Comparison of protection and viral shedding following vaccination with Newcastle disease virus strains of different genotypes used in vaccine formulation.

Newcastle disease (ND) is a devastating disease and cause high t mortality and morbidity in poultry and nonpoultry avian species worldwide. An intensive vaccination program against ND is a routine practice in Pakistan like other developing countries, but still frequent outbreaks have been recorded in the field. In this study, vaccines prepared from ND viruses corresponding to four different genotypes were compared, to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced the amount of challenge virus shed. In the experiment, 1-day-old pathogen-free Hubbard chicks were divided into five groups and all groups except control were received live LaSota vaccine. The chicks were re-vaccinated at day 5 and were given oil-adjuvanted inactivated vaccines prepared from one of four different inactivated NDV strains including SFR-55 (genotype-VIIi), Chicken-12 (XIIIb), Mukteswar (III), and LaSota (II), and control group was treated with PBS only. Pre- and post-challenge serum was collected from all groups and tested for antibody against NDV using hemagglutination inhibition (HI) assay. After challenged with virulent SFR-55, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for viral shedding. All the vaccines induced high immune response, and all the groups except the control induced > 84% protection against vNDV challenge. The vaccine genetically and antigeneically similar with challenge NDV strain reduced oral shedding significantly as compared to mismatched strains. From the present study, it was concluded that genotype-matched vaccine has potential to result in better protection by limiting the viral shedding.

Draft Genome Sequences of Three Ochrobactrum spp. Isolated from Different Avian Hosts in Pakistan.

Here, we present the draft genome sequences of three Ochrobactrum sp. strains with multidrug-resistant properties, isolated in 2015 from a pigeon and two chickens in Pakistan.

Repeated isolation of virulent Newcastle disease viruses in poultry and captive non-poultry avian species in Pakistan from 2011 to 2016.

Virulent viruses of the panzootic Avian avulavirus 1 (AAvV-1) of sub-genotype VIIi were repeatedly isolated (2011-2016) from commercial chickens and from multiple non-poultry avian species in Pakistan. These findings provide evidence for the existence of epidemiological links between Newcastle disease outbreaks in commercial poultry and infections with virulent AAvV-1 strains in other avian species kept in proximity to poultry. Our results suggest that the endemicity of Newcastle disease in Pakistan involves multiple hosts and environments.

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses.

BACKGROUND: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. METHODS: In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform. RESULTS: Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25-30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2-3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. CONCLUSIONS: This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.

Complete Genome Sequence of a Velogenic Newcastle Disease Virus Strain Isolated from a Clinically Healthy Exotic Parakeet (Melopsittacus undulatus) in Pakistan.

The complete genome sequence of a virulent Newcastle disease virus (vNDV) strain isolated from an exotic parakeet (Melopsittacus undulatus) is described here. The virulent strain parakeet/Pak/R-Pindi/SFR-16/2016 was isolated from a bird reared as a pet in the province of Punjab in the northern region of Pakistan in 2016. Phylogenetic analysis classified the isolate as a member of NDV class II, subgenotype VIIi, in genotype VII.

Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan.

Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI.

H9N2 low pathogenic avian influenza in Pakistan (2012-2015).

Significant economic losses from deaths and decreased egg production have resulted from H9N2 low pathogenic avian influenza virus (LPAIV) infections in poultry across North Africa, the Middle East and Asia. The H9N2 LPAIVs have been endemic in Pakistani poultry since 1996, but no new viruses have been reported since 2010. Because novel genotypes of Pakistani H9N2 contain mammalian host-specific markers, recent surveillance is essential to better understand any continuing public health risk. Here the authors report on four new H9N2 LPAIVs, three from 2015 and one from 2012. All of the viruses tested in this study belonged to Middle East B genetic group of G1 lineage and had PAKSSR/G motif at the haemagglutinin cleavage site. The mammalian host-specific markers at position 226 in the haemagglutinin receptor-binding site and internal genes suggest that Pakistan H9N2 viruses are still potentially infectious for mammals. Continued active surveillance in poultry and mammals is needed to monitor the spread and understand the potential for zoonotic infection by these H9N2 LPAIVs.

Antibacterial activity of herbal extracts against multi-drug resistant Escherichia coli recovered from retail chicken meat.

Increasing incidence rate of multiple drug resistance in Escherichia coli (E. coli) due to extensive uses of antibiotics is a serious challenge to disease treatment. Contaminated retail chicken meat is one of the major sources of spread of multi drug resistant (MDR) E. coli. Current study has been conducted to study the prevalence of MDR E. coli in retail chicken meat samples from Lahore city of Pakistan and it was found that 73.86% of E. coli isolates have MDR pattern. In vitro evaluation of antibacterial activity of crude ethanolic extracts of six herbs against MDR E. coli phenotypes has revealed that clove and cinnamon have maximum zones of inhibition as compared to other herbal extracts. Mint and coriander gave the intermediate results while garlic and kalonji showed the least antibacterial activity against the MDR E. coli phenotypes using the agar well diffusion technique. Average Minimum Inhibitory Concentrations (MICs) for clove, mint, cinnamon, coriander, kalonji and garlic extracts were 1.15, 1.38, 0.5, 1.99, 2.41, 8.60 mg/mL respectively using the broth micro dilution method. The results obtained in present study were revealed that crude ethanol extracts of selected herbs have had significant antibacterial activity. Hence they can be used as promising alternatives of antimicrobials against MDR E. coli species and can be used for cooked food preservation.

Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan.

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features.

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in 1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.

Phylogenetic and biological characterization of Newcastle disease virus isolates from Pakistan.

Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matrix gene assay, designed to detect all avian paramyxovirus 1, did not detect four of the isolates. A new matrix gene test that detected all isolates was developed. Phylogenetic analysis and pathotyping confirmed that virulent viruses of different genotypes are circulating in Pakistan.

Molecular characterization of Pakistani field isolates of infectious bursal disease virus.

The reverse transcriptase-polymerase chain reaction followed by restriction fragment length polymorphism (RT-PCR/RFLP) technique was used to identify and characterize Pakistani field isolates of infectious bursal disease virus (IBDV). These isolates have caused heavy losses to the poultry industry (mortality up to 60%) during the period between 1999 and 2005. Ten samples (five local isolates and five commercial vaccines) were examined for IBDV. Nine samples were positive for IBDV as evidenced by the amplification of the 743-bp region of the VP2 gene by RT-PCR. The RT-PCR products were subjected to restriction enzyme digestion with BstNI, MboI, and SspI. The RFLP profiles of all samples on digestion with the MboI enzyme yielded a fragment size of 229 and 362 bp except for vaccine strain Bursine Plus, which yielded a profile of 229 and 480 bp. However, digestion with BstNI yielded two distinct RFLP patterns. The first profile was detected in field isolates ML-1/SPVC/2001 and NP2/SPVC/2002 with four fragments of 119, 154, 172, and 209 bp, resembling RFLP profiles of molecular group 4 isolates. NL-3/SPVC/2003, NK-4/SPVC/2004, and NPK-5/SPVC/2005 generated a different RFLP profile with fragments of 119, 172, and 424 bp, resembling the profiles of molecular group 6 isolates. However, all the field and vaccine strains showed the absence of SspI restriction sites in their genome. It can be concluded that the Pakistani isolates can be grouped in molecular groups 4 and 6 of IBDV.