Akbar Dastjerdi

Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century.

The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.

Complete Genome Sequences of Elephant Endotheliotropic Herpesviruses 1A and 1B Determined Directly from Fatal Cases

ABSTRACT A highly lethal hemorrhagic disease associated with infection by elephant endotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephant husbandry. We have used high-throughput methods to sequence the genomes of the two genotypes that are involved in most fatalities, namely, EEHV1A and EEHV1B (species Elephantid herpesvirus 1, genus Proboscivirus, subfamily Betaherpesvirinae, family Herpesviridae). The sequences were determined from postmortem tissue samples, despite the data containing tiny proportions of viral reads among reads from a host for which the genome sequence was not available. The EEHV1A genome is 180,421 bp in size and consists of a unique sequence (174,601 bp) flanked by a terminal direct repeat (2,910 bp). The genome contains 116 predicted protein-coding genes, of which six are fragmented, and seven paralogous gene families are present. The EEHV1B genome is very similar to that of EEHV1A in structure, size, and gene layout. Half of the EEHV1A genes lack orthologs in other members of subfamily Betaherpesvirinae, such as human cytomegalovirus (genus Cytomegalovirus) and human herpesvirus 6A (genus Roseolovirus). Notable among these are 23 genes encoding type 3 membrane proteins containing seven transmembrane domains (the 7TM family) and seven genes encoding related type 2 membrane proteins (the EE50 family). The EE50 family appears to be under intense evolutionary selection, as it is highly diverged between the two genotypes, exhibits evidence of sequence duplications or deletions, and contains several fragmented genes. The availability of the genome sequences will facilitate future research on the epidemiology, pathogenesis, diagnosis, and treatment of EEHV-associated disease.

Incursion of RHDV2-like variant in Great Britain.

RABBIT haemorrhagic disease virus (RHDV) causes an acute, fulminating and generally fatal disease in the European rabbit ( Oryctolagus cuniculus ). It was first discovered in China in 1984 and then confirmed in the UK in 1992, at …

Replication of hepatitis E virus in three-dimensional cell culture.

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR

This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six elephants from a UK collection were sampled weekly over a period of 11 weeks for this study. The herd prevalence of elephant endotheliotropic herpesvirus-1 (EEHV-1) was 100 per cent by PCR. The virus DNA was detected in all the sampling sites; however, the prevalence of virus DNA in the conjunctiva swabs was higher. In addition, Asian elephants from two continental European collections were sampled once and one animal tested positive on a trunk wash. The virus from this animal was phylogenetically typed as EEHV-1A based on 231 nucleotides of the terminase gene.

Porcine Epidemic Diarrhea Virus among Farmed Pigs, Ukraine

An outbreak of porcine epidemic diarrhea occurred in the summer of 2014 in Ukraine, severely affecting piglets <10 days of age; the mortality rate approached 100%. Full genome sequencing showed the virus to be closely related to strains reported from North America, showing a sequence identity of up to 99.8%.

First Fatality Associated with Elephant Endotheliotropic Herpesvirus 5 in an Asian Elephant: Pathological Findings and Complete Viral Genome Sequence

Infections of Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus (EEHV) can cause a rapid, highly lethal, hemorrhagic disease, which primarily affects juvenile animals up to the age of four years. So far, the majority of deaths have been attributed to infections with genotype EEHV1 or, more rarely, EEHV3 and EEHV4. Here, we report the pathological characteristics of the first fatality linked to EEHV5 infection, and describe the complete viral DNA sequence. Gross post-mortem and histological findings were indistinguishable from lethal cases previously attributed to other EEHV genotypes, and the presence of characteristic herpesviral inclusions in capillary endothelial cells at several sites was consistent with the diagnosis of acute EEHV infection. Molecular analysis confirmed the presence of EEHV5 DNA and was followed by sequencing of the viral genome directly from post-mortem material. The genome is 180,800 bp in size and contains 120 predicted protein-coding genes, five of which are fragmented and presumably nonfunctional. The seven families of paralogous genes recognized in EEHV1 are also represented in EEHV5. The overall degree of divergence (37%) between the EEHV5 and EEHV1 genomes, and phylogenetic analysis of eight conserved genes, support the proposed classification of EEHV5 into a new species (Elephantid herpesvirus 5).

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses.

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.

Lack of evidence for circovirus involvement in bovine neonatal pancytopenia

THE newly described syndrome of bovine neonatal pancytopenia (BNP) (previously known as idiopathic haemorrhagic diathesis or bleeding calf syndrome), reported in the UK by [Penny and others (2009)][1], has caused considerable animal health and welfare interest across Europe. The detection in calves

Equine Encephalosis Virus in Israel

We wish to draw your attention to an outbreak of equine encephalosis virus (EEV) in Israel. Between October 2008 and January 2009, a febrile horse disease was observed in more than 60 equine premises across the country. Clinical signs included: raised body temperatures, unrest, decreased appetite, edema of the neck, legs, lips and eyelids, accelerated pulse and breathing rates, and congested mucosae. The morbidity ranged from 2% to 100% but there were no fatalities. In the affected stables, the disease infected all breeds, ages and sexes and the duration of illness lasted from between 7 and 30 days. The initial diagnostic investigation was performed by the Kimron Veterinary Institute (KVI), Israel, on nasal swabs, blood and serum samples from affected animals. The tests for WNV and EHV-1 were negative. Initial serological results indicated that the disease was equine viral arteritis (EAV), but no EAV virus could be isolated and PCR tests were also negative. The virus isolates and original sample material were passed on to the Veterinary Laboratories Agency (VLA) in the UK for further investigation. Based on the clinical signs and possible suspect diagnosis, the VLA performed PCR tests for EAV, Togaviruses (Getah, WEE, EEE, VEE, Ross River), and Flaviviruses (Tick Borne Encephalitis, WN, Usutu, Louping ill, Dengue, Japanese encephalitis), with all tests being negative. Using a novel DNA array technique, with subsequent RT-PCR and sequence analysis, the virus was finally identified as EEV. While further characterization of the virus is underway and an extended description of the investigation will be provided in due course, we feel obliged to alert the scientific community of this important disease and the implications involved. Equine encephalosis virus is member of the Orbivirus genus, thus transmitted by midges of the Culicoides family and is related to African horse sickness; all equids (horses, donkeys, zebras) being susceptible to infection. This disease has never been diagnosed in Israel or anywhere else North of Southern Africa before (van Niekerk et al., 2003). In the future, it will now be important to distinguish EEV from other diseases such as EAV and Getah, which can have similar clinical signs. In this context, we would wish to recommend further testing of equines in the region, in order to obtain a better knowledge of the distribution of this virus.