Development of Paper-Based Fluorescent Molecularly Imprinted Polymer Sensor for Rapid Detection of Lumpy Skin Disease Virus.

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.

A longitudinal study of Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in humans in 2012. Since then, 2605 cases and 937 associated deaths have been reported globally. Camels are the natural host for MERS-CoV and camel to human transmission has been documented. The relationship between MERS-CoV shedding and presence of neutralizing antibodies in camels is critical to inform surveillance and control, including future deployment of camel vaccines. However, it remains poorly understood. The longitudinal study conducted in a closed camel herd in Egypt between December 2019 and March 2020 helped to characterize the kinetics of MERS-CoV neutralizing antibodies and its relation with viral shedding. RESULTS: During the 100-day longitudinal study, 27 out of 54 camels (50%) consistently tested negative for presence of antibodies against MERS-CoV, 19 (35.2%) tested positive and 8 (14.8%) had both, positive and negative test results. Fourteen events that could be interpreted as serological indication of probable infection (two seroconversions and twelve instances of positive camels more than doubling their optical density ratio (OD ratio) in consecutive samples) were identified. Observed times between the identified events provided strong evidence (p = 0.002) against the null hypothesis that they occurred with constant rate during the study, as opposed to clustering at certain points in time. A generalized additive model showed that optical density ratio (OD ratio) is positively associated with being an adult and varies across individual camels and days, peaking at around days 20 and 90 of the study. Despite serological indication of probable virus circulation and intense repeated sampling, none of the tested nasal swab samples were positive for MERS-CoV RNA, suggesting that, if the identified serological responses are the result of virus circulation, the virus may be present in nasal tissue of infected camels during a very narrow time window. CONCLUSIONS: Longitudinal testing of a closed camel herd with past history of MERS-CoV infection is compatible with the virus continuing to circulate in the herd despite lack of contact with other camels. It is likely that episodes of MERS-CoV infection in camels can take place with minimal presence of the virus in their nasal tissues, which has important implications for future surveillance and control of MERS-CoV in camel herds and prevention of its zoonotic transmission.

Development of multiplex gold nanoparticles biosensors for ultrasensitive detection and genotyping of equine herpes viruses.

Gold nanoparticles (GNPs) biosensors can detect low viral loads and differentiate between viruses types, enabling early diagnosis and effective disease management. In the present study, we developed GNPs biosensors with two different capping agent, citrate-GNPs biosensors and polyvinylpyrrolidone (PVP)-GNPs biosensors for detection of EHV-1 and EHV-4 in multiplex real time PCR (rPCR). Citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-1 with mean Cycle threshold (Ct) 11.7 and 9.6, respectively and one copy as limit of detection, while citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-4 with mean Ct 10.5 and 9.2, respectively and one copy as limit of detection. These findings were confirmed by testing 87 different clinical samples, 4 more samples were positive with multiplex GNPs biosensors rPCR than multiplex rPCR. Multiplex citrate-GNPs and PVP-GNPs biosensors for EHV-1 and EHV-4 are a significant breakthrough in the diagnosis of these virus types. These biosensors offer high sensitivity and specificity, allowing for the accurate detection of the target viruses at very low concentrations and improve the early detection of EHV-1 and EHV-4, leading to faster control of infected animals to prevent the spread of these viruses.

West Nile virus: The current situation in Egypt.

Background and Aim: Due to climatic changes, arthropod-borne viruses have become a global health concern. In Egypt, West Nile virus (WNV) was initially detected in humans in 1950 and then in 1951, 1954, 1968, and 1989. Although WNV infection has been recorded in numerous Middle Eastern countries, its prevalence among the equine population in Egypt is unknown. This study aimed to investigate the current situation of vector-borne WNV in Egypt, estimate its seroprevalence, and assess the associated risk factors. Materials and Methods: We screened 1100 sera samples and nasal swabs from the same equids, 156 mosquito pools, and 336 oropharyngeal and cloacal swabs from migratory birds for WNV. The sera were investigated for the presence of immunoglobulin G (IgG) and immunoglobulin M (IgM) against WNV-prE. Real-time reverse transcription-polymerase chain reaction was used to detect WNV RNA in the nasal swab samples, mosquito pools, and migratory birds' oropharyngeal and cloacal swabs. Results: The seroprevalence showed positive IgG in sera samples collected from different districts. The data showed that horses were 1.65-fold more susceptible than donkeys, with male being 1.45 times more susceptible than females. Moreover, the tested equids samples were divided into three groups based on their age: <5 years, 5-10 years, and >10 years. The 5-10-year group was 1.1 and 1.61 times more vulnerable to infection than the <5- and >10 year groups. All the sera samples were negative for IgM. The nasal swabs from equids, oropharyngeal and cloacal swabs from migratory birds, and mosquito samples tested negative for WNV by molecular detection. Conclusion: Based on the obtained data, we recommend that effective control programs should be implemented to enable epidemiological investigations and understand the current situation of WNV in Egypt.

Circulating foot-and-mouth disease virus serotype A African-genotype IV in Egypt during 2022.

Background and Aim: Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Materials and Methods: Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Results: Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35-75) and the immunogenic determinants of the G-H loop of VP1 (residues 100-146 and 161-175). Conclusion: The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes.

Interspecies transmission of SARS CoV-2 with special emphasis on viral mutations and ACE-2 receptor homology roles.

COVID-19 outbreak was first reported in 2019, Wuhan, China. The spillover of the disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to a wide range of pet, zoo, wild, and farm animals has emphasized potential zoonotic and reverse zoonotic viral transmission. Furthermore, it has evoked inquiries about susceptibility of different animal species to SARS-CoV-2 infection and role of these animals as viral reservoirs. Therefore, studying susceptible and non-susceptible hosts for SARS-CoV-2 infection could give a better understanding for the virus and will help in preventing further outbreaks. Here, we review structural aspects of SARS-CoV-2 spike protein, the effect of the different mutations observed in the spike protein, and the impact of ACE2 receptor variations in different animal hosts on inter-species transmission. Moreover, the SARS-CoV-2 spillover chain was reviewed. Combination of SARS-CoV-2 high mutation rate and homology of cellular ACE2 receptors enable the virus to transcend species barriers and facilitate its transmission between humans and animals.

The potential role of migratory birds in the transmission of pathogenic Campylobacter species to broiler chickens in broiler poultry farms and live bird markets.

BACKGROUND: Campylobacter species (spp.) are one of the most important zoonotic bacteria possessing potential hazards for animal and human health worldwide. Migratory birds are implicated as significant carriers for microbes and a play very important role in the dissemination of Campylobacter to broiler chickens and their environment. The purpose of this investigation was to detect the prevalence, antibiotic resistant patterns, virulence and diversity of pathogenic Campylobacter spp. in 7 migratory bird species (Northern shoveler, Common pochard, Common teal, Northern pintail, Eared Grebe, Great Crested Grebe and Garganey) and broiler chickens that were collected from broiler poultry farms and live bird markets. RESULTS: The prevalence of Campylobacter was 12.5% (25/200), of which 15% (15/100) was recovered from 5 migratory bird species only and 10% (10/100) from broiler chickens. At the level of migratory birds, eight isolates (53.3%) were Campylobacter jejuni (C. jejuni) and 7 isolates (46.7%) were Campylobacter coli (C. coli) meanwhile, in broiler chickens C. jejuni and C. coli were 50% (5/10) for each. All isolated strains had phenotypic resistance to doxycycline, while all of the isolates were susceptible to amikacin. The multidrug resistance to three, four or five antimicrobial classes was found in 72% (18/25) of the isolated strains. The multiantibiotic resistance index between the examined isolates was 0.22-0.77, with 10 antibiotic resistance patterns. The virulence of isolated Campylobacter strains (from both migratory birds and broiler chicken birds) was detected by targeting the VirB11, ciaB and iam genes which were recorded at 16%, 52% and 100%, respectively. Additionally, 100% and 84% of the antibiotic resistance genes were identified as tetA and BlaOXA-61, respectively. CONCLUSIONS: The results of this study revealed the diversity between all the isolated strains from migratory birds and their similarity to broiler chicken isolates. The findings of the present study highlight the impact of migratory birds visiting Egypt and other countries on pathogenic Campylobacter spp. carrying pathogenic virulence and resistance genes, necessitating the application of biosecurity measures to prevent migratory birds from entering farms during their migration period.

Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

SARS-CoV-2 infection of companion animals in Egypt and its risk of spillover.

BACKGROUND: Reverse zoonoses occur because of interactions between humans and animals. Homology of ACE-2 cell receptors in different hosts and high mutation rate of SARS-CoV-2 enhance viral transmission among species. OBJECTIVES: This study aimed to investigate spillover of SARS-CoV-2 between humans and companion animals. METHODS: A cross-sectional study was constructed using nasopharyngeal/oropharyngeal swabs, serum and blood samples collected from 66 companion animals (33 cats and 33 dogs) that were in contact with SARS-CoV-2-positive owners from December 2020 to March 2021. Swabs were screened by rRT-PCR and some positive cases were confirmed by partial spike gene sequencing. Clinical pathology and pathological studies were also performed. RESULTS: Our findings revealed that 30% of cats (10/33) and 24% of dogs (8/33) were SARS-CoV-2 positive. While 33% of these animals were asymptomatic (6/18), 28% showed mild respiratory signs (5/18) and 39% displayed severe respiratory signs (7/18) including 4 dead cats 40% (4/10). Partial spike gene sequencing of 6 positive samples collected in December 2020 were identical to SARS-CoV-2 that was detected in humans in Egypt in that time frame. Clinical pathology findings revealed thrombocytopenia, lymphocytopenia, as well as elevated levels of D-dimer, LDH, CRP, and ferritin. Post-mortem and histopathological examinations illustrated multisystemic effects. CONCLUSIONS: There is a potential occurrence of SARS-CoV-2 spillover between humans and pet animals. IMPACTS: The present study highlighted the potential occurrence of SARS-CoV-2 spillover between humans and their companion animals. Biosecurity measures should be applied to decrease spread of SARS-CoV-2 among humans and pet animals.

Molecular detection and phylogenetic analysis of newly emerging foot-and-mouth disease virus type A, Lineage EURO-SA in Egypt in 2022.

A newly emerging and exotic foot-and-mouth disease virus (FMDV) caused a recent outbreak of serotype A in Egypt in 2022, which affected cattle and water buffalo. Previous phylogenetic studies on FMDV circulating in Egypt have mainly focused on genomic regions encoding the structural proteins which determine FMDV serotype. No study has yet determined structural proteins sequences of the newly emerging Europe-South America (EURO-SA) lineage which was recently isolated from Egypt during a routine surveillance in 2022. The objective of the current study was to analyze the structural proteins of the Venezuelan type which belongs to EURO-SA. The new isolate was related to serotype A lineage Euro-South America. Phylogentic analyses have reveled that the newly isolated lineage samples were closely related to reported sequences that have been identified in Venzuela and Colombia. Analysis of structural protein sequences revealed the recent isolates belong to prototype strain A24 Cruzeiro. Notably, nucleotide sequences of the Egyptian isolate was related to Venezuelan, Brazilian, and Colombian strains with identity not exceeding 90%. The divergence which appears in the genetic identity of the Egyptian A/EURO-SA lineage from other related strains may be attributed to the absence of Euro-SA lineage sequence from Egypt. The present study is the first report on the detection of EURO-SA lineage in Egypt. The recent detection of the EURO-SA lineage samples may be explained due to imported animals from Colombia or Brazil which share geographical borders with Venezuela. The findings of the present study highlight the significance of continuous monitoring of FMDV in Egypt for newly emerging FMDVs.

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Phenotypic and Genotypic Screening of Colistin Resistance Associated with Emerging Pathogenic Escherichia coli Isolated from Poultry.

Chickens continue to be an important reservoir of zoonotic multidrug-resistant illnesses. Antimicrobial resistance correlated with colistin has emerged as a critical concern worldwide in the veterinary field and the public health sector. The current study investigated the prevalence of multidrug-resistant avian pathogenic Escherichia coli among chicken farms in three Egyptian governorates, focusing on colistin resistance assessment. A total of 56 Escherichia coli isolates were recovered out of 120 pooled samples obtained from diseased chicken broilers (46.7%). The E. coli isolates were serotyped to nine different serotypes; the highest incidence was for O125 (n = 18). The E. coli isolates demonstrated multidrug-resistant patterns against 10 antibiotics, especially clindamycin, tetracycline, streptomycin and ampicillin, by 100, 100, 96.4 and 92.9%, respectively. On the other hand, colistin resistance was 41.1% using AST. All E. coli isolates displayed positive colistin resistance growth on chromogenic medium, but only 25% represented this positivity via MIC estimation and Sensititre kit. PCR results revealed that all isolates harbored mcr-1, but no isolates harbored the other 2-5 mcr genes. In conclusion, the study demonstrated the emergence of multidrug-resistant, especially colistin-resistant, E. coli among chicken broiler flocks, and mcr-1 is the master gene of the colistin resistance feature.

Mutations of the SARS-CoV-2 Spike Glycoprotein Detected in Cats and Their Effect on Its Structure and Function.

The high frequency of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) mutations and homology of the Angiotensin-Converting Enzyme-2 (ACE2) cell receptors in various hosts help the virus transcend species barriers. In this study, we investigated the mutations of the SARS-CoV-2 spike glycoprotein detected in cats and their effect on its structure and function. Interestingly, some of these mutations are reported here in cats for the first time. Structural analysis showed seven residue substitutions in the spike glycoprotein. Four of the detected mutations are located on the spike surface, which are critical interaction points for neutralizing antibodies. Furthermore, three of the reported mutations could facilitate viral binding to the ACE2 host receptor, influence S1/S2 cleavage, destabilize the ß-hairpin structure of the S2 and enhance viral infectivity. Structural modeling and phylogenic analysis of the ACE2 receptor provided an indication of the binding capacity of the virus to the specific cell receptors of different species and hosts. The presented work highlights the effects of the residue substitutions on viral evasion, infectivity and possibility of SARS-CoV-2 spillover between humans and cats. In addition, the work paves the way for in-depth molecular investigation into the relationship between SARS-CoV-2 receptor binding and host susceptibility.

Impact of udder infections on biochemical composition of milk in context of pesticides exposure.

Background and Aim: Environmental contaminants such as pesticides have shown immunomodulatory effects that can make animals highly susceptible to pathogenic invasion. The current work aims to study the incidence of udder infections in a single dairy herd of 160 cows in Qalyoubia Governorate, in relation to the potential intoxication of dairy cattle with organochlorine (OCs) pesticides. The study also aims to investigate the impact of udder infections on milk composition. Materials and Methods: The dairy herd was screened for udder infections using the California mastitis test and measurement of somatic cell count (SCC), followed by bacteriological and molecular analysis. In parallel, the milk samples were also tested for residues of 15 OCs compounds using gas chromatographic analysis. Results: The examined herd showed a high prevalence of mastitis (37.5%) and Mycoplasma was identified as the main bacterial pathogen. OCs residues were detected in milk of 45 cows out of 160 with a higher incidence in mastitic (43.3%) than in healthy cows (19%). Further, the biochemical analysis of milk showed a significant drop in major electrolytes combined with a significant rise in blood-borne electrolytes (Na and Cl) and total protein. This was more extreme in the case of Mycoplasam mastitis compared to non-Mycoplasma mastitis. In addition, Mycoplasma mastitic milk revealed a high level of malondialdehyde associated with reduced antioxidant enzymes (glutathione peroxidase, superoxide dismutase and catalase), compared to non-Mycoplasma mastitis. Conclusion: Mycoplasma mastitis was shown to be associated with increased SCC and, in turn, appeared significantly correlated with increased biochemical changes in milk, indicating the serious impact of Mycoplasma mastitis on the dairy industry. Our data also show a strong correlation between increased SCC and biochemical changes in milk, suggesting that tested biochemical parameters might serve as potential biomarkers for the early detection of mastitis. The study also suggested a potential relationship between poisoning with OCs and susceptibility to bacterial udder infections. However, further studies are required to examine the immune status of a dairy herd in relation to the level of OCs in cow's blood, as well as the water sources used, grass forage and soil.

Multidrug-Resistant and Genetic Characterization of Extended-Spectrum Beta-Lactamase-Producing E. coli Recovered from Chickens and Humans in Egypt.

Colonization of food chain animals such as chickens with extended-spectrum ß-lactamases (ESBL) poses a major health threat to human. The current study aimed to determine the phenotypic and genotypic relationship between ESBL-producing E. coli from diseased human and chickens in Egypt. A total of 56 out of 120 chicken farms (46.7%) and 9 human samples (100%) were phenotypically and genotypically identified with at least one ESBL-phenotype/gene. Chicken isolates showed a high proportion of beta lactamase from CTX-M group 9 > TEM > PER families, followed by CTX-M group 1 > SHV > GES > OXA group10 > VEB > OXA group2 families, while human isolates only contained the CTX-M family. A high incidence of ESBL genes from the CTX-M family was recognized in both human and chicken isolates. Furthermore, nucleotide identity showed high similarity between chicken and human isolates. In conclusion, the current study traced phenotypes and genotypes of ESBL-producing E. coli from chickens and human samples in Egypt, reporting degrees of similarity that suggest potential zoonotic transmission. Our data highlighted the significant importance of chicken as a major food source not only in Egypt but all over the world in the spreading of ESBL-producing E. coli to human.

Molecular detection, phylogenetic analysis and genetic diversity of recently isolated foot-and-mouth disease virus serotype A African topotype, Genotype IV.

BACKGROUND: Surveillance for circulating emerging diseases of economic importance has a major role in the rapid response to major pathogen outbreaks. Foot-and-mouth disease virus (FMDV) is one of the significant endemic viruses in Egypt. FMDV is periodically investigated for monitoring evolution and emergence of new variants. The genetic characterization of foot-and-mouth disease (FMD) virus serotype A responsible for recent outbreaks of FMD in Egypt was determined. METHODS: Samples were collected from different locations and virus isolation was performed using BHK-21 cells. Viral RNA was extracted and samples were screened for FMDV using real-time RT-PCR. DNA sequence analysis was performed and computational and bioinformatics analyses were used to determine the substitution rates and phylogenetic relationship. RESULTS: Sequence and phylogenetic analyses of full-length 1D region of FMDV samples collected from different governorates in 2020 showed close similarity to Egyptian FMDV strains from serotype A-African topotype-G-IV with genetic variation of 6.5%. Recently isolated FMDV strains showed high genetic variations from locally used vaccine strains in the major antigenic sites of VP1 region. CONCLUSIONS: Although, efforts made by the veterinary authorities to implement an effective mass vaccination plan, the recently detected FMDV strains in this study could not be subtyped using the FMDV primers routinely used for molecular serotyping. These dissimilarities raise the alarm for reconsideration of the FMDV isolates used in vaccine manufacture. Clearly close monitoring of FMD in Egypt is urgently required to define the risks of future outbreaks and to ensure appropriate control measures against FMD major outbreaks.

Molecular Characterization of Newly Emerging Foot-and-Mouth Disease Virus Serotype SAT 2 of Lib-12 Lineage Isolated from Egypt.

An outbreak of foot-and-mouth disease virus (FMDV) serotype SAT 2 occurred in Egypt in 2018, which affected cattle and water buffalo. Previous phylogenetic studies on FMDV circulating in Egypt have mainly focused on genomic regions encoding structural proteins which determine FMDV serotype. So far, none of these studies have analyzed the open reading frame (ORF) sequence of Egyptian SAT 2/Lib-12 lineage. The present study aimed to analyze and identify the ORF genome sequence of Lib-12 lineage which belongs to FMDV serotype SAT 2 topotype VII in Egypt. The protocol workflow was optimized and tested using a representative field isolate of FMDV/SAT 2/Lib-12 from a bovine tongue sample collected in 2018 from Ismailia governorate (SAT2/EGY/Ismailia/2018). The protocol was based on reverse transcription polymerase chain reaction with multiple overlapping primers, amplicons sequencing, and assembly to complete the ORF consensus sequence. Alignments of the sequence fragments formed consensus genome sequence of 7219 nucleotides in length. The complete nucleotide sequence of the Egyptian isolate was related to Ethiopian, Nigerian, and Ghanaian strains, with identity not exceeding 95%. The divergence in the genetic identity of the Egyptian SAT 2/Lib-12 lineage from other Egyptian strains and Libyan isolates was 7%, and this may be attributed to the absence of the Lib-12 lineage ORF sequence from Egypt and Libya in the database. The present study significantly advances knowledge of the molecular analysis of FMDV SAT 2 and the design of vaccine selection for FMDV SAT 2 in Egypt. The study protocol could be applied to other FMDV serotypes.

Molecular characterization and pathological identification of a novel strain of delta papillomavirus-4 (bovine papillomavirus-2) in Egypt.

BACKGROUND AND AIM: Bovine papillomaviruses (BPV) are a heterogeneous group of oncoviruses, distributed globally, which produce major economic losses. In the current study, we compared the results of different diagnostic approaches and compared the strains identified in this study with previously characterized strains at local and international levels. MATERIALS AND METHODS: Samples of skin warts were collected from five bovines with generalized papillomatosis from two Egyptian provinces, Menya and Ismailia, in 2020. Electron microscopy, molecular characterization, histopathological, and immunohistochemical examination were performed. RESULTS: BPV was detected using electron microscopy in the collected samples. Using molecular characterization, BPV-2 was successfully identified for 1st time in Egypt. The strain has 99.6% identity with the BPV-2 reference strains obtained from GenBank. These results were supported by histopathology and immunohistochemistry examination. Partial nucleotide sequences of the L1 gene were submitted to GenBank with accession numbers MW289843 and MW289844. CONCLUSION: BPV-2 was reported for 1st time in the current study. The strain was identified grossly, microscopically, and pathologically and confirmed using molecular approaches. All results were consistent. The sequence analysis revealed that this strain has high sequence similarity to the reference Deltapapillomavirus-4, BPV-2 strains from Brazil and China.

Diagnosis of naturally occurring lumpy skin disease virus infection in cattle using virological, molecular, and immunohistopathological assays.

BACKGROUND AND AIM: Lumpy skin disease (LSD) is a contagious viral disease that has great economic losses among Egyptian breeding flocks. The present study was designed to compare the results of different diagnostic approaches used for the diagnosis of LSD virus (LSDV). MATERIALS AND METHODS: A total of 73 skin nodule samples were collected from suspected infected cattle with LSDV from some Egyptian governorates during 2019 and 2020. Trials for virus isolation (VI) and identification on embryonated chicken eggs (ECEs) were conducted. Molecular detection, histopathological, and immunohistochemical examination were also conducted. RESULTS: The virus was isolated into ECEs, and 58 samples of 73 were positive and gave a characteristic pock lesion on the chorioallantoic membrane. Twenty-two representative nodular skin specimens of the 58 positive samples were selected to be used for molecular, histopathological, and immunohistochemistry (IHC) diagnosis. Conventional polymerase chain reaction succeeded in detecting LSDV DNA in all tested 22 skin nodule samples. Histological examination of skins of different cases revealed various alterations depending on the stage of infection. IHC was used as a confirmatory test for detecting LSDV antigen in the tissues of the skin nodules of infected cattle using specific anti-LSDV antibodies. Lumpy skin viral antigen was detected within the cytoplasm of the epidermal basal cells layer and prickle cell and within the cytoplasm of the hair follicles' epithelial outer and inner roots. CONCLUSION: This study confirmed the prevalence of LSDV infection in different Egyptian governorates during 2019 and 2020. In addition, histopathology and IHC could be potential methods to confirm Lumpy skin disease infection besidesVI and molecular detection.

Temporal Dynamics of Influenza A(H5N1) Subtype before and after the Emergence of H5N8.

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015-2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.