Emergence of the novel infectious bursal disease virus variant in vaccinated poultry flocks in Egypt.

RESEARCH HIGHLIGHTS: New variant IBDV which emerged in Egypt clustered with Chinese nVarIBDV.nVarIBDV spread subclinically across a wide geographic area.Mutation at 321 represents capsid's most exposed part, a defining feature.Antigenically modified vvIBDV still circulating in Egypt with typical lesions.

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.

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.

Highly Pathogenic Avian Influenza Virus (H5N8) Clade 2.3.4.4 Infection in Migratory Birds, Egypt.

We isolated highly pathogenic avian influenza virus (H5N8) of clade 2.3.4.4 from the common coot (Fulica atra) in Egypt, documenting its introduction into Africa through migratory birds. This virus has a close genetic relationship with subtype H5N8 viruses circulating in Europe. Enhanced surveillance to detect newly emerging viruses is warranted.

The sequence of the full spike S1 glycoprotein of infectious bronchitis virus circulating in Egypt reveals evidence of intra-genotypic recombination.

Infectious bronchitis virus (IBV) continues to circulate worldwide, with a significant impact on the poultry industry and affecting both vaccinated and unvaccinated flocks. Several studies have focused on the hypervariable regions (HVRs) of the spike gene (S1); however, genetic and bioinformatics studies of the whole S1 gene are limited. In this study, the whole S1 gene of five Egyptian IBVs was genetically analyzed. Phylogenetic analysis revealed that the Egyptian IBVs are clustered within two distinct groups: the classic group resembling the GI-1 genotype (vaccine strains) and the variant group (field strains) of the GI-23 genotype. The variant genotype was divided into two distinct subgroups (Egy/var I and Egy/var II) resembling the Israeli variants IS/1494 and IS885 strain, respectively. Significant amino acid sequence differences between the two subgroups, especially in the epitope sites, were identified. A deletion at position 63 and an I69A/S substitution mutation associated with virus tropism were detected in the receptor-binding sites. The deduced amino acid sequence of HVRs of the variant subgroups indicated different genetic features in comparison to the classic vaccine group (H120 lineage). The Egyptian variant IBVs also contained additional N-glycosylation sites compared to the classical viruses. Recombination analysis gave evidence for distinct patterns of origin by recombination throughout the S1 gene, suggesting that the recent virus IBV-EG/1586CV-2015 emerged as a recombinant of two viruses from the variant groups Egy/var I and Egy/var II, providing another example of intra-genotypic recombination among IBVs and the first example of recombination within the GI-23 genotype. Our data suggest that both mutation and recombination may be contributing to the emergence of IBV variants. Moreover, we found that the commercially used vaccines are genotypically distant from the circulating field strains. Hence, continuous follow-up of the current vaccine strategy is highly recommended for better control and prevention of infectious bronchitis virus in the poultry sector in Egypt.

The matrix gene of influenza A H5N1 in Egypt, 2006-2016: molecular insights and distribution of amantadine-resistant variants.

Large-scale sequence analysis of Matrix (M) gene and its coding proteins M1 and M2 was performed for 274 highly pathogenic avian influenza viruses H5N1 circulated in Egypt from 2006 to 2016. The aim is to study the amantadine-resistant markers distribution and to estimate the evolutionary rate. 246 viruses were obtained from the Global Initiative on Sharing All Influenza Data base, and 28 additional viruses were sequenced. Maximum clade credibility (MCC) phylogenetic tree revealed that the M gene has evolved into two different lineages. Estimated Evolutionary analysis showed that the M2 protein possessed higher evolutionary rates (3.45 × 10-3) than the M1 protein (2.73 × 10-3). M gene encoding proteins revealed significant markers described to be associated with host tropism and increase in virulence: V15I, N30D, and T121A in M1 and L55F in M2 protein. Site analysis focusing attention on the temporal and host distribution of the amantadine-resistant markers was carried out and showed that vast majority of the M2 amantadine-resistant variants of clade 2.2.1.1 (n = 90) is N31 marker, in addition to G27 (n = 7), A27 (n = 5), I27 (n = 1), and S30 (n = 1). In 2010-2011, amantadine resistant frequency increased considerably resembling more than half of the resistant variants. Notably, all viruses of clade 2.2.1.1 possessed amantadine-resistant marker. However, almost all current circulating viruses in Egypt of clade 2.2.1.2 from 2014 to 2016 did not carry any amantadine-resistant markers.

SARS-CoV-2 seroprevalence determination in pets and camels in Egypt using multispecies enzyme-linked immunosorbent assay.

The coronavirus disease 2019 (COVID-19) pandemic has translated into a worldwide economic recession and public health crisis. Bats have been incriminated as the main natural host for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of the COVID-19 pandemic. However, the reservoir and carrier hosts of the virus remain unknown. Therefore, a cross sectional serosurvey study was performed to estimate antibodies to SARS-CoV-2. To assess IgM antibodies to SARS-CoV-2 nucleocapsid protein (NP), a SARS-CoV-2 Double Antigen Multispecies diagnostic enzyme-linked immunosorbent assay kit was used. The seropositive samples were confirmed and validated by measuring IgG antibody titers in sera. The enrolled animals were from different locations in the Giza governorate, Egypt, and were sampled at the time of the pandemic; they comprised 92 companion animals and 92 domestic camels. The study established that 4.76% (1/21 clinical samples) of dogs, 7.69% of cats (1/13 shelter samples) and 1.08% (1/92) of camels, had measurable SARS-CoV-2 NP IgM antibodies. All IgM-seropositive samples were IgG positive with a measurable titer of 34.5, 28.6, and 25.8 UI/mL for dog, cat, and camels, respectively. According to our best knowledge, this study was the first to assess SARS-CoV-2 seroprevalence in the specific animals investigated in Egypt. These results may herald a promising epidemiological role for pet animals and camels in SARS-CoV-2 virus maintenance. Thus, our study's results ought to be confirmed with a nationwide seroprevalence study, and further studies are required to clarify whether these animals act as active or passive carriers.

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.

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.

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.

Evidence for origin of lavender foal syndrome among Egyptian Arabian horses in Egypt.

BACKGROUND: Lavender foal syndrome (LFS) is a fatal hereditary condition that is inherited in an autosomal recessive pattern. This detrimental mutation is more common in Arabian foals of Egyptian origin than foals from other bloodlines. Heterozygous horses are carriers of the LFS trait and appear normal, while recessive homozygous foals died shortly after birth due to serious complications. In Egypt, in 2014, an Egyptian foal died after manifestations of neurological signs and abnormal coat colour as LFS signs. Therefore, it is important to identify LFS carriers in the population of Arabian horses in Egypt and to encourage improvement of the Arabian horse industry in Egypt by constructing a breeding system based on genetic background in order to avoid mating between carriers and reduce financial losses from deaths of affected foals. OBJECTIVES: To establish a PCR-based test for detecting the MYO5A gene mutation causing LFS in the registered Arabian horse population in Egypt prior to breeding. STUDY DESIGN: Cross sectional survey (n = 170) plus targeted sampling (n = 30). METHODS: A total of 200 samples were collected from an Arabian farm in Egypt and some of them were traced for LFS based on the farm records. The LFS genotypes were identified using the PCR-RFLP technique, fragment analysis followed by sequence analysis. RESULTS: The overall mutated allele and genotype frequencies (N/L) were 0.08 and 16%, respectively. CONCLUSION: The observed frequency of heterozygotes suggests foals affected with LFS will be produced among Arabian horses in Egypt. Therefore, screening of the entire population for this mutation should be undertaken in the breeding program.

Immunogenicity and Cross-Protective Efficacy Induced by an Inactivated Recombinant Avian Influenza A/H5N1 (Clade 2.3.4.4b) Vaccine against Co-Circulating Influenza A/H5Nx Viruses.

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

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.

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.

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.

Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt.

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt is a major concern since this subtype is widely distributed in different countries in the Middle East, here we describe the full genetic characterization of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was previously isolated from a clinically normal quail flock in Giza, Egypt in May 2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated Egyptian virus showed the highest similarity with one group of recent Israeli strains (97 %) circulating from 2006-2010. Sequence homology and phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong Kong/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that circulated in the region from 2006, especially with one group of recent Israeli strains. However, phylogenetic analysis of the internal genes like PB2, NP, and PA genes identified possible reassortment events for these genes with singular Israeli strains. This study indicates progressive evolution of this subtype in the Middle East region and possible mechanism of virus adaptation in land-based poultry like in quails.

Effect of cocirculation of highly pathogenic avian influenza H5N1 subtype with low pathogenic H9N2 subtype on the spread of infections.

Widespread prevalence of avian influenza H9N2 subtype in the Middle East region and its detection in Egypt in quail in early summer 2011 added another risk factor to the Egyptian poultry industry in addition to highly pathogenic H5N1 subtype. This situation increases the need for further surveillance and investigation of H9N2 viruses in commercial and household chickens. This work describes detection and genetic characterization of recently isolated H9N2 viruses from chicken flocks. Parallel detection and genetic characterization of H5N1 viruses from infections in poultry has also been done to compare the prevalence of the two subtypes in close geographic locations in Egypt. Phylogenetic analysis of the HA gene showed that the Egyptian isolates of H9N2 were grouped together within the quail/Hong Kong/G1/97-like lineage, similar to the circulating viruses in the Middle East, with very close phylogeny to the Israeli viruses. The prevalence of H5N1 viruses from cases recorded in poultry in the nearby areas revealed a marked decrease in disease incidence in commercial broilers but an increased incidence in household birds. The genetic characterization of the H5N1 viruses indicated predominance of the classic 2.2.1 subclade, with evolution of new viruses and no detection for the variant 2.2.1.1 subclade. The cocirculation of the two subtypes, H5N1 and H9N2, of avian influenza may affect the limit of spread and the epizootiologic pattern of the infections for both subtypes, especially when different vaccination and biosecurity approaches are applied in the field level.

Application of Loop-Mediated Isothermal Amplification (LAMP) in Sex Identification of Parrots Bred in Egypt.

Over 400 of the 3800 tropical avian species are endangered or threatened. One of many solutions to conserve animal biodiversity is breeding animals in zoos or private animal farms. Animal breeding programs are difficult to implement in species with sexual monomorphism, such as parrots. Molecular biology methods offer a solution to determine the sex of these species. Therefore, in this study, we aimed to test the performance of PCR and LAMP techniques on sex identification for 21 parrot species belonging to three families, i.e., Psittacidae, Cacatuidae, and Psittaculidae. We established a protocol for DNA isolation from feathers in our laboratory and found optimal conditions for PCR and LAMP. We showed that the LAMP method with the use of the PSI-W primers set, developed by Centeno-Cuadros, functions in 17 previously untested species. Moreover, we found that further improvements are required in universal LAMP primers for the detection of parrot DNA, which are necessary for confirmation of the male sex. The LAMP method also proved to be more sensitive for female sex identification in contrast to the reference PCR test. Therefore, we conclude that LAMP is a suitable method for the routine diagnostic sex identification of parrots.

Development and evaluation of one-step real-time RT-PCR assay for improved detection of foot-and-mouth disease virus serotypes circulating in Egypt.

Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RT-qPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.

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.