ruminants Virus Isolated from Outbreaks in Punjab, Pakistan

Peste des Petits Ruminants (PPR) is an important viral disease of small ruminants and is endemic in Pakistan. In the following study, samples from two outbreaks of PPR in goats have been subjected to laboratory investigations. The Peste des Petits Ruminants virus (PPRV) genome was detected using both conventional and real-time PCR. Genetic characterization of the local PPRV field isolates was conducted by sequencing 322 bp of the fusion (F) gene and 255 bp of the nucleoprotein (N) gene. The phylogenetic tree based on the F gene clustered samples from both outbreaks into lineage 4 along with other Asian isolates, specifically into subcluster 1 along with isolates from Middle East. Analysis of N gene revealed a different pattern. In this case, the Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which probably represents the true geographical pattern of virus circulation. This is the first report presenting the phylogenetic tree based on N gene as well as performing a parallel comparison of the trees of F and N gene together from Pakistani isolates. The results of this study shed light on the PPRV population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology. Such understanding is essential in any efforts to control the number and impact of outbreaks that are occurring in endemic countries such as Pakistan, especially in the current scenario where OIE and FAO are eager to control and subsequently eradicate PPR from the globe, as has been achieved for Rinderpest.

Avian influenza A(H10N7) virus involvement in mass mortality of harbour seals (Phoca vitulina) in Sweden, March through October 2014

We provide the first scientific report of influenza A virus involvement in a mass mortality event among harbour seals (Phoca vitulina) off the west coast of Sweden. Avian influenza A (H10N7) virus was detected in the lungs of two affected animals. This subtype has not been reported in seals to date, nor has influenza A-associated mortality been reported in seals in Europe. Circulation of avian influenza viruses in mammals may have implications for public health.

Molecular Biology and Pathogenesis of Peste des Petits Ruminants Virus

Peste des Petits Ruminants (PPR) virions are enveloped, pleomorphic particles containing a genome of single stranded RNA that is enclosed in a ribonucleoprotein core. The PPRV genome is 15,948 nucleotides (nts) long, which is the longest of all the morbillivirus members except for a recently described feline morbillivirus, which is revealed to be 16,050 bases long due to unusually long 50 trailer sequence. The genome of PPRV encodes for eight genes in the order 30-N–P/C/V-M-F-HN-L-50. The mean diameter of PPR virions (400–500 nm) is slightly larger than rinderpest virus (RPV) (300 nm). As a typical feature for all members of the genus morbillivirus, the PPRV genome length follows the ‘‘rule of six’’, but carries a certain degree of flexibility by accommodation of +1, +2 and -1 nts, which is a unique property of PPRV among morbilliviruses. In this chapter, all of the known features of the PPRV genome structure and biology are discussed. Additionally, all of the structural and nonstructural proteins are described comprehensively.

Simultaneous Genotyping of All Hemagglutinin and Neuraminidase Subtypes of Avian Influenza Viruses by Use of Padlock Probes

ABSTRACT A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005

BackgroundAlthough the important role of the non-structural 1 (NS) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Europe is incomplete. In this study we determined the subtypes and prevalence of influenza A viruses present in mallards in Northern Europe and further analysed the NS gene of these isolates in order to obtain a more detailed knowledge about the genetic variation of NS gene of influenza A virus in their natural hosts.ResultsA total number of 45 influenza A viruses of different subtypes were studied. Eleven haemagglutinin- and nine neuraminidase subtypes in twelve combinations were found among the isolated viruses. Each NS gene reported here consisted of 890 nucleotides; there were no deletions or insertions. Phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present at the same time in the same geographic location in the mallard populations in Northern Europe. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. The degree of variation within the alleles is very low. In our study allele A viruses displays a maximum of 5% amino acid divergence while allele B viruses display only 2% amino acid divergence. All the viruses isolated from mallards in Northern Europe possessed the typical avian ESEV amino acid sequence at the C-terminal end of the NS1 protein.ConclusionOur finding indicates the existence of a large reservoir of different influenza A viruses in mallards population in Northern Europe. Although our phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present in the mallards populations in Northern Europe, allele B viruses appear to be less common in natural host species than allele A, comprising only about 13% of the isolates sequenced in this study.