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In the current study, we are investigating the viral causes of some respiratory clinical signs in some animals belongs to the family Equidae in eastern Saudi Arabia (ESA) during winter- 2019. We observed the progression of severe respiratory clinical signs among some horses, donkeys, and ponies in the ESA. Animals showed rapid respiration, fever, nasal discharges (started as serous then changed into mucopurulent with the progression of the infection per some animals). We conducted a longitudinal study to monitor the progression of this outbreak. We conducted molecular surveillance for the influenza virus Type-A using real-time PCR and regular RT-PCR. We also conducted a serosurveillance of the virus in sera of the tested animals using the commercially available enzyme-linked immunosorbent assay (ELISA). The molecular detection of the Influenza virus type-A virus from nasal swabs of the affected animals using the real-time PCR results clearly showing that 35.1% of the tested horses, donkeys, and ponies were positives. Further confirmation was achieved by reporting the seroconversion of some of the affected animals. Several attempts were conducted to isolate the circulating influenza strains using the embryonated chicken eggs were unsuccessful. This was based on the absence of any amplicons in the harvested embryonated egg fluids using some oligonucleotides for the common influenza virus genes (HA, NA, M, and N). Meanwhile, ELISA results revealed the detection of the antibodies in sera of horses and donkeys 72.9%. Seroconversion was reported in many animals several weeks after the onset of the outbreak. Taken together all these pieces of evidence, we confirm an influenza virus type-A outbreak among the tested animals during winter 2019.

Middle East respiratory syndrome coronavirus (MERS‐CoV) is one of the recently identified zoonotic coronaviruses. The one‐hump camels are believed to play important roles in the evolution and transmission of the virus. The animal‐to‐animal, as well as the animal‐to‐human transmission in the context of MERS‐CoV infection, were reported. The camels shed the virus in some of their secretions, especially the nasal tract. However, there are many aspects of the transmission cycle of the virus from animals to humans that are still not fully understood. Rodents played important roles in the transmission of many pathogens, including viruses and bacteria. They have been implicated in the evolution of many human coronaviruses, especially HCoV‐OC43 and HCoV‐HKU1. However, the role of rodents in the transmission of MERS‐CoV still requires more exploration. To achieve this goal, we identified MERS‐CoV that naturally infected dromedary camel by molecular surveillance. We captured 15 of the common rodents (rats, mice, and jerboa) sharing the habitat with these animals. We collected both oral and rectal swabs from these animals and then tested them by the commercial MERS‐CoV real‐time‐PCR kits using two targets. Despite the detection of the viral shedding in the nasal swabs of some of the dromedary camels, none of the rodents tested positive for the virus during the tenure of this study. We concluded that these species of rodents did not harbor the virus and are most unlikely to contribute to the transmission of the MERS‐CoV. However, further large‐scale studies are required to confirm the potential roles of rodents in the context of the MERS‐CoV transmission cycle, if any.

Dromedary camels are playing essential roles in the evolution and transmission of MERS-CoV. MERS-CoV shedding in some dromedary camel secretions, particularly nasal swabs, were studied in more detail. However, the roles of viral shedding in saliva and ocular secretions are still required further detailed studies. We performed a longitudinal study on a farm of dromedary camel herd from 10th March until 7th April, 2019, in eastern Saudi Arabia. This is a closed management herd including a large number of colour-based breed animals and include animals of both sexes. We collected saliva and ocular swabs from 18% of the target animal population. Detection of the MERS-CoV-RNAs in these samples was conducted by the real-time PCR technique. We detected the viral RNAs in the saliva of and conjunctival swabs of some of the tested animals at 33%, 77% and 88% during the three-time points, respectively. Moreover, we also detected the viral RNAs in the conjunctival swabs at 11%, 22% and 33% at similar time intervals. Our results are suggesting the possibility of MERS-CoV shedding in the saliva and the ocular discharges of the infected dromedary camels. This explains, at least in part, the mechanism of transmission of MERS-CoV from animals to humans. More studies are needed for a better understanding of the transmission of MERS-CoV from animals to humans; thus, the risk of virus spread can be mitigated.

MERS-CoV usually causes respiratory and renal failure in some patients, which may be the underlying cause of death. Dromedary camels are the only known reservoir of the virus until now. They shed the virus in their body secretions thus potentiate a risk for human infection. MERS-CoV tropism and replication is mainly affected by the presence of certain receptor ligands on the target tissues. The dipeptidyl peptidase-4 (DPP-4) is believed to act as receptors for MERS-CoV. The main objective of this study was to determine the expression levels of the DPP-4 in various organs of some naturally infected camels. We conducted a surveillance study to identify some positive MERS-CoV infected camels. Three positive animals identified by the Real time PCR. Our results are clearly showing the high level of expression of the DPP-4 in various organs of these animals' particularly nasal turbinate, trachea, and lungs. The expression level may explain at least in part the pathogenesis of MERS-CoV in these organs. These findings confirm the pivotal roles of the DPP4 in the context of the MER-CoV infection in dromedary camels. Further studies are needed for a better understanding of the molecular pathogenesis of MER-CoV infection.

Background The natural MERS-CoV infection in dromedary camels is understudied. Recent experimental studies showed no obvious clinical signs in the infected dromedary camels. Aim To study the pathological changes associated with natural MERS-CoV infection in dromedary camels. Methods Tissues from three MERS-CoV positive animals as well as two negative animals were collected and examined for the presence of pathological changes. The screening of the animals was carried out first by the rapid agglutination test and then confirmed by the RT-PCR. The selected animals ranged from six to twelve months in age. The sensitivity of the latter technique was much higher in the detection of MERS-CoV than the Rapid test (14 out of 75 animals positive or 18% versus 31 out of 75 positive or 41%). Results MERS-CoV induced marked desquamation of the respiratory epithelium accompanied by lamina propria and submucosal mononuclear cells infiltration, epithelial hyperplasia in the respiratory tract, and interstitial pneumonia. Ciliary cell loss was seen in the trachea and turbinate. In addition, degeneration of glomerular capillaries with the complete destruction of glomerular tufts that were replaced with fibrinous exudate in renal corpuscles in the renal cortex were noticed. Expression of the MERS-CoV-S1 and MERS-CoV-N proteins was revealed in respiratory tract, and kidneys. Conclusion To our knowledge, this is the first study describing the pathological changes of MERS-CoV infection in dromedary camels under natural conditions. In contrast to experimental infection in case of spontaneous infection interstitial pneumonea is evident at least in some affected animals.

In the current study, we are investigating the viral causes of some respiratory clinical signs in some animals belongs to the family Equidae in eastern Saudi Arabia (ESA) during winter- 2019. We observed the progression of severe respiratory clinical signs among some horses, donkeys, and ponies in the ESA. Animals showed rapid respiration, fever, nasal discharges (started as serous then changed into mucopurulent with the progression of the infection per some animals). We conducted a longitudinal study to monitor the progression of this outbreak. We conducted molecular surveillance for the influenza virus Type-A using real-time PCR and regular RT-PCR. We also conducted a serosurveillance of the virus in sera of the tested animals using the commercially available enzyme-linked immunosorbent assay (ELISA).The molecular detection of the Influenza virus type-A virus from nasal swabs of the affected animals using the real-time PCR results clearly showing that 35.1% of the tested horses, donkeys, and ponies were positives. Further confirmation was achieved by reporting the seroconversion of some of the affected animals. Several attempts were conducted to isolate the circulating influenza strains using the embryonated chicken eggs were unsuccessful. This was based on the absence of any amplicons in the harvested embryonated egg fluids using some oligonucleotides for the common influenza virus genes (HA, NA, M, and N). Meanwhile, ELISA results revealed the detection of the antibodies in sera of horses and donkeys 72.9%. Seroconversion was reported in many animals several weeks after the onset of the outbreak. Taken together all these pieces of evidence, we confirm an influenza virus type-A outbreak among the tested animals during winter 2019.

We detected Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in 305/1,131 (27%) camels tested at an abattoir in Al Hasa, Eastern Province, Saudi Arabia, during January 2016-March 2018. We characterized 48 full-length MERS-CoV genomes and noted the viruses clustered in MERS-CoV lineage 5 clade B.

Dromedary camels remain the currently identified reservoir for the Middle East respiratory syndrome coronavirus (MERS-CoV). The virus is released in the secretions of the infected camels, especially the nasal tract. The virus shedding curve through the nasal secretions was studied. Although human transmission of the virus through the respiratory tract of close contact people with dromedary reported previously, the exact mechanism of transmission is still largely unknown. The main goal of this study was to check the possibility of MERS-CoV shedding in the exhaled air of the infected camels. To achieve this goal, we conducted a follow-up study in one of the dromedary camel herds, December 2018–April 2019. We tested nasal swabs, breath samples from animals within this herd by the real-time PCR. Our results showed that some of the tested nasal swabs and breath were positive from 24 March 2019 until 7 April 2019. The phylogenetic analysis of the obtained S and N gene sequences revealed the detected viruses are clustering together with some human and camel samples from the eastern region, especially from Al-Hufuf city, as well as some samples from Qatar and Jordon. These results are clearly showing the possibility of shedding of the virus in the breath of the infected camels. This could explain, at least in part, the mechanism of transmission of MERS-CoV from animals to humans. This study is confirming the shedding of MERS-CoV in the exhaled air of the infected camels. Further studies are needed for a better understanding of the MERS-CoV.

West Nile virus (WNV) is an important emerging zoonotic arbovirus giving rise to clinical syndromes of varying severity in humans and horses. Culex mosquitoes are the main vector. Although WNV has been reported in many countries in the Middle East and Asia, little is known about its prevalence in equine populations in the Arabian Peninsula. We have carried out a serological study on 200 horses to assess WNV infection in the Eastern and Central regions of Saudi Arabia in 2013-2015. Sera were tested for the presence of WNV antibodies in parallel using a commercial enzyme-linked immunosorbent assay (ELISA) kit and microneutralization (MN) tests. In comparison with the MN assay used as "gold standard," we find the ELISA had a sensitivity of 94.7% and specificity of 80.1%. The prevalence of WNV neutralizing antibody ranged from 5 (17.3%) of 29 sera collected in Riyadh up to 15 (55.6%) of 27 sera collected from Al-Qateef. These findings highlight the need to be aware of the possibility of WNV disease in humans and horses presenting with central nervous system disease in the Kingdom of Saudi Arabia.

BACKGROUND Equine influenza virus (EIV) is one of the main causes of viral respiratory affections in horses. Little is known about the prevalence of EIV in Saudi Arabia especially the H3N8 serotype. OBJECTIVES To assess prevalence of equine influenza in horse populations in Eastern and Central Saudi Arabia. STUDY DESIGN Cross-sectional study. METHODS We collected 145 sera, 323 nasal and 323 rectal swabs from horses from six major cities in Eastern and Central regions. None of the horses were vaccinated against EIV. Sera were tested in ELISA assays for influenza A type-specific antibodies and by haemagglutination inhibition (HI) tests using equine H3N8. The swabs were tested by RT-qPCR assay targeting a conserved region of the influenza A matrix gene that detects influenza A viruses of all subtypes. RESULTS None of the swabs had detectable influenza A virus RNA. Of the 145 serasamples tested by ELISA, 81 (55.9%) were positive and 98 (67.6%) of 145 sera tested by HI tests were positive for equine H3. MAIN LIMITATIONS Our failure to detect and sequence any EIV prevents identification of the lineage of virus that circulates in the Kingdom of Saudi Arabia. CONCLUSIONS These results confirm that EIV H3N8 is circulating in Saudi Arabia and should be considered as a possible cause when investigating horses with respiratory disease in Saudi Arabia.

African horse sickness virus (AHSV) is one of the most devastating viral diseases of the family Equidae. Infection with AHSV threatens not only the Saudi equine industry but also the equine industry worldwide. This is due to the high morbidity and mortality rates among the infected population of up to 100%. The World Organisation for Animal Health (OIE) lists AHSV among its notifiable diseases; this requires Member Countries to monitor the situation with regard to AHSV very carefully in order to avoid the spread of the virus. The OIE also suggests the systematic monitoring of AHSV in the equine population at regular intervals. The main aim of the current study is to perform molecular and serological surveillance on different horse populations in eastern and central regions of Saudi Arabia. To achieve this aim, the authors collected 361 serum samples, 103 whole blood samples and 323 swabs from Al-Hasa, Dammam, Al-Jubail, Al-Qateef, Riyadh and Al-Qassim. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect AHSV antibodies and commercial real-time reverse transcriptase-polymerase chain reaction (RT-PCR) kits were used to detect AHSV nucleic acids in blood and swabs. The results of this study demonstrate the absence of anti-AHSV antibodies in the sera of tested animals. Furthermore, no viral nucleic acids were detected in the collected blood and swab samples, as evaluated by real-time AHSV-RT-PCR. Moreover, all tested samples collected during 2014-2016 were negative for AHSV. This confirms that the horse populations studied in the eastern and central regions of Saudi Arabia during 2014-2016 were AHSV free.

The Middle East respiratory syndrome coronavirus (MERS-CoV) presents an ideal example for developing One Health concepts. Dromedary camels are the principal reservoir for the virus. Infected camels shed the virus in body secretions, particularly nasal discharges. MERS-CoV has the potential to remain active in the environment for some time under optimum conditions of temperature and humidity. This shedding sustains the virus in endemic communities and thus contact with camels is considered a major risk factor for human infection. Reducing virus shedding from camels will have a great positive impact on reducing the human risk of infection. Our main objective is to highlight the potential aspects of reducing virus shedding from camels to the environment, thereby reducing the possibility of human infection. We will focus on the potential roles of camel markets, camel shows, importation, transportation and grazing in the amplification and shedding of the virus, providing some novel concepts for the control approaches for the MERS-CoV. Keywords: MERS-CoV, One Health, Dromedary camel, Human, Transmission, Shedding

The currently known animal reservoir for MERS-CoV is the dromedary camel. The clinical pattern of the MERS-CoV field infection in dromedary camels is not yet fully studied well. Some pathological changes and the detection of the MERS-CoV antigens by immunohistochemistry have been recently reported. However, the nature of these changes by the scanning electron microscope (SEM) was not revealed. The objective of this study was to document some changes in the respiratory organs induced by the natural MERS-CoV infection using the SEM. We previously identified three positive animals naturally infected with MERS-CoV and two other negative animals. Previous pathological studies on the positive animals showed varying degrees of alterations. MERS-CoV-S and MERS-CoV-Nc proteins were detected in the organs of positive animals. In the current study, we used the same tissues and sections for the SEM examination. We established a histopathology lesion scoring system by the SEM for the nasal turbinate and trachea. Our results showed various degrees of involvement per animal. The main observed characteristic findings are massive ciliary loss, ciliary disorientation, and goblet cell hyperplasia, especially in the respiratory organs, particularly the nasal turbinate and trachea in some animals. The lungs of some affected animals showed signs of marked interstitial pneumonia with damage to the alveolar walls. The partial MERS-CoV-S gene sequencing from the nasal swabs of some dromedary camels admitted to this slaughterhouse confirms the circulating strains belong to clade-B of MERS-CoV. These results confirm the respiratory tropism of the virus and the detection of the virus in the nasal cavity. Further studies are needed to explore the pathological alterations induced by MERS-CoV infection in various body organs of the MERS-CoV naturally infected dromedary camels.

Background Avian influenza viruses are still causing major concern not only to the poultry industry but also to human health across the globe. The live poultry markets and the small-scale local breeding of various species of birds in backyards are still playing important roles in the sustainability of most virulent influenza viruses, especially H5N8. Methods The authors investigated an outbreak of highly pathogenic avian influenza H5N8 in backyard flocks in Al Ahsa, Eastern Saudi Arabia that occurred in 2017–2018. Results A range of poultry including chickens, ostriches, ducks, pigeons and turkeys were clinically affected. Phylogenetic analysis suggested that this was a common source outbreak caused by a virus closely related to H5N8 viruses causing outbreaks elsewhere in Saudi Arabia in early 2018. Conclusions Small backyard flocks are still contributing to the epidemiology and transmission of H5N8.

Summary Background Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection causing severe viral pneumonia, with index cases having resided in or recently travelled to the Arabian peninsula, and is a global concern for public health. Limited human-to-human transmission, leading to some case clusters, has been reported. MERS-CoV has been reported in dromedary camels but phenotypic characterisation of such viruses is limited. We aimed to compare MERS-CoV isolates from dromedaries in Saudi Arabia and Egypt with a prototype human MERS-CoV to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. Methods We characterised MERS-CoV viruses from dromedaries in Saudi Arabia and Egypt and compared them with a human MERS-CoV reference strain. We assessed viral replication kinetics and competence in Vero-E6 cells (rhesus monkey), tissue tropism in cultures of ex-vivo human bronchial and lung tissues, and cytokine and chemokine induction, gene expression, and quantification of viral RNA in Calu-3 cells (human respiratory tract). We used mock-infected tissue as negative controls for ex-vivo experiments and influenza A H5N1 as a positive control for cytokine and chemokine induction experiments in Calu-3 cells. Findings We isolated three dromedary strains, two from Saudi Arabia (Dromedary/Al-Hasa-KFU-HKU13/2013 [AH13] and Dromedary/Al-Hasa-KFU-HKU19D/2013 [AH19D]), and one from Egypt (Dromedary/Egypt-NRCE-HKU270/2013 [NRCE-HKU270]). The human and dromedary MERS-CoV strains had similar viral replication competence in Vero-E6 cells and respiratory tropism in ex-vivo cultures of the human respiratory tract, and had similar ability to evade interferon responses in the human-respiratory-tract-derived cell line Calu-3. Interpretation The similarity of virus tropism and replication competence of human and dromedary MERS-CoV from the Arabian peninsula, and genetically diverse dromedary viruses from Egypt, in ex-vivo cultures of the human respiratory tract suggests that dromedary viruses from Saudi Arabia and Egypt are probably infectious to human beings. Exposure to zoonotic MERS-CoV is probably occurring in a wider geographical region beyond the Arabian peninsula. Funding King Faisal University, Egyptian National Research Centre, Hong Kong Food and Health Bureau, National Institute of Allergy and Infectious Diseases, and European Community Seventh Framework Program.

A prospective study of a dromedary camel herd during the 2013-14 calving season showed Middle East respiratory syndrome coronavirus infection of calves and adults. Virus was isolated from the nose and feces but more frequently from the nose. Preexisting neutralizing antibody did not appear to protect against infection.

The pseudoparticle virus neutralisation test (ppNT) and a conventional microneutralisation (MN) assay are specific for detecting antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) when used in seroepidemiological studies in animals. Genetically diverse MERS-CoV appear antigenically similar in MN tests. We confirm that MERS-CoV was circulating in dromedaries in Saudi Arabia in 1993. Preliminary data suggest that feral Australian dromedaries may be free of MERS-CoV but larger confirmatory studies are needed.