Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

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.

Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe, Asia, and Africa.

BACKGROUND: The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. RESULTS: Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014-2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. CONCLUSIONS: We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions.

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.

Complete Genome Sequence of a Virulent Newcastle Disease Virus Strain Isolated from a Clinically Healthy Duck (Anas platyrhynchos domesticus) in Pakistan.

Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks (Anas platyrhynchos domesticus) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II.

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.

Complete Genome Sequence of a Recent Panzootic Virulent Newcastle Disease Virus from Pakistan.

The genome sequence of a new strain of Newcastle disease virus (NDV) (chicken/Pak/Quality Operations Lab/SFR-611/13) is reported here. The strain was isolated from a vaccinated chicken flock in Pakistan in 2013 and has panzootic features. The genome is 15,192 nucleotides in length and is classified in subgenotype VIIi of genotype VII, class II.

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.

Characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008.

Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006 to 2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of the HA genes, these isolates belong to clade 2.2 and both the HA and NA are closely related to each other (nucleotide identities above 99.0%) and to other Middle Eastern H5N1 AIV isolates (nucleotide identities above 98.0%). The phylogenetic data suggest that the virus in both epornitics of H5N1 HPAIV in commercial poultry in the Karachi region of Pakistan between 2006 and 2008 were from a very closely related source, however, there is inadequate epidemiological data to determine what the reservoir was for the virus between the 2006 and 2007 outbreaks other than that there was a single introduction into the region.

H7 avian influenza virus vaccines protect chickens against challenge with antigenically diverse isolates.

Vaccination has been a critical tool in the control of some avian influenza viruses (AIV) and has been used routinely in Pakistan to help control sporadic outbreaks of highly pathogenic (HP) H7 AIV since 1995. During that time, several AIV isolates were utilized as inactivated vaccines with varying degrees of success. In order to evaluate which H7 AIV strains may serve as optimal vaccines for diverse H7 AIVs from Pakistan we conducted vaccination-challenge studies with five H7 vaccines against challenge with two HPAIVs: A/chicken/Murree/NARC-1/1995 H7N3 and A/chicken/Karachi/SPVC-4/2004 H7N3. To further characterize the isolates antigenic cartography was used to visually demonstrate the antigenic relationships among the isolates. All vaccines provided similar protection against mortality, morbidity and shedding of challenge virus from the respiratory tract. However, some minor (not statistically significant) differences were observed and correlated with antibody levels induced by the vaccine prior to challenge.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004.

BACKGROUND: Avian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis. RESULTS: Phylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct. CONCLUSIONS: Although rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

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.