Neil LeBlanc

Genetic Characterization of Peste des Petits Ruminants Virus, Sierra Leone

To the Editor: Peste des petits ruminants (PPR) is a highly infectious disease of small ruminants, characterized by high rates of illness and death and caused by a single-stranded RNA virus (peste des petits ruminants virus [PPRV]). PPRV can be divided into 4 genetically distinct lineages based on the nucleocapsid (N) gene (1). The lineages correlate well with geographic distribution of the virus, with lineages I and II mainly restricted to western and central Africa, lineage III to eastern Africa and the Arabian peninsula, and lineage IV to Southeast Asia, the Middle East, and more recently northern Africa (2). n nPPRV is endemic to most of western Africa, and considered a major constraint on the livestock industry. In Sierra Leone, a country bordered by Guinea, Liberia, and the Atlantic Ocean, and having high goat and sheep populations, PPRV is believed to be the cause of outbreaks of respiratory disease with high death rates. Inadequate veterinary infrastructure and diagnostic capacity, exacerbated by the civil war in 1991–2002, however, has prevented confirmation. In this study, we confirmed presence of PPRV in Sierra Leone, which led to the official report of PPR to the World Organisation for Animal Health (Paris, France). n nThe study was conducted in April 2009 as part of a training mission organized at Teko Central Veterinary Laboratory, Makeni, Sierre Leone, by the World Organisation for Animal Health Collaborating Centre for Biotechnology–based Diagnosis of Infectious Diseases in Veterinary Medicine (www.sva.se/oie-cc) in collaboration with the Food and Agriculture Organization–Emergency Center for Transboundary Animal Diseases, Bamako, Mali. During the training, blood and serum samples were collected from goats (n = 9) and sheep (n = 1) from 2 smallholders with suspected outbreaks of PPR in the area around Makeni in central Sierra Leone. In addition, serum from 5 goats with respiratory disease was sampled at a livestock market in Kabala 100 km north of Makeni. n nSerologic testing was performed at Teko. All serum samples (n = 15) were tested for PPRV antibodies by using a commercial ELISA (BDSL, Ayrshire, UK; 3); 12 (80%) of the samples were positive for PPRV. n nBlood samples were collected on Nobuto filter strips (Advantec MFS Inc., Tokyo, Japan) and transported to the BioSafety Level 3 laboratory at the National Veterinary Institute, Uppsala, Sweden, for nucleic acid detection (4,5). RNA was eluted from the blood impregnated filter strips and screened for PPRV by using real-time RT-PCR specific for the N gene (6). Viral RNA was detected in 13 (87%) of the samples, with most of the positive samples indicating high viral load (cycle threshold <20). n nFor determination of the genetic lineage of detected viruses, RNA from all samples was subjected to PCR amplification of a 351-bp segment of the N gene by using the NP3/NP4 primer pair (7), but with a modified protocol using the One-Step RT-PCR kit (QIAGEN, Hilden, Germany) (5). Amplified PCR products were separated by electrophoresis, gel extracted, purified, and processed for sequencing by using ABI PRISM BigDye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. n nN gene sequences were obtained from 10 (67%) of the samples, and showed 83%–100% nt identity level compared with sequences available in GenBank using the BLASTn tool (www.ncbi.nlm.nih.gov/blast) and 93%–100% identity between each other. Phylogenetic analysis was performed with 4 representative sequences (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:JN602079-JN602082,start_term:JN602079,end_term:JN602082,start_term_id:371444676,end_term_id:371444682}}JN602079-JN602082) from this study by using neighbor-joining and the Kimura 2-parameter model in MEGA5 (CEMI, Tempe, AZ, USA), including N gene sequences representing all 4 lineages. n nThe PPR viruses from Sierra Leone clustered in lineage II with viruses from Mali, Nigeria, and Ghana, and could further be distinguished into 2 clusters (Figure). One virus from Kabala clustered closely with viruses from Mali (Mali 99/1), whereas all others showed 100% identity with a virus from Nigeria (Nig/75/1), in many countries used as vaccine virus strain. In Sierra Leone at the time, however, PPR vaccination was not being performed, suggesting that obtained sequences originated from circulating field viruses related to Nig/75/1 rather than being vaccine derived. This suggestion was strongly supported by the clinical presentation typical of PPR. Surprisingly, no relationship was found with PPRV strains so far described from Guinea, the immediate neighboring country and closest livestock trading partner, or those from Senegal, Guinea-Bissau, Cote d’Ivoire, and Burkina Faso, which constitute lineage I. n n n nFigure n nMajority rule consensus tree of peste des petits ruminants viruses (PPRV) based on the variable region of the N gene (255 bp), constructed using the neighbor-joining method and the Kimura-2-parameter model in MEGA5 (www.megasoftware.net). Numbers indicate ... n n n nIn conclusion, we confirm the presence of PPRV in Sierra Leone, and provide genetic characterization of detected viruses, knowledge that is fundamental for control, prevention, and in the long run, eradication of the disease. The detection of 2 different sublineages at the livestock market in Kabala shows how markets can serve as mixing vessels, and also gives evidence of at least 2 separate introductions of PPRV into the country, underlining the transboundary nature of the disease, particularly in regions with uncontrolled livestock movements. Since this study, an official vaccination program based on Nigeria/75/1 has been launched.

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.

Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses.

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5 biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

Advances in viral disease diagnostic and molecular epidemiological technologies

The early and rapid detection and characterization of specific nucleic acids of medico–veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Development of a real-time RT-PCR assay based on primer–probe energy transfer for the detection of all serotypes of bluetongue virus

A real-time RT-PCR assay based on the primer-probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mutations in the probe region. Furthermore, melting curve analysis following immediately PCR confirms specific probe hybridization and can reveal mutations in the probe region by showing a difference in the melting point. The assay sensitivity was in the range of 10-100 target copies and the specificity tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging strains.

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10⁹ to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻⁵. The detection limit was 10⁻⁵ dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field.

A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses

The genus pestivirus contains four recognized species: classical swine fever virus, border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars. Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europe. Since all the members of the pestivirus genus can infect swine, differential diagnosis using traditional methods poses some problems. Antibody tests may lack specificity due to cross-reactions, antigen capture ELISAs may have low sensitivity, and virus isolation may take several days or even longer time to complete. PCR-based tests overcome these problems for the most part, but in general lack the multiplexing capability to detect and differentiate all the pestiviruses simultaneously. The assay platform described here addresses all of these issues by combining the advantages of real-time PCR with the multiplexing capability of microarray technology. The platform includes a TaqMan real-time PCR designed for the universal detection of pestiviruses and a microarray assay that can use the amplicons produced in the real-time PCR to identify the specific pestivirus.

African swine fever outbreak on a medium-sized farm in Uganda: biosecurity breaches and within-farm virus contamination

In Uganda, a low-income country in east Africa, African swine fever (ASF) is endemic with yearly outbreaks. In the prevailing smallholder subsistence farming systems, farm biosecurity is largely non-existent. Outbreaks of ASF, particularly in smallholder farms, often go unreported, creating significant epidemiological knowledge gaps. The continuous circulation of ASF in smallholder settings also creates biosecurity challenges for larger farms. In this study, an on-going outbreak of ASF in an endemic area was investigated on farm level, including analyses of on-farm environmental virus contamination. The study was carried out on a medium-sized pig farm with 35 adult pigs and 103 piglets or growers at the onset of the outbreak. Within 3xa0months, all pigs had died or were slaughtered. The study included interviews with farm representatives as well as biological and environmental sampling. ASF was confirmed by the presence of ASF virus (ASFV) genomic material in biological (blood, serum) and environmental (soil, water, feed, manure) samples by real-time PCR. The ASFV-positive biological samples confirmed the clinical assessment and were consistent with known virus characteristics. Most environmental samples were found to be positive. Assessment of farm biosecurity, interviews, and the results from the biological and environmental samples revealed that breaches and non-compliance with biosecurity protocols most likely led to the introduction and within-farm spread of the virus. The information derived from this study provides valuable insight regarding the implementation of biosecurity measures, particularly in endemic areas.