Phylogenetic characterization of circulating Dengue and Alkhumra Hemorrhagic Fever viruses in western Saudi Arabia and lack of evidence of Zika virus in the region: A retrospective study, 2010-2015.

Flaviviruses represent a global public health concern. They consist of ∼70 viruses with almost half of them causing human diseases with unspecified febrile illnesses. Cities in western Saudi Arabia are endemic for viruses (DENV) with sporadic infections due to Alkhumra hemorrhagic fever virus (AHFV). They also represent a major destination for travelers coming for annual religious pilgrimages (Hajj and Umrah) from all over the world. However, whether other flaviviruses are circulating is not known because of the limited number of surveillance studies. Here, we retrospectively screened 690 samples for flaviviruses in samples from patients with unexplained febrile illnesses between 2010 and 2015 in western Saudi Arabia using a pan-flaviviruses RT-PCR assay. Despite Zika virus RNA was not detected, this study confirms circulation and/or sporadic spread of DENV-2, DENV-3, and AHFV, higher prevalence of DENV-2, and a role for visitors from DENV endemic countries in DENV importation into the Kingdom. Further analysis also showed very low genetic diversity of AHFV confirming its slow microevolution. Accordingly, continuous and prospective surveillance for flaviviruses using such assay are warranted in Saudi Arabia which receives millions of Muslims annually to implement effective control measures in light of the global widespread and outbreaks of several flaviviruses.

Comparison of RT-PCR assay and virus isolation in cell culture for the detection of Alkhumra hemorrhagic fever virus.

Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was isolated originally from Saudi Arabia in 1994-1995. The main tests used for the detection of AHFV are the real time (rt) RT-PCR and virus isolation in cell culture. In the present study the detection of AHFV by rtRT-PCR was compared with virus isolation in BHK-21, HEp-2, and LLC-MK2 cell lines. AHFV suspensions grown in BHK-21, HEp-2, and LLC-MK2 cell lines were serially diluted 10-fold from 10(-1) to 10(-11) . Samples from each dilution were used to inoculate four cell culture tubes and were also examined by the rtRT-PCR for AHFV RNA. Fifteen non-inoculated cell culture samples (five from each cell line) were included blindly in both tests. Thus, a total of 132 AHFV-positive and 15 negative control samples were tested. The rtRT-PCR could detect the viral RNA in all diluted specimens up to and including the 10(-10) dilution (40 specimens for each cell line), whereas, cell cultures were positive in 70% of specimens for BHK-21, 65% for LLC-MK2, and 45% for HEp-2 at this dilution. None of the three cell cultures nor the rtRT-PCR was positive at 10(-11) dilution. The specificity and positive predictive values of virus isolation compared to rtRT-PCR were each 100%, whereas the negative predictive values were 29.4% for BHK-21, 26.3% for LLC-MK2, and 18.5% for HEp-2. In conclusion, the rtRT-PCR is more sensitive than virus isolation for detecting AHFV.

The Biological and Ecological Features of Northbound Migratory Birds, Ticks, and Tick-Borne Microorganisms in the African-Western Palearctic.

Identifying the species that act as hosts, vectors, and vehicles of vector-borne pathogens is vital for revealing the transmission cycles, dispersal mechanisms, and establishment of vector-borne pathogens in nature. Ticks are common vectors for pathogens causing human and animal diseases, and they transmit a greater variety of pathogenic agents than any other arthropod vector group. Ticks depend on the movements by their vertebrate hosts for their dispersal, and tick species with long feeding periods are more likely to be transported over long distances. Wild birds are commonly parasitized by ticks, and their migration patterns enable the long-distance range expansion of ticks. The African-Palearctic migration system is one of the world's largest migrations systems. African-Western Palearctic birds create natural links between the African, European, and Asian continents when they migrate biannually between breeding grounds in the Palearctic and wintering grounds in Africa and thereby connect different biomes. Climate is an important geographical determinant of ticks, and with global warming, the distribution range and abundance of ticks in the Western Palearctic may increase. The introduction of exotic ticks and their microorganisms into the Western Palearctic via avian vehicles might therefore pose a greater risk for the public and animal health in the future.

Human Alkhumra hemorrhagic Fever: Emergence, history and epidemiological and clinical profiles.

Alkhumra hemorrhagic fever (AHF) is a severe, often fatal hemorrhagic disease in humans. It is caused by Alkhumra hemorrhagic fever virus (AHFV), a newly described flavivirus first isolated in 1995 in Alkhumra district, south of Jeddah City, Saudi Arabia. It is transmitted from infected livestock animals to humans by direct contact with infected animals or by tick bites. In the recent past, the incidence of AHF has increased, with a total of 604 confirmed cases have been reported in Saudi Arabia between 1995 and 2020. Yet, no specific treatment or control strategies have been developed and implemented against this infection. Hence, the likelihood of increased prevalence or the occurrence of outbreaks is high, particularly in the absence of appropriate prevention and control strategies. This narrative review presents an overview of the current knowledge and future concerns about AHF globally.

Ultrastructural Features of Alkhumra Hemorrhagic Fever Virus Infection of Cells Under In Vivo and In Vitro Conditions.

Alkhumra hemorrhagic fever virus (AHFV) is an emerging novel flavivirus that was discovered in Saudi Arabia in 1995. The virus has since caused several outbreaks in the country that resulted in case fatality rates ranging from 1% to 25%. Meager information has been published on the ultrastructural features of the virus on cells under in vitro or in vivo conditions. The present electron microscopic study examined and compared the intracellular growth of the AHFV on the LLC-MK2 cells and brain cells of new born Wistar rats, inoculated intracerebrally. The cytopathological changes in both cell systems were noted, and localization of the virus particles in different cellular components was observed. Both apoptotic and lytic cell interactions were seen in the electron micrographs of both the LLC-MK2 and the rat brain cells. The results were discussed in relation to similar situations reported for other virus members of the genus Flavivirus.

Comparative genome analysis of Alkhumra hemorrhagic fever virus with Kyasanur forest disease and tick-borne encephalitis viruses by the in silico approach.

Alkhumra hemorrhagic fever virus (AHFV), a relatively new member of the Flaviviruses, was discovered in Saudi Arabia 23 years ago. AHFV is classified in the tick-borne encephalitis virus serocomplex, along with the Kyasanur forest disease virus (KFDV) and tick-borne encephalitis virus (TBEV). Currently, very little is known about the pathologies of AHFV. In this study, using the available genome information of AHFV, KFDV and TBEV, we have predicted and compared the following aspects of these viruses: evolution, nucleotide and protein compositions, recombination, codon frequency, substitution rate, N- and O-glycosylation sites, signal peptide and cleavage site, transmembrane region, secondary structure of 5' and 3' UTRs and RNA-RNA interactions. Additionally, we have modeled the 3D protease and RNA-dependent RNA polymerase structures for AHFV, KFDV and TBEV. Recombination analysis showed no evidence of recombination in the AHFV genome with that of either KFDV or TBEV, although single break point analysis showed that nucleotide position 7399 (in the NS4B) is a breakpoint location. AHFV, KFDV and TBEV are very similar in terms of codon frequency, the number of transmembrane regions, properties of the polyprotein, RNA-RNA interaction sequences, NS3 protease and NS5 polymerase structures and 5' UTR structure. Using genome sequences, we showed the similarities between these closely- related viruses on several different areas.

Alkhurma hemorrhagic fever virus.

Alkhurma hemorrhagic fever virus (AHFV) was first isolated in Jeddah, Saudi Arabia, in the 1990s from the blood of a butcher. Subsequently, the virus was recognized in many patients in Saudi Arabia and rarely from Egypt and Djibouti. In this review, we summarize the current literature on AHFV globally with special focus on Saudi Arabia.

Electron Microscopy of Alkhumra Hemorrhagic Fever Virus.

Alkhumra hemorrhagic fever virus (AHFV) is a newly described zoonotic flavivirus that was first isolated during 1994-1995 from the Alkhumra district south of Jeddah, Saudi Arabia. Subsequently, the virus was also isolated from Makkah city (2001-2003) and Najran (2008-2009), Saudi Arabia. The virus causes acute febrile illness with hepatitis, hemorrhagic manifestations, and encephalitis. A case fatality rate of 25% was reported among hospitalized patients. Although several biological and molecular characteristics of the virus have been published, no data are available on electron microscopic features of the virus. In this article, we describe the morphological features and metrics of the AHFV particles under electron microscopy, and localization of the virus particles in brain cells of newborn Wistar rats and in Rhesus monkey (Macaca mulatta) kidney epithelial cells (LLC-MK2). Virus particles in both the LLC-MK2 cells and the rat brain cells showed dark hexagonal core (capsid) and a translucent envelope. The mean diameter of the enveloped virus particle was 40.59 ± 1.29 nm in the rat brain cells (n = 154) and 40.97 ± 1.40 nm in the LLC-MK2 cells (n = 105; p > 0.05). The virus particles, both in vitro and in vivo, were enclosed into cytoplasmic vesicles. In conclusion, the shape, size, and diameter of the AHFV particle lie within the framework of the genus Flavivirus, family Flaviviridae.

Growth Characteristics of Alkhumra Hemorrhagic Fever Virus in Mammalian Cell Lines.

BACKGROUND: Alkhumra hemorrhagic fever virus (AHFV) is a flavivirus that was discovered in 1995 in Saudi Arabia. Clinical manifestations of AHFV infection include hemorrhagic fever, hepatitis, and encephalitis with a reported mortality rate as high as 25%. There are no published data on the growth characteristics of AHFV in mammalian cell lines. The objective of this study was to examine the ability of AHFV to grow and propagate in four of the commonly used mammalian cell culture lines and to determine the virus growth curve characteristics in each. MATERIALS AND METHODS: Human epidermoid carcinoma (HEp-2), LLC-MK2, Madin-Darby canine kidney (MDCK), and Vero cell lines were inoculated with AHFV. The virus production by each cell line was determined by growth curve studies. Mean titers were calculated and expressed as median tissue culture infective dose per mL (TCID50/mL). RESULTS: AHFV grew and propagated to variable titers in the employed cell lines. The highest mean titers were observed in the LLC-MK2, followed by the MDCK, Vero, and HEP-2, in descending order. CONCLUSIONS: The growth curve studies showed that AHFV can propagate in the four types of cell lines to variable titers. LLC-MK2 cells are superior to MDCK, Vero, and HEP-2 for propagation of AHFV.

Propagation and titration of Alkhumra hemorrhagic fever virus in the brains of newborn Wistar rats.

Alkhumra hemorrhagic fever virus (AHFV) is a novel flavivirus identified first in Saudi Arabia. In this study, successful propagation of AHFV in the brains of newborn Wistar rats is described and the median rat lethal dose (RLD50) is determined. AHFV-RNA-positive human sera diluted 1:10 were injected intracerebrally into 16, ≤24h old rats. Post-inoculation, the rats were observed daily for 30 days. Brains of moribund rats were tested for AHFV-RNA using RT-PCR and cultured in LLC-MK2 cells. The titer of the isolated virus was determined and expressed in median tissue culture infectious dose (TCID50). To determine the RLD50, AHFV brain suspension was 10-fold diluted serially and each dilution was inoculated in the cerebral hemispheres of 10 rats for a total of 90 rats. Three days post-inoculation, the rats developed tremor, irritability, convulsion, opisthotonus, and spastic paresis starting in the hind limbs and ascending to involve the whole body. All infected rats died within 3-7 days with histopathologically confirmed meningoencephalitis. AHFV-RNA was detected in the brains of all infected rats and the virus titer was 10(9.4) RLD50/ml. The virus titer in LLC-MK2 was 10(8.2) TCID50/ml. In conclusion, AHFV was propagated successfully to high titers in the brains of newborn Wistar rats.