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.

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.

Complete genome sequencing and genetic characterization of Alkhumra hemorrhagic fever virus isolated from Najran, Saudi Arabia.

BACKGROUND: Alkhumra hemorrhagic fever virus (AHFV) is a newly described flavivirus first isolated in 1994-1995 from the Alkhumra district south of Jeddah, Saudi Arabia. Subsequently, the virus was also isolated from Makkah (2001-2003) and Najran (2008-2009), Saudi Arabia. METHODS: The full-length genome of an AHFV strain isolated from patients in Najran (referred to as AHFV/997/NJ/09/SA) was PCR amplified and sequenced, and compared with the sequences of 18 other AHFV strains previously isolated from Jeddah and Makkah, dengue virus (DENV), Kyasanur forest disease virus (KFDV), Langat virus, Omsk hemorrhagic fever virus (OHFV), and tick-borne encephalitis virus (TBEV). RESULTS: The RNA of the AHFV/997/NJ/09/SA strain was found to have 10,546 nucleotides encoding for a single 3,416-amino acid polyprotein, whereas the previously reported AHFV strains were composed of 10,685-10,749 nucleotides. The AHFV/997/NJ/09/SA strain showed about 99% homology with the previously reported AHFV strains. The KFDV, Langat virus, TBEV, and OHFV isolates formed a separate cluster with a variable homology. The most important variations were observed in the core protein and NS4a gene sequences of two AHFV isolates. CONCLUSION: The variation in the number of nucleotides and phylogenetic analysis with the other AHFV isolates could have resulted from recombination of circulating virus strains.

Thermal inactivation of Alkhumra hemorrhagic fever virus.

The physico-chemical and biological characteristics of Alkhumra hemorrhagic fever virus (AHFV) are not yet known. The present study describes the thermal stability of this virus at different temperatures for different periods. The kinetics of thermal inactivation were studied, linear regressions were plotted, the Arrhenius equation was applied, and the activation energy was calculated accordingly. Titers of the residual virus were determined in median tissue culture infective dose (TCID50), and the rate of destruction of infectivity at various temperatures was determined. Infectivity of AHFV was completely lost upon heating for 3 minutes at 60 °C and for 30 min at 56 °C. However, the virus could maintain 33.2 % of its titer after heating for 60 min at 45 °C and 32 % of its titer after heating for 60 min at 50 °C. In conclusion, AHFV is thermo-labile, and its inactivation follows first-order kinetics.

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.

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.

Susceptibility of tick cell lines to infection with Alkhumra haemorrhagic fever virus.

BACKGROUND: Although Alkhumra haemorrhagic fever virus (AHFV) has been isolated from ticks, epidemiological data suggest that it is transmitted from livestock to humans by direct contact with animals or by mosquito bites, but not by ticks. This study was carried out to assess the ability of the virus to replicate in tick cells in vitro. METHODS: AHFV was inoculated into cell lines derived from the hard ticks Hyalomma anatolicum (HAE/CTVM9) and Rhipicephalus appendiculatus (RAE/CTVM1) and the soft tick Ornithodoros moubata (OME/CTVM24). Inoculated cells were directly examined every week for 4 weeks by real-time reverse transcription PCR and by IFAT using polyclonal antibodies. RESULTS: AHFV RNA was detected in all three inoculated tick cell lines throughout the 4-week observation period at levels up to almost twice that of the inoculum, but none of them exhibited a cytopathic effect. AHFV antigen could be detected in all three cell lines by IFAT. Titration of tick cell culture suspension in LLC-MK2 cells yielded AHFV titres of 10(6.6) 50% tissue culture infective dose (TCID50)/ml for OME/CTVM24 and 10(5.5) TCID50/ml for RAE/CTVM1 cells after 4 weeks of culturing; no viable virus was detected in HAE/CTVM9 cells. CONCLUSION: This is the first description of propagation of AHFV in tick cells.

Outbreak of viral hemorrhagic fever caused by dengue virus type 3 in Al-Mukalla, Yemen.

BACKGROUND: Investigations were conducted by the authors to explore an outbreak of viral hemorrhagic fever (VHF) reported in 2010 from Al-Mukalla city, the capital of Hadramout in Yemen. METHODS: From 15-17 June 2010, the outbreak investigation period, specimens were obtained within 7 days after onset of illness of 18 acutely ill patients hospitalized with VHF and 15 household asymptomatic contacts of 6 acute cases. Additionally, 189 stored sera taken from acutely ill patients with suspected VHF hospitalized in the preceding 12 months were obtained from the Ministry of Health of Yemen. Thus, a total of 222 human specimens were collected; 207 specimens from acute cases and 15 specimens from contacts. All samples were tested with RT-PCR for dengue (DENV), Alkhumra (ALKV), Rift Valley Fever (RVFV), Yellow Fever (YFV), and Chikungunya (CHIKV) viruses. Samples were also tested for DENV IgM, IgG, and NS1-antigen. Medical records of patients were reviewed and demographic, clinical, and laboratory data was collected. RESULTS: Of 207 patients tested, 181 (87.4%) patients were confirmed to have acute dengue with positive dengue NS1-antigen (97 patients, 46.9%) and/or IgM (163 patients, 78.7%). Of the 181 patients with confirmed dengue, 100 (55.2%) patients were IgG-positive. DENV RNA was detected in 2 (1%) patients with acute symptoms; both samples were molecularly typed as DENV type 3. No other VHF viruses were detected. For the 15 contacts tested, RT-PCR tests for the five viruses were negative, one contact was dengue IgM positive, and another one was dengue IgG positive. Of the 181 confirmed dengue patients, 120 (66.3%) patients were males and the median age was 24 years. The most common manifestations included fever (100%), headache (94.5%), backache (93.4%), malaise (88.4%), arthralgia (85.1%), myalgia (82.3%), bone pain (77.9%), and leukopenia (76.2%). Two (1.1%) patients died. CONCLUSIONS: DENV-3 was confirmed to be the cause of an outbreak of VHF in Al-Mukalla. It is important to use both IgM and NS1-antigen tests to confirm acute dengue particularly under the adverse field conditions, where proper storage and transportation of specimens are missing, which substantially reduce the sensitivity of the RT-PCR for detecting DENV RNA.

Trypsin-dependent hemagglutination of erythrocytes of a variety of mammalian and avian species by Alkhumra hemorrhagic fever virus.

Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was discovered in 1994-1995 in Saudi Arabia. Clinical manifestations of AHFV infection include hemorrhagic fever, hepatitis, and encephalitis, with a reported mortality rate as high as 25 %. Biological characteristics of this virus have not been well defined. Agglutination of erythrocytes (hemagglutination) is a laboratory tool for studying the attachment of viruses to cellular receptors. The envelope protein contains sites for attachment to host receptors to initiate the process of infection and is thus an essential component of the virion. In the present study, we examined the ability of AHFV to agglutinate erythrocytes of 13 mammalian and avian species (human group O+, camel, cow, sheep, goat, rabbit, guinea pig, mouse, rat, chicken, duck, goose and turkey) with and without trypsin-treatment. Without trypsin treatment, AHFV failed to agglutinate erythrocytes of all examined species. Following trypsin treatment, AHFV agglutinated erythrocytes of five species, namely, goose, human group O+, rat, guinea pig, and mouse, in descending order of sensitivity. This trypsin-dependent hemagglutination test has potential for use in serological and functional studies of AHFV.

Superiority of the buffy coat over serum or plasma for the detection of Alkhumra virus RNA using real time RT-PCR.

RT-PCR to detect Alkhumra virus (ALKV) RNA in plasma or serum has been the standard practice to confirm this infection in the first seven days of illness. In this study, RT-PCR detection of viral RNA from the plasma, serum, and buffy coat (BC) was compared to virus isolation. Plasma, serum, and BC were obtained from seven patients with clinically suspected ALKV infection in Najran, Saudi Arabia. Baby hamster kidney (BHK-21) and rhesus monkey kidney (LLC-MK2) cell culture monolayers were used for virus isolation. Real-time RT-PCR was used to confirm ALKV infection and to detect viral RNA directly from plasma, serum, and BC. ALKV was isolated from five of the seven patients. The virus was isolated from all three specimen types (plasma, serum, and BC) of the five confirmed patients. ALKV RNA was detected directly by RT-PCR in BC in all five (100%) culture-positive patients and in plasma or serum in only four (80%) of the five patients. Three of the five patients for whom ALKV RNA was detected in BC also had detectable viral RNA in plasma and serum. In the remaining two patients with detectable ALKV RNA in the BC, the plasma was positive but the serum was negative in one patient, whereas the serum was positive and the plasma was negative in the other patient. The use of real-time RT-PCR to detect ALKV RNA in the BC was superior to using plasma and serum and equivalent to virus isolation.

Successful propagation of Alkhumra (misnamed as Alkhurma) virus in C6/36 mosquito cells.

Epidemiological data suggest that Alkhumra (misnamed as Alkhurma) virus (ALKV) is transmitted from livestock animals to humans by direct contact with animals or by the mosquito bites, but not by ticks. To assess the ability of the virus to replicate in mosquito cells, serum and plasma of seven acutely febrile patients with clinically suspected ALKV infection reported in Najran, Saudi Arabia in 2009 were inoculated onto Aedes albopictus mosquito cells (C6/36) and directly examined with ALKV-RNA-specific real time RT-PCR as well as indirect immunfluorescence assay (IFA) using ALKV-specific polyclonal antibodies. The isolated virus was titrated in the mammalian rhesus monkey kidney cells (LLC-MK2). Five of the seven specimens were RT-PCR- and culture-positive demonstrating cytopathic effects in the form of cell rounding and aggregation appearing on day 3 post inoculation with syncytia eventually appearing on day 8 post inoculation. Identification of ALKV-RNA in the cell culture was confirmed with RT-PCR and IFA. The virus titre was 3.2×10(6) tissue culture infective dose 50 (TCID(50)) per mL. Three more viral passages were successfully made in the C6/36 cells. This is the first description of propagation of ALKV in mosquito cells.

Alkhumra (Alkhurma) virus outbreak in Najran, Saudi Arabia: epidemiological, clinical, and laboratory characteristics.

OBJECTIVE: After its first appearance in Alkhumra district of Jeddah in 1994-1995, and then in Makkah in 2001-2003, the new hemorrhagic fever virus, known as Alkhumra (misnamed as Alkhurma) virus (ALKV), has subsequently been reported from Najran, in the south border of Saudi Arabia. METHODS: This is a descriptive cohort study summarizing the epidemiological, clinical, and laboratory characteristics of ALKV infected patients diagnosed in Najran from 1 August 2003 through 31 December 2009. RESULTS: A total of 148 suspected cases were reported, of which 78 (52.7%) cases were laboratory confirmed; 2 cases in 2003, 1 case in 2004, 4 cases in 2005, 1 case in 2007, 12 cases in 2008, and 58 cases in 2009. The cases were reported year round but 64.1% (50/78) of them occurred in the summer time. Twenty-five (32.1%) cases occurred as clusters in 5 families. The virus seemed to be transmitted from livestock animals to humans by direct contact with these animals and likely by mosquito bites. Ticks did not seem to be involved in the transmission of infection from animals to humans. Clinical and laboratory features included fever (100%), headache (85.9%), malaise (85.9%), arthralgia (83.3%), anorexia (82.1%), myalgia (82.1%), backache (71.8%), nausea and vomiting (71.8%), chills (60.3%), retro-orbital pain (55.1%), diarrhea (51.3%), abdominal pain (48.7%), hemorrhagic manifestations (25.6%), central nervous system manifestations (23.1%), leucopenia (87.7%), elevated liver enzymes (85.7%), prolonged partial thromboplastin time (52.6%), thrombocytopenia (46.2%), elevated creatine kinase level (45.7%), and elevated lactate dehydrogenase (25.0%). CONCLUSION: ALKV infection has now been recognized outside its original boundaries in Saudi Arabia which may herald its identification in other countries.