Identification of a novel lineage of Crimean-Congo haemorrhagic fever virus in dromedary camels, United Arab Emirates.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus causing Crimean-Congo haemorrhagic fever (CCHF), a disease reported to have a high fatality rate in numerous countries. The virus is geographically widespread due to its vector, and numerous wild and domestic animals can develop asymptomatic infection. Serological and limited molecular evidence of CCHFV has previously been reported in Camelus dromedarius (the dromedary, or one-humped camel) in the United Arab Emirates (UAE). In this study, 238 camel samples were screened for CCHFV RNA where 16 camel samples were positive for CCHFV by RT-PCR. Analysis of full-length CCHFV genome sequences revealed a novel lineage in camels from the UAE, and potential reassortment of the M segment of the genome.

Gangrenous mastitis in dromedary camels in UAE caused by Streptococcus agalactiae.

BACKGROUND: Mastitis is a disease of economic concern that affects dairy industry worldwide. This study aimed to investigate and identify possible etiologies encountered in an episode of acute gangrenous mastitis in lactating she-camels in Al Dhafra region, Abu Dhabi Emirate, United Arab Emirates (UAE). Beside the routine clinical examination, conventional bacteriological methods were used to isolate and identify possible aerobic/anaerobic bacterial or fungal pathogens from cultured milk samples collected from the mastitic she-camels. Moreover, quantitative real-time polymerase chain reaction (qPCR) was used for the detection of Mycoplasma agalactiae and Mycoplasma bovis strains, and the 16S rRNA gene was sequenced to confirm the isolation. The isolates were also tested for their susceptibility to antimicrobials. RESULTS: Acute gangrenous mastitis is reported in the dromedary camel herd with about 80% morbidity rate among lactating she-camels exhibited acute, painful hard swelling of affected teat, quarter or entire udder. About 41.7% of the infected animals were stamped out for culling due to complete or partial amputation of udder quarters. Streptococcus agalactiae was the sole isolated organism (6 isolates). The antimicrobial susceptibility testing revealed that, the Streptococcus agalactiae isolates were sensitive to both penicillin and ampicillin. Comparison of the 16S rRNA gene sequencing results by BLASTN confirmed the presence of Streptococcus agalactiae with high confidence (100% identity). Phylogenetic analysis indicated clustering of one isolate (CMAUAE accession number; MN267805.1) with Streptococcus agalactiae that infects multi-hosts including humans, while strains (CMBUAE to CMFUAE with accession numbers; MN267806.1 to MN267810.1 respectively) clustered with Streptococcus agalactiae that infects humans. No Mycoplasma spp was detected by qPCR analysis. CONCLUSIONS: In the present study, the Streptococcus agalactiae was found to be the main cause of acute gangrenous mastitis in dromedary camels in UAE. More research should be done to investigate other possible causes of clinical or subclinical mastitis in dromedary camels in UAE.

Preparation of the porcine secretory component and a monoclonal antibody against this protein.

Secretory component (SC) is a component of secretory IgA that is designated sIgA to distinguish it from IgA. The monoclonal antibody (MAb) against SC has been shown to be an excellent tool for the detection of the level of sIgA and for the evaluation of the efficacy of mucosal immunity. To prepare a monoclonal antibody against porcine SC, a recombinant porcine SC was expressed and purified. To develop this recombinant SC, the gene encoding the porcine SC was ligated into the pCold I vector. The recombinant vector was then transformed into Escherichia coli BL 21 (DE3), and gene expression was successfully induced by isopropyl-ß-D-thiogalactoside (IPTG). After affinity purification with Ni-NTA resin and gel recovery, the recombinant SC protein was used to immunize BALB/c mice. Finally, three hybridoma cell lines showing specific recognitions of both recombinant SC and native SC were used as stable secretors of MAbs against porcine SC and were confirmed to have no reaction to porcine IgA or IgG. The successful preparations of recombinant SC protein and MAbs provide valuable materials that can be used in the mucosal infection diagnosis for porcine disease and mucosal immune evaluation for porcine vaccine, respectively.

A tripeptide (NSK) inhibits Japanese encephalitis virus infection in vitro and in vivo.

Japanese encephalitis virus (JEV) is a major pathogen that can cause acute viral encephalitis in both humans and animals. Domain III of the viral envelope protein (EDIII) is involved in binding to host cell receptor(s) to facilitate virus entry. Our previous study showed that the loop3 peptide of EDIII possesses antiviral activity against JEV infection. In this paper, we demonstrate that three residues (NSK) in loop3 are responsible for the antiviral activity of loop3 peptide. In vitro experiments showed that the tripeptide NSK could inhibit JEV infection in both BHK-21 and Neuro-2A cells by inhibiting attachment of JEV to the cells, with IC50 values of 8 µM and 6.5 µM, respectively. In vivo experiments showed that the tripeptide could increase the survival of mice challenged with JEV to 75 % when administrated intracerebrally. Therefore, this tripeptide may serve as the basis for the development of novel antiviral agents against Japanese encephalitis virus infection.

Foot and Mouth Disease Vaccine Matching and Post-Vaccination Assessment in Abu Dhabi, United Arab Emirates.

Despite the annual vaccination of livestock against foot and mouth disease (FMD) in the United Arab Emirates (UAE), outbreaks of the disease continue to be reported. The effective control of field outbreaks by vaccination requires that the vaccines used are antigenically matched to circulating field FMD viruses. In this study, a vaccine matching analysis was performed using the two-dimensional virus neutralization test (VNT) for three field isolates belonging to the O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages collected from different FMD outbreaks that occurred within the Abu Dhabi Emirate in 2021 affecting Arabian oryx (Oryx leucoryx), goat, and sheep. In addition, post-vaccination antibodies in sheep and goats were measured using solid-phase competitive ELISA (SPCE) for FMDV serotypes A and O at five months after a single vaccine dose and a further 28 days later after a second dose of the FMD vaccine. An analysis of vaccine matching revealed that five out of the six vaccine strains tested were antigenically matched to the UAE field isolates, with r1-values ranging between 0.32 and 0.75. These results suggest that the vaccine strains (O-3039 and O1 Manisa) included in the FMD vaccine used in the Abu Dhabi Emirate are likely to provide protection against outbreaks caused by the circulating O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages. All critical residues at site 1 and site 3 of VP1 were conserved in all isolates, although an analysis of the VP1-encoding sequences revealed 14-16 amino acid substitutions compared to the sequence of the O1 Manisa vaccine strain. This study also reports on the results of post-vaccination monitoring where the immunization coverage rates against FMDV serotypes A and O were 47% and 69% five months after the first dose of the FMD vaccine, and they were increased to 81 and 88%, respectively, 28 days after the second dose of the vaccine. These results reinforce the importance of using a second booster dose to maximize the impact of vaccination. In conclusion, the vaccine strains currently used in Abu Dhabi are antigenically matched to circulating field isolates from two serotype O clades (O/ME-SA/PanAsia-2/ANT-10 sublineage and O/ME-SA/SA-2018 lineage). The bi-annual vaccination schedule for FMD in the Abu Dhabi Emirate has the potential to establish a sufficient herd immunity, especially when complemented by additional biosecurity measures for comprehensive FMD control. These findings are pivotal for the successful implementation of the region's vaccination-based FMD control policy, showing that high vaccination coverage and the wide-spread use of booster doses in susceptible herds is required to achieve a high level of FMDV-specific antibodies in vaccinated animals.

Molecular Detection of Candidatus Anaplasma camelii in Naturally Infected Dromedary Camels (Camelus dromedarius) in Abu Dhabi Emirate, United Arab Emirates, 2019-2023.

The recent emergence of anaplasmosis in camels has raised global interest in the pathogenicity and zoonotic potential of the pathogen causing it and the role of camels as reservoir hosts. In the United Arab Emirates (UAE), molecular studies and genetic characterization of camel-associated Anaplasma species are limited. This study aimed to characterize molecularly Anaplasmataceae strains circulating in dromedary camels in the UAE. Two hundred eighty-seven whole-blood samples collected from dromedary camels across regions of the Abu Dhabi Emirate were received between 2019 and 2023 at the Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) veterinary laboratories for routine diagnosis of anaplasmosis. The animals were sampled based on field clinical observation by veterinarians and their tentative suspicion of blood parasite infection on the basis of similar clinical symptoms as those caused by blood parasites in ruminants. The samples were screened for Anaplasmataceae by PCR assay targeting the groEL gene. Anaplasmataceae strains were further characterized by sequencing and phylogenetic analysis of the groEL gene. Thirty-five samples (35/287 = 12.2%) tested positive for Anaplasmataceae spp. by PCR assay. Nine positive samples (9/35 = 25.7%) were sequenced using groEL gene primers. GenBank BLAST analysis revealed that all strains were 100% identical to the Candidatus A. camelii reference sequence available in the GenBank nucleotide database. Phylogenetic analysis further indicated that the sequences were close to each other and were located in one cluster with Candidatus A. camelii sequences detected in Saudi Arabia, Morocco, and the UAE. Pairwise alignment showed that the UAE sequences detected in this study were completely identical and shared 100% identity with Candidatus A. camelii from Morocco and Saudi Arabia and 99.5% identity with Candidatus A. camelii from the UAE. This study demonstrates the presence of Candidatus A. camelii in UAE dromedary camels. Further critical investigation of the clinical and economical significance of this pathogen in camels needs to be carried out.

Molecular Epidemiology of Foot-and-Mouth Disease Viruses in the Emirate of Abu Dhabi, United Arab Emirates.

Foot-and-mouth disease (FMD) is an endemic disease in the United Arab Emirates (UAE) in both wild and domestic animals. Despite this, no systematic FMD outbreak investigation accompanied by molecular characterisation of FMD viruses (FMDVs) in small ruminants or cattle has been performed, and only a single report that describes sequences for FMDVs in wildlife from the Emirate has been published. In this study, FMD outbreaks that occurred in 2021 in five animal farms and one animal market in the Emirate of Abu Dhabi were investigated. Cases involved sheep, goats, and cattle, as well as Arabian oryx (Oryx leucoryx). Twelve samples were positive for FMDV via RT-qPCR, and four samples (Arabian oryx n = 1, goat n = 2, and sheep n = 1) were successfully genotyped using VP1 nucleotide sequencing. These sequences shared 88~98% identity and were classified within the serotype O, Middle East-South Asia topotype (O/ME-SA). Phylogenetic analysis revealed that the Arabian oryx isolate (UAE/2/2021) belonged to the PanAsia-2 lineage, the ANT-10 sublineage, and was closely related to the FMDVs recently detected in neighbouring countries. The FMDV isolates from goats (UAE/10/2021 and UAE/11/2021) and from sheep (UAE/14/2021) formed a monophyletic cluster within the SA-2018 lineage that contained viruses from Bangladesh, India, and Sri Lanka. This is the first study describing the circulation of the FMDV O/ME-SA/SA-2018 sublineage in the UAE. These data shed light on the epidemiology of FMD in the UAE and motivate further systematic epidemiological studies and genomic sequencing to enhance the ongoing national animal health FMD control plan.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo.

Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 µg/ml and a 50 % inhibitory concentration (IC(50)) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.

Isolation and genetic characterization of MERS-CoV from dromedary camels in the United Arab Emirates.

Background: The study of coronaviruses has grown significantly in recent years.Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in various cell types, and quick development has been made of assays for its growth and quantification. However, only a few viral isolates are now available for investigation with full characterization. The current study aimed to isolate MERS-CoV from nasal swabs of dromedary camels and molecularly analyze the virus in order to detect strain-specific mutations and ascertain lineage classification. Methods: We isolated the virus in Vero cells and adapted it for in vitro cultivation. The isolates were subjected to complete genome sequencing using next-generation sequencing followed by phylogenetic, mutation, and recombination analysis of the sequences. Results: A total of five viral isolates were obtained in Vero cells and adapted to in vitro cultures. Phylogenetic analysis classified all the isolates within clade B3. Four isolates clustered close to the MERS-CoV isolate camel/KFU-HKU-I/2017 (GenBank ID: MN758606.1) with nucleotide identity 99.90-99.91%. The later isolate clustered close to the MERS-CoV isolate Al-Hasa-SA2407/2016 (GenBank ID: MN654975.1) with a sequence identity of 99.86%. Furthermore, the isolates contained several amino acids substitutions in ORF1a (32), ORF1ab (25), S (2), ORF3 (4), ORF4b (4), M (3), ORF8b (1), and the N protein (1). The analysis further identified a recombination event in one of the reported sequences (OQ423284/MERS-CoV/dromedary/UAE-Al Ain/13/2016). Conclusion: Data presented in this study indicated the need for continuous identification and characterization of MERS-CoV to monitor virus circulation in the region, which is necessary to develop effective control measures. The mutations described in this investigation might not accurately represent the virus's natural evolution as artificial mutations may develop during cell culture passage. The isolated MERS-CoV strains would be helpful in new live attenuated vaccine development and efficacy studies.

Pathological, microscopic, and molecular diagnosis of paratuberculosis/John's disease in naturally infected dromedary camel (Camelus dromedarius).

Background and Aim: Paratuberculosis (PTB) or John's disease is a chronic disease of ruminants impeding the reproduction and productivity of the livestock sector worldwide. Since there is a lack of pathological studies explaining the nature and development of the disease in camels, this study aimed to highlight the anatomopathological changes of PTB in camels, which may help in verifying and validating some diagnostic tests used to detect the etiology of the disease in camel tissues. Materials and Methods: In August 2017, at Alselaa border's Veterinary Clinic of Al Dhafra Region, Western Abu Dhabi, UAE, one imported culled she-camel of 2 years old was subjected to clinical, microscopic, and anatomopathological investigations along with real-time quantitative polymerase chain reaction (q-PCR) to confirm the infection and correlate between clinical signs and pathological lesions of the PTB in dromedary camels. Results: Clinically, typical clinical signs compliant with the pathognomonic gross and histologic lesions of PTB were seen in naturally infected dromedary camel. As presumptive diagnosis microscopically, acid-fast coccobacillus bacterium clumps were demonstrated in direct fecal smears as well as in scraped mucosal and crushed mesenteric lymph node films, and in histopathological sections prepared from a necropsied animal and stained by Ziehl-Neelsen stain. Free and intracellular acid-fast clump phagosomes were further confirmed as Mycobacterium avium subsp. paratuberculosis by q-PCR. Conclusion: Clinical signs and pathological lesions of paratuberculosis in a dromedary camel were found to be similar to those of the other susceptible hosts.

A Clinical, Pathological, Epidemiological and Molecular Investigation of Recent Outbreaks of Peste des Petits Ruminants Virus in Domestic and Wild Small Ruminants in the Abu Dhabi Emirate, United Arab Emirates.

(1) Background: Peste des petits ruminants (PPR) is a highly contagious animal disease affecting small ruminants, leading to significant economic losses. There has been little published data on PPR virus (PPRV) infection in the United Arab Emirates (UAE); (2) Methods: four outbreaks reported in goats and Dama gazelle in 2021 were investigated using pathological and molecular testing; (3) Results: The infected animals showed symptoms of dyspnea, oculo-nasal secretions, cough, and diarrhea. Necropsy findings were almost similar in all examined animals and compliant to the classical forms of the disease. Phylogenetic analysis based on N gene and F gene partial sequences revealed a circulation of PPRV Asian lineage IV in the UAE, and these sequences clustered close to the sequences of PPRV from United Arab Emirates, Pakistan, Tajikistan and Iran; (4) Conclusions: PPRV Asian lineage IV is currently circulating in the UAE. To the best of our knowledge, this is a first study describing PPRV in domestic small ruminant in the UAE.

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity.

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.

Pathology and Molecular Epidemiology of Fowl Adenovirus Serotype 4 Outbreaks in Broiler Chicken in Abu Dhabi Emirate, UAE.

BACKGROUND: Fowl adenovirus serotype 4 (FAdV-4), causing inclusion body hepatitis (IBH) and hydropericardium hepatitis syndrome (HPS), is responsible for the significant economic losses in poultry industry worldwide. This study describes FAdV disease and molecular characteristics of the virus as the first report in UAE. METHODOLOGY: Clinical, necropsy, histopathology, qPCR and phylogenetic analysis of hexon gene were used to diagnose and characterize the virus. RESULTS: The age of the infected broiler chicken was 2-4 weeks. The morbidity and mortality rates ranged between 50 and 100% and 44 and 100%, respectively. Clinically, sudden onset, diarrhea, anemia and general weakness were recorded. At necropsy, acute necrotic hepatitis, with swollen, yellowish discoloration, enlarged and friable liver; hydropericarditis with hydropericardium effusions; and enlarged mottled spleen were observed. Histopathology examination revealed degeneration and necrosis, lymphocytic infiltration and inclusion bodies. The qPCR analysis detected the virus in all samples tested. Hexon gene sequence analysis identified FAdV serotype 4, species C as the major cause of FAdV infections in UAE in 2020, and this strain was closely related to FAdV-4 circulating in Saudi Arabia, Pakistan, Nepal and China. CONCLUSION: The serotype 4, species C, was the common FAdV strain causing IBH and HPS episodes in the region. This result may help design effective vaccination programs that rely on field serotypes.

Antibiotics Resistance in Escherichia coli Isolated from Livestock in the Emirate of Abu Dhabi, UAE, 2014-2019.

Escherichia coli (E. coli) is a zoonotic pathogen that showed growing resistance to antibiotics. No descriptive analysis highlights the threat of antimicrobial-resistant (AMR) of E. coli among livestock in the United Arab Emirates (UAE). Herein, we conducted phenotypic and genotypic resistance studies on E. coli isolates from livestock samples in the Emirate of Abu Dhabi based on routine diagnosis between the periods 2014-2019. Bacterial culture and disk diffusion methods were used for bacterial isolation and phenotypic resistance analysis. Resistance mechanism was studied by PCR targeting the most commonly resistance genes: ampicillin (bla SHV , bla CMY , and blaTEM-1B), tetracyclines (tetA and tetB), co-trimoxazole [sulfamethoxazole (sul1, sul2, and sul3) + trimethoprim (dfrA1 and dfrA17)], aminoglycosides [aph(3")-Ia, aph(6)-Id, and aac(3)-IV], and fluoroquinolones (qnrA and aac(6')-Ib-cr). Analysis of 165 E. coli isolates showed resistant to ampicillin, tetracycline, co-trimoxazole, gentamicin, and enrofloxacin by 157/165 (95.4%), 154/165 (93.6%), 141/165 (86%), 139/165 (85%), and 135/165 (82.7%), respectively. Predominant resistance gene/s detected by PCR were bla CMY (119/160, 72%) and blaTEM-1B (154/160, 96.3%) for ampicillin; tetA (162/164, 98.8%) and tetB (112/164, 68.3%) for tetracyclines; sul2 (156/164, 95%), sul3 (138/164, 84%), and dfra17 (74/164, 44.5%) for co-trimoxazole; aph(3")-Ia (134/164, 82.1%) and aph(6)-Id (161/164, 98.2%) for aminoglycosides; and aac(6')-Ib-cr (61/61, 100%) for enrofloxacin. Both phenotypic and genotypic analyses revealed that all E. coli isolates were multidrug-resistant (resistance to 3, 4, and 5 antibiotics classes by 3.6%, 57.6%, and 38.8%, respectively) carrying one or more resistance gene/s for the same antibiotic. PCR profiling confirmed the presence of resistance genes corresponding to their antibiotic profile. Results of the study will highlight the knowledge based on E. coli AMR related to livestock in UAE that may call for interventions.

Pathology, bacteriology and molecular studies on caseous lymphadenitis in Camelus dromedarius in the Emirate of Abu Dhabi, UAE, 2015-2020.

Caseous lymphadenitis (CLA) or pseudotuberculosis is a chronic zoonotic bacterial disease caused by Corynebacterium pseudotuberculosis, which affects livestock and humans. This study aimed to describe the pathology, bacteriology and confirm the identity of the pathogen by 16S rRNA gene sequencing in Camelus dromedarius. A total of 12 camels with suspected CLA in three regions of Abu Dhabi Emirate (Abu Dhabi, Al Ain and Al Dhafra), United Arab Emirate (UAE) were subjected to clinical and postmortem examinations from January 2015 to December 2020. Clinically, camels were emaciated and showed the presence of external caseous abscesses suggestive of CLA. Postmortem examination showed multiple abscesses of variable sizes with caseous material encapsulated by fibrous tissue in the liver, lungs, muscle, and lymph nodes. Following clinical and postmortem examination, blood, pus and different tissue samples were collected for subsequent analysis. Histopathological examination of all organs stained with Hematoxylin and Eosin (H&E) indicated a central caseo-necrotic core that was admixed with bacterial colonies and infiltration of chronic inflammatory cells, surrounded by a pyogenic membrane, and an outer fibrous connective tissue capsule. Bacterial culture identified the isolates of Corynebacterium pseudotuberculosis biotype ovis strain, and these isolates were shown to be sensitive to all antibiotics tested (penicillin, ampicillin, Co-trimoxazole, enrofloxacin and tetracycline). Moreover, the identity of the isolates was confirmed by partial sequencing of the 16S rRNA gene which showed a 100% identity to Corynebacterium pseudotuberculosis. Phylogenetic analysis based on 16S rRNA gene sequence clearly differentiates Corynebacterium pseudotuberculosis from other species of Corynebacterium. Briefly, this study provided the basic information for infection of Corynebacterium pseudotuberculosis in Camels and will help in controlling of this pathogen in the region.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

Field investigation and phylogenetic characterization of orf virus (ORFV) circulating in small ruminants and Pseudocowpoxvirus (PCPV) in dromedary camels of eastern Sudan.

In this study, livestock herders in eastern Sudan were interviewed through structured questionnaire involved 14046 animals in 151 herds (87 camel herds, 51 sheep and 13 goats) from June to September of 2016 in Showak area of Gadarif State to get some epidemiological information on contagious ecthyma (CE) infection. 102 suspected cases of CE were investigated (38 sheep, 22 goats and 42 camels) by a second questionnaire focusing on age and sex of affected animals beside number and localization of the lesions. Representative tissue samples of scab lesion scrapings were collected from a total of 36 suspected sheep, goats and camels for DNA extraction to identify PPV by quantitative real-time PCR and gel-based PCR, then a PCR protocol was used to obtain DNA fragment of B2L gene from six DNAs (2 from each animal species) for sequencing. Phylogenetic tree based on nucleotide sequences was constructed and all data were analyzed statistically. Obtained result has shown morbidity rate of 23.8% and a case fatality rate of 4.7 % in overall investigated animals resulting in a significant economic loss. Within individual herd, the morbidity rate varied from 5.6 to 42.8%, while the case fatality rate ranged between 0 and 33.3%. Camels accounted for the highest case fatality rate with 6.5% compared to sheep and goats which their rates were 2.8% and 1.3%, respectively. 93% of the affected animals were young less than one-year-old. The prevalence of CE was high in the rainy season compared to winter and summer. Out of 36 scab materials collected from sheep, goats, and camels, 24 gave positive specific amplification in real-time PCR and 21 in the gel-based PCR. DNA sequencing confirmed the PCR results. All sequences had a high G + C content of 62.6-63.9%. A BLAST search also revealed that the studied sheep PPV (SPPV) isolates shared 99.08% nucleotide sequence intragroup identity, 96.88-97.27% identity with the goat PPV (GPPV) isolates and together they belong to the Orf virus (ORFV) species, while the camel PPV (CPPV) isolates are close to the Pseudocowpoxvirus (PCPV) species of the PPV genus and share 92.51-93.62 % identity with the GPPV isolates. In conclusion the present study demonstrated that the gross lesion produced by PPV in sheep, goats and camels is generally similar, yet the PPVs circulating in eastern Sudan in camels (PCPV) are genetically distinct from those affecting sheep and goats (ORFV). Contagious ecthyma in eastern Sudan causes significant morbidities and mortalities and control measures, guided by the results of this investigation ought to be implemented.

More cell culture passaged Camelpox virus sequences found resembling those of vaccinia virus.

Background: Camelpox is the most infectious and economically important disease of camelids that causes significant morbidity and mortality rates. Several live attenuated vaccines against Camelpox virus (CMLV) are produced worldwide by passaging field isolates in cell culture. Sequence of a high passage Saudi isolate of CMLV was previously found closely resembled Vaccinia virus (VACV). Aim: To determine whether other high cell culture passage CMLV isolates are genetically resemble VACV and further to explore the possible mechanism of the resemblance. Methods: We performed polymerase chain reaction and DNA sequence analysis of A-type inclusion body protein (ATIP), L1R, and open reading frame (ORF) 185 genes on different cell culture passage levels of a field isolate, two high passage vaccines, wild-type, and reference strains of CMLV. Results: We demonstrate that additional two high passage attenuated vaccine candidate from Sudan and UAE likewise contain sequences resembling VACV more than CMLV. Furthermore, sequence analysis of the ATIP gene of selected virus passages in cell culture revealed that the shift to VACV-like occurred between passage 11 and 20 and up to the 10th passage the genome still resembles wild-type virus. This observation was further confirmed by recombination analysis which indicated recombination events at ATIP and ORF185 genes occurred at higher passages. Conclusion: We confirmed that the cell culture passage CMLV turns to resemble VACV after cell culture passage and concluded that the resemblance may not be a result of contamination or misidentification as previously thought but could be due to recombination events that occurred during the passage process.

Development of oriC-plasmids for use in Mycoplasma hyorhinis.

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 × 10-4 and 2.0 × 10-5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas.