Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV

Detection of capripoxvirus DNA using a field-ready nucleic acid extraction and real-time PCR platform

Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real-time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory-based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory-based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory-based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings

Cardiac tamponade and paroxysmal third-degree atrioventricular block revealing a primary cardiac non-Hodgkin large B-cell lymphoma of the right ventricle: a case report

<p>Abstract</p> <p>Introduction</p> <p>Primary cardiac lymphoma is rare.</p> <p>Case Presentation</p> <p>We report the case of a 64-year-old non-immunodeficient Caucasian man, with cardiac tamponade and paroxysmal third-degree atrioventricular block. Echocardiography revealed the presence of a large pericardial effusion with signs of tamponade and a right ventricular mass was suspected. Scanner investigations clarified the sites, extension and anatomic details of myocardial and pericardial infiltration. Surgical resection was performed due to the rapid impairment of his cardiac function. Analysis of the pericardial fluid and histology confirmed the diagnosis of non-Hodgkin large B-cell lymphoma. He was treated with chemotherapy.</p> <p>Conclusion</p> <p>The prognosis remains poor for this type of tumor due to delays in diagnosis and the importance of the site of disease.</p

Outbreaks of foot-and-mouth disease in Libya and Saudi Arabia during 2013 due to an exotic O/ME-SA/Ind-2001 lineage virus

Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia.N.J. Knowles, K. Bachanek-Bankowska, J. Wadsworth, V. Mioulet, B. Valdazo-González, I.M. Eldaghayes, A.S. Dayhum, A.M. Kammon, M.A. Sharif, S. Waight, A.M. Shamia, S. Tenzin, U. Wernery, S. Grazioli, E. Brocchi, S. Subramaniam, B. Pattnaik, and D.P. Kin

Scanning mutagenesis identifies critical residues in the rinderpest virus genome promoter

Short regions at the 3′ and 5′ ends of the genome of Rinderpest virus (RPV) contain signals that regulate transcription of the viral genome, known as the genome promoter and the (complement to the) antigenome promoter, respectively. An RPV minigenome construct carrying the CAT coding sequence was used as a reporter to investigate residues in the 3′-terminal region of the genome important for these functions. Single-base scanning mutagenesis showed that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader had an extremely inhibitory effect on transcription and/or encapsidation of the minigenome, with CAT expression reduced to 0–10% of control values. Changes in any of the other first 22 nucleotides reduced the efficiency of the minigenome to 20–80% of the wild-type control, with the exception of nucleotides 16, 17 and 20, where mutations did not affect CAT expression significantly. Mutagenesis in blocks identified critical residues in positions 23–26, but changes to leader residues 27–48 had no major effect on CAT expression. A region of about 16 nucleotides (49–65) located around the start of the nucleocapsid gene, including the intergenic triplet CTT, was identified as essential for minigenome function. Mutations further into the nucleocapsid gene (nt 66–89) had a moderate effect (CAT activity 20–60% of control), while at least one critical residue was found in positions 93–96. The importance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G79 was found to be optimal, though not critical, while a purine was required at 85 and 91. Although G97 is conserved in morbilliviruses, all bases were equally effective at this position.</jats:p

Serial passage of foot-and-mouth disease virus in sheep reveals declining levels of viraemia over time

If an infectious agent is to maintain itself within a closed population by means of an unbroken serial chain of infections, it must maintain the level of infectiousness of individuals through time, or termination of the transmission chain is inevitable. One possible cause of diminution in infectiousness along serial chains of transmission may be that individuals are unable to amplify and transmit comparable levels of the infectious agent. Here, the results are reported of a novel experiment designed specifically to assess the effects of serial passage of foot-and-mouth disease virus (FMDV) in experimental groups of sheep. A virus isolate taken from an epidemic of foot-and-mouth disease (FMD) characterized by rapid fade-out of infection was passed serially through four groups of sheep housed in an isolation unit. Although it was not possible to measure individual infectiousness directly, blood virus load from infected individuals was quantified using a real-time PCR assay and used as an underlying indicator of the level of infection. The results of this assay concurred well with those of the traditional tissue-culture assay and were shown to be highly repeatable. The level of peak viraemia was shown to fall significantly with the time of infection and with passage group, both in terms of the group mean and regression analysis of individual values, suggesting that this isolate of FMDV may, under certain conditions, be unable to maintain itself indefinitely in susceptible sheep populations. The results of these experiments are discussed in terms of the epidemiology of FMD in sheep