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.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
•Emerging virulent NDV strains from a new sub-genotype have panzootic characteristics.•ND outbreaks in three countries are linked by common sub-genotypes.•Ancestral NDV strains from wild birds ...suggest the existence of unknown reservoirs.
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 in1982. 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.
•Virulent NDVs from sub-genotype VIIi were repeatedly isolated from different species in Pakistan during 2011–2016.•Nearly identical sequences suggest the existence of epidemiological links between ...these viruses.•Clearly distinct phylogenetic branches suggest separate simultaneous evolution at more than one site or host.•Repeated isolation of NDV in different hosts and locations suggests a complex multiple components endemic cycle in Pakistan.
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.
Efficacy of live attenuated and inactivated oil emulsion infectious bursal disease virus vaccines in broiler chicks Lone, N.A. (Karakoram International Univ., Gilgit-Baltistan (Pakistan). Dept. of Environmental Sciences); Rehmani, S.F. (University of Veterinary and Animal Sciences, Lahore (Pakistan). WTO Quality Operation Lab.); Khan, T.A. (Karachi Univ. (Pakistan). Dept. of Physiology) ...
Pakistan veterinary journal,
(Oct-Dec 2012), Letnik:
32, Številka:
4
Journal Article
Recenzirano
Odprti dostop
This study was carried out with the aims to evaluate the efficacy of indigenous live and inactivated Infectious bursal disease virus (IBDV) vaccines in broilers. Two hundred and fifty (250), ...a-day-old broiler chicks divided into five groups (A-E) were immunized with live and inactivated vaccine at varying ages. Live vaccine was given to group A (at 8 days post hatch), B (at 8, 15 days post hatch), C (at 8, 15 and 23 days post hatch) and D (at 8 days post hatch). In addition group D received at booster dose of inactivated at 21 days of age, while group E served as control. Antibody titers were measured via Agar Gel Precipitation (AGP) test and ELISA, while the degree of protection against the virulent strains of IBDV was also recorded. Results showed that vaccine program adopted for group C and D produced significantly (P less than 0.05) higher antibody titer as compared to other groups. While a significant (P less than 0.05) difference in antibody titers was observed between group A and B while no considerable antibodies were detected in group E. The response to challenge dose was recorded as the difference of lesions in bursa, pectoral muscles or other visceral organs with the exception of group C and D. The study suggests that broiler chicks may be vaccinated at days 8, 15 and 23 with live attenuated vaccine or live attenuated vaccine followed by inactivated vaccine at days 8 and 21 that could provide an adequate protection against the virulent form of IBDV.
Characterization of newcastle disease virus isolated during 1995-2009 from suburbs of Karachi-Pakistan Khan, T.A. (Karachi Univ. (Pakistan). Dept. of Physiology); Rehmani, S.F. (University of Veterinary and Animal Sciences, Lahore (Pakistan). WTO Quality Operation Lab.); Ahmed, A. (Dalian Medical Univ. (China). Dept. of Biotechnology) ...
Pakistan journal of zoology,
04/2012, Letnik:
44, Številka:
2
Journal Article
Recenzirano
Newcastle disease (ND) which is caused by Avian Paramyxovirus type I is among the major concerns of poultry production worldwide. In developing countries especially where backyard poultry are not ...monitored for infection, ND remains a threat. The use of vaccines (live and inactivated) for the last 50 years in South Asia has not been able to control the infection. The poultry mortality rate worsened after the emergence of Avian Influenza in commercial poultry during early 1990's. The present study was conducted on 84 samples during 19952069, from commercial poultry flocks in the suburbs of Karachi to isolate and characterize strains of Newcastle disease virus circulating in the region. The samples were passaged in 9-10 day old chicken embryonated eggs. Haemagglutinating activity was observed in eighty samples which were cross checked using ND virus (NDV) polyclonal serum employed in haemagglutination inhibition assay and only forty seven were positive for NDV. The infective allantoic fluid was later used for pathotyping of isolates by employing biological characterization tools. Results demonstrated that the majority of isolates were either mesogenic or velogenic strains based on the mean death time and intra cerebral pathogenicity indices. Molecular identification was done using reverse transcription polymerase chain reaction (RT-PCR) performed using viral RNA extracted from infective allantoic fluid. Three sets of previously reported primers were employed and primer set BK1/BK2 was found to be the most sensitive. The results suggest that Newcastle disease remains endemic and requires similar attention to that given to highly pathogenic avian influenza.
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.
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.
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