Ausama Yousif

Marine and brackish water aquacultures are rapidly expanding in the Mediterranean basin. In this context, Egypt recently received a shipment of a 1.5 million juvenile gilthead seabream (Sparus aurata L.) from European Mediterranean facility. Within a few weeks of their arrival, 95% of the imported fish developed nodules on their skin and fins that lasted for several months. This study was undertaken to describe the clinical disease course, to identify the causative agent, and to investigate its origin. Preliminary diagnosis based on gross lesions and postmortem examination suggested lymphocystis disease (LCD), caused by the lymphocystis disease virus (LCDV; genus Lymphocystivirus, family Iridoviridae). Histopathological and ultrastructural features were typical of LCDV infections. PCR followed by sequencing and phylogenetic analysis of a 306-bp fragment of the major capsid protein (MCP) gene demonstrated the presence of LCDV genotype I, originally associated with LCD in Northern European countries, with 99.7% and 100% nucleotide and deduced amino acid identity values, respectively. LCDV genotype I has neither been reported in this species nor in the region. Regardless of the source of infection, findings of this study add to existing knowledge about the ecology of LCDV genotype I and its host range.

Live-attenuated (LA), and inactivated adjuvant (IA) camelpox virus (CMLV) vaccines are produced in several countries worldwide. A tissue culture attenuated CMLV isolated (Jouf-78) is used to produce an LA vaccine in Saudi Arabia (Hafez et al., 1992). DNA extracts from the Saudi LA vaccine were used as positive controls for a routine ATIP PCR produced fragments longer than 881 bp. PCR-amplified ATIP sequences were similar to vaccinia virus (VACV) Lister strain. PCR and sequence analysis of two extracellular enveloped virus (EEV)-specific (A33R and B5R), and two intracellular mature virus (IMV) (L1R and A27L) othrologue genes from the vaccine DNA extracts confirmed the finding. CMLV sequences were not detected in vaccine DNA extracts. A VACV Lister strain imported from Switzerland was used in control experiments during initial testing of the Saudi LA vaccine. High antigenic similarity between VACV and CMLV, and a possible contamination event during production may have caused this issue. Environmental and health impact studies were recommended because early VACV vaccines produced in some European countries contained nonhighly attenuated strains that were not adequately screened for adventitious agents.

Potentially pathogenic orthopoxviruses (OPVs) persist in nature and re-emerge for reasons we do not fully understand. New information pertaining to Orthopoxvirus (OPV) persistence in nature would significantly improve surveillance and control programs. In a recent investigation of a Camelpox virus (CMLV) outbreak in Eastern Saudi Arabia, atypical minute pox-like skin lesions (AMPL) persisted on 42.9% of convalescent camels (8.8% of herd) for more than a year after the onset of clinical signs. In order to investigate whether AMPL were related to CMLV infection, AMPL homogenates were inoculated on the chorioallantoic membranes (CAM) of specific-pathogen-free (SPF) embryonating chicken eggs (ECE). Live CMLV was recovered from AMPL homogenates. The sequences of the ATIP gene of viruses isolated in the beginning of the outbreak, and one year later from AMPL were identical, and similar to the Kazakhstan isolate CMLV M-96. Virus identity was confirmed by sequence analysis of the CMLV A33R, A27L, B5R, and L1R orthologue genes. Uninfected adult camels that came in contact with animals showing AMPL became infected within two weeks. Since AMPL were easily missed by veterinarians and camel drivers, it was concluded that CMLV survival in persistent skin lesions may be a key mechanism in maintaining the virus in previously infected camel herds during inter-epizootic periods.

The use of embryonating chicken eggs in preparation of avian virus vaccines is the principle cause for contamination with Chicken anemia virus (CAV). Identification of CAV in contaminated vaccines relies on the expensive, tedious, and time-consuming practice of virus isolation in lymphoblastoid cell lines. The experience of the last 2 decades indicates that polymerase chain reaction is extending to replace most of the classic methods for detection of infectious agents. In the present report, a simple, rapid, and accurate polymerase chain reaction method for detection of CAV in poultry vaccines is described. Oligonucleotide primers homologous to highly conserved sequences of the VP1 gene were used to amplify a fragment of 676 bp. The developed assay was specific for detecting CAV from different sources, with no cross reactivity with many avian viruses. No inter- and intra-assay variations were observed. The analytical sensitivity of the test was high enough to detect 5 TCID50 (50% tissue culture infective dose) of the virus per reaction; however, different factors related to the vaccine matrix showed considerable effects on the detection limit. In conclusion, this method may represent a suitable alternative to virus isolation for identification of CAV contamination of poultry virus vaccines.

Molecular diagnostic investigations of orthopoxvirus (OPV) infections are performed using a variety of clinical samples including skin lesions, tissues from internal organs, blood and secretions. Skin samples are particularly convenient for rapid diagnosis and molecular epidemiological investigations of camelpox virus (CMLV). Classical extraction procedures and commercial spin-column-based kits are time consuming, relatively expensive, and require multiple extraction and purification steps in addition to proteinase K digestion. A rapid non-enzymatic procedure for extracting CMLV DNA from dried scabs or pox lesions was developed to overcome some of the limitations of the available DNA extraction techniques. The procedure requires as little as 10 mg of tissue and produces highly purified DNA [OD260/OD280 ratios between 1.47 and 1.79] with concentrations ranging from 6.5 to16 μg/ml. The extracted CMLV DNA was proven suitable for virus-specific qualitative and, semi-quantitative PCR applications. Compared to spin-column and conventional viral DNA extraction techniques, the two-step extraction procedure saves money and time, and retains the potential for automation without compromising CMLV PCR sensitivity.