Zoonotic origin and transmission of Middle East respiratory syndrome coronavirus in the UAE.

Since the emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, there have been a number of clusters of human-to-human transmission. These cases of human-to-human transmission involve close contact and have occurred primarily in healthcare settings, and they are suspected to result from repeated zoonotic introductions. In this study, we sequenced whole MERS-CoV genomes directly from respiratory samples collected from 23 confirmed MERS cases in the United Arab Emirates (UAE). These samples included cases from three nosocomial and three household clusters. The sequences were analysed for changes and relatedness with regard to the collected epidemiological data and other available MERS-CoV genomic data. Sequence analysis supports the epidemiological data within the clusters, and further, suggests that these clusters emerged independently. To understand how and when these clusters emerged, respiratory samples were taken from dromedary camels, a known host of MERS-CoV, in the same geographic regions as the human clusters. Middle East respiratory syndrome coronavirus genomes from six virus-positive animals were sequenced, and these genomes were nearly identical to those found in human patients from corresponding regions. These data demonstrate a genetic link for each of these clusters to a camel and support the hypothesis that human MERS-CoV diversity results from multiple zoonotic introductions.

Peste des petits ruminants infection in domestic ruminants in Sudan.

The existence of peste des petits ruminants (PPR) in domestic ruminants and camels in Sudan during 2008-2012 was investigated. Lung tissues and serum samples were randomly collected from sheep, goats, cattle, and camels at different areas of Sudan. A total of 12,384 serum samples were collected from clinically healthy 7413 sheep, 1988 camels, 1501 cattle, 1459 goats, and 23 gazelles at different areas in the Sudan. They were examined for PPR antibodies using competitive ELISA (cELISA). The overall detected seroprevalence of PPR in tested sera was 49.4%; seroprevalence values within species were 67.1, 48.2, 25.8, 2.1, and 21.7% in sheep, goat, cattle, camels, and gazelles, respectively. The highest seroprevalence (68.1%) was observed in sera collected from Darfur states, then the central states (54.3%). A total of 1276 lung tissue samples (623 sheep, 324 cattle, 220 camels, and 109 goats) were collected. The majority of lung samples were collected from clinically healthy animals that showed lesions on PM in slaughterhouses (95%) and during PPR outbreaks; samples were tested for PPR antigen using immunocapture ELISA (IcELISA). PPR antigen was detected in 233 out of the 1276 tested samples (18.3%). Positive results were observed in samples collected from clinically healthy and diseased animals. The observed prevalence values in each species were 33.6, 21.1, 15.4, and 12.3% in camel, goat, sheep, and cattle, respectively. PPR antigen was detected in samples from different areas; however, the highest prevalence (63.9%) was found in samples collected from the eastern states, then Khartoum state (28%). Trials for virus isolation were done in different cell cultures. Out of 30 IcELISA-positive samples inoculated in primary bovine and ovine kidney cells, Vero cells, the PPR virus was successfully isolated from 15 (eight sheep, five camels, and two goats) samples in the three cell culture types. Using RT-PCR, PPRV nucleic acid was detected in all 25 IcELISA-positive tested samples.

Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan.

A reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 1412 bp of the fusion protein gene (F gene) of four Newcastle disease virus (NDV) isolates; two velogenic (TY-1/90 and DIK-90) and two lentogenic isolates (Dongla 88/1 and GD.S.1). Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1) suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

Development and evaluation of a live attenuated camelpox vaccine from a local field isolate of the virus.

A strain of camelpox virus (CMLV) isolated in the Sudan was attenuated by serial passage in Vero cell monolayers for use as a future vaccine strain. The safety and potency of passage 115 virus (designated Sudan CMLV/115) was tested. Camels inoculated with CMLV/115 showed no clinical disease or skin lesions, developed low-level antibodies and cell-mediated immune response and resisted challenge with virulent wild-type CMLV. Field testing of the candidate vaccine showed that the developed vaccine induces immune response and is safe for young and pregnant camels.

Characterization of the complete genomes of Camelus dromedarius papillomavirus types 1 and 2.

Camel papillomatosis has been described previously, but the genome of the suspected papillomavirus (PV) has not been identified. An outbreak of papillomatosis occurred in a dromedary farm of 55 animals in Sudan during August 2009. The disease was only present in young animals aged about 3-7 months, of which 44 % (11/25) were affected with lesions, mainly on the lips and lower jaw. This study reports for the first time the complete genomes of Camelus dromedarius papillomavirus types 1 (CdPV1) and 2 (CdPV2), isolated from a cauliflower-like nodule and a round oval raised nodule, respectively. Pairwise comparisons of their L1 nucleotide sequences revealed 69.2 % identity, and phylogenetic analyses suggested that these two PV types are grouped within the genus Deltapapillomavirus. Both viruses were isolated from fibropapillomas, although no putative E5 proteins homologous to that of bovine papillomavirus type 1 were identified. The genetic information will be useful for evolutionary studies of the family Papillomaviridae, as well as for the development of diagnostic methods for surveillance of the disease in dromedaries.

Respiratory syncytial virus infection of camels (Camelus dromedaries).

This study aimed to investigate the occurrence of respiratory syncytial virus (RSV) infections in camels in Sudan. A total of 272 camel lung specimens showing pneumonia were collected from slaughter houses at four different areas in Sudan, additionally 8 specimens were collected from outbreaks of respiratory infection in camels. Using sandwich ELISA kits for RSV antigen detection 4 out of 280 tested lungs (1.4%) were positive, all were from Central Sudan (Tambool slaughter house). FAT was used to confirm the ELISA positives. Polymerase chain reaction RT/PCR was applied for the detection of RSV genome in camel lungs; 1 out of 4 ELISA positives was positive by RT/PCR. Using indirect ELISA kits 135 out of 495 (27.3%) camel sera showed antibodies to RSV, highest prevalence was observed in Western (33.5%) then Central (31.6%) and Eastern Sudan (23.5%). Based on the manufacturer specified calculations for OD readings, most of positive sera (90/135) were low reactive (1+). This is the first report for the detection of RSV antigen, genome and antibody in camels in Sudan.

Respiratory infection of camels associated with parainfluenza virus 3 in Sudan.

This study was undertaken to investigate the role of parainfluenza virus 3 (PIV3) in respiratory infection of camels. A total of 273 lung specimens from camels with pneumonia lesions were collected from slaughterhouses in four different areas of Sudan. In addition, eight specimens were collected from outbreaks of respiratory infection in camels. Using antigen detection sandwich ELISA kits, six out of the 281 specimens tested were positive for the PIV3 antigen (2.1%); the highest prevalence was noted in Eastern Sudan (4.2%), then in Central and Northern Sudan (1.4%). The direct immunofluorescent test (FAT) was used to confirm the positive reactions for PIV3 by ELISA. The polymerase chain reaction (RT-PCR) was applied for the detection of the PIV3 genome in lungs of camels; two out of four samples which were positive by the PIV3 ELISA were also positive by RT-PCR. Virus isolation was attempted for PIV3 in MDBK cells; four specimens yielded cytopathic virus when inoculated onto the cell culture. The cytopathic effect (CPE) consisted of cell rounding, multinucleated cells, sloughing and elongation of cells, and some syncytia were observed on the 3rd to 7th day post-inoculation. Using commercially available indirect ELISA kits for antibodies to PIV3, 495 camel sera were tested, and the seroprevalence detected was 82.2%. The highest seroprevalence was observed in Central (92.6%), then in Eastern (92.2%) and Central to South Sudan (82.5%); the lowest prevalence was found in Northern Sudan (64.8%).

Detection of camel pox and vaccinia viruses by polymerase chain reaction.

PCR following two methods of DNA extraction was used to confirm the growth of camel pox virus (CPV) and vaccinia virus in cell culture and chorioallantoic membrane. Results were compared with the commonly used neutralization test. The first method of DNA extraction was accomplished by using viral DNA in tissue culture supernatant and Chorioallantoic membrane, which was released by initial heating for 15 min at 99 degrees C followed by ordinary PCR. In the second method DNA was extracted by using DNA Isolation Kit from tissue culture supernatant and used as a template. Rapid identification and differentiation of CPV and Vaccinia virus were achieved by PCR and this assay proved to be fast and feasible, and can be an alternative to orthodox serological methods.

Natural exposure of Dromedary camels in Sudan to infectious bovine rhinotracheitis virus (bovine herpes virus-1).

The occurrence of bovine herpes virus-1 (BHV-1) in camels was studied. A total of 186 pneumonic camel lungs were collected from slaughter houses at four different areas in Sudan during 2000-2006. Using sandwich ELISA 1.6% of 186 tested lungs were found positive for BHV-1 antigen, all were from Tambool at Central Sudan. Direct fluorescent antibody test (FAT) was used to confirm the BHV-1 ELISA positives, all ELISA positives were also positive. PCR was used to detect BHV-1 genome with three positive results. BHV-1 was isolated from two camel lungs in MDBK cells. Isolates were identified using ELISA and FAT. Indirect ELISA was used to detect antibodies to BHV-1 in 260 camel sera; 76.9% were found positive. Highest prevalence was observed in sera from Kordofan (84%) then Blue Nile (80%) and Tambool (76.3%). This is the first report for the detection of BHV-1 antigen, genome using PCR, isolation in cell culture and antibodies in camels in Sudan.

Observations on the use of Komarov strain of Newcastle disease vaccine in the Sudan.

Broilers at 18 days old were vaccinated with a single dose of the K strain of Newcastle disease vaccine administered by different routes. The serological results obtained by haemagglutination inhibition at weekly intervals post-vaccination showed that birds vaccinated intranasally (i/n) or intramuscularly (i/m) had antibody titres higher than those vaccinated by the oral method. Furthermore, when challenged 3 weeks after vaccination, those broilers vaccinated i/n or i/m had 75% and 70% resistance respectively, while those vaccinated by the oral or spray methods failed to withstand the challenge.

Lumpy skin disease: observations on the recent outbreaks of the disease in the Sudan.

Outbreaks of lumpy skin disease in imported Friesian and indigenous cattle which occurred in the Khartoum State during the period of 1989-1991, are described. The disease was diagnosed from clinical findings, isolation and identification of the virus and from electron microscopy. Clinical findings included pyrexia, nasal discharge, appearance of multiple skin nodules of varying sizes, oedema of legs and brisket and abortion. The disease in purebred Friesian cattle was severe with a morbidity rate of 37.9% and a mortality rate of 4.2% while it was rather mild in indigenous cattle.

Salmonella enteritidis infection in the Sudan.

Twenty-one Salmonella enteritidis isolates were recovered from several poultry farms in three states in the Sudan over an eighteen-month period. The infection was disseminated from a distributing company which had imported infected fertilized eggs and parent stock. The Sudan S. enteritidis epidemic which devastated many poultry farms during 1990 was attributable and concurrent to that in Europe in particular and throughout the world in general.

Pathogenic properties of Newcastle disease virus isolates in the Sudan.

Six Newcastle disease virus (NDV) isolates were obtained from disease outbreaks on different poultry farms in the Sudan between 1988 and 1991. The pathogenic properties of these isolates were studied in comparison to those of strain Herts 33/56. All the isolates were similar in that they killed chicken embryos quickly, in mean death time (MDT) and embryo lethal dose 50 per cent (ELD50), had higher intracerebral pathogenicity indices (ICPI), and produced viscerotropic lesions in the infected chickens. The field isolates had the characteristics of the velogenic viscerotopic strains of NDV. The pathogenesis of infection caused by one of the isolates was studied. The virus was first detected in different organs and in oral and cloacal swabs on the third day after infection.