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.

Alkhumra hemorrhagic fever virus (AHFV) is a newly described flavivirus associated with hemorrhagic fever that was first identified in Saudi Arabia. It is a member of the tick-borne encephalitis group in the genus Flavivirus of the family Flaviviridae. It was first isolated in 1994-1995 from 6 patients living in the Alkhumra district in Jeddah, the main seaport in western Saudi Arabia [1]. From 2001 to 2003, Madani [2] described a series of 20 confirmed cases in the holy city of Makkah, 75 km from the Alkhumra district in Jeddah, and proposed the name ‘Alkhumra' be given to the virus after the geographic location from which it was originally isolated. The virus has sometimes been mistakenly referred to as ‘Alkhurma virus' [2,3,4,5]. The International Committee on Taxonomy of Viruses (ICTV) has recently corrected this mistake and approved the name ‘Alkhumra' as the correct name of the virus [6]. From 2003 to 2007, 8 confirmed cases of AHFV infections were sporadically reported from Najran in southern Saudi Arabia [3]. Subsequently, an outbreak of AHFV infection occurred in Najran in 2008-2009 with 70 confirmed cases reported [3]. Reports of AHFV came only from Saudi Arabia until 2010 when two travelers returning to Italy from southern Egypt were confirmed to be infected with AHFV [7].

Despite host differences and distinct geographic distribution, AHFV and Kyasanur forest disease virus (KFDV) have high sequence homology. It has been speculated that AHFV emerged from KFDV. Previous phylogenetic tree analysis of AHFV and KFDV relied on partial gene sequences of envelope, NS5, and NS3 genes [8,9]. Complete coding sequence of AHFV (strain 1176) showed that it is composed of 10,248 nucleotides [10]. A better understanding of the AHFV full-length genome and information and analysis of variability at the nucleotide and amino acid level would provide insight into its evolution and possibilities of emergence of new strains. The objective of this study was to determine the complete genome sequence of the AHFV strain isolated from patients in Najran, and its genetic characteristics, variability, possibility of new strain emergence, spread, and relationships with other tick-borne flaviviruses, namely, KFDV, Langat virus, Omsk hemorrhagic fever virus (OHFV), and tick-borne encephalitis virus (TBEV), as well as the mosquito-borne dengue virus (DENV).

Collection of Samples

Blood samples were collected from 7 acutely febrile patients with suspected viral hemorrhagic fever in Najran from March 18 to April 4, 2009 when an AHFV outbreak was initially recognized in this region. The blood samples were collected within 3-7 days after the onset of the illness when the patients were still febrile. Whole blood was separately collected from each of the 7 patients and placed in EDTA (ethylenediaminetetraacetic acid) and plain vacutainers. The specimens collected in EDTA vacutainers were spun in a cooled centrifuge at 252 g for 10 min and the plasma was collected and stored in 0.5-ml aliquots at -86°. The blood samples in the plain vacutainers were left to clot and the serum was separated by low-speed cooled centrifugation and stored at -20°. The specimens were subsequently transported on dry ice in IATA-compliant transport containers from Najran to the Special Infectious Agents Unit, a biosafety level 3 virology laboratory, at King Fahd Medical Research Centre, King Abdulaziz University, Jeddah, Saudi Arabia.

Viral RNA Extraction and RT-PCR

Viral RNA was extracted from the samples using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted viral RNA materials were subjected to RT-PCR using a pair of primers (forward primer, AHFV S1 5′-GTGAGTGGCGCTTTGTTTG TA, and reverse primer, AHFV R 5′-CCCCCTTTCCTTTAAGGACG), and 5′-nuclease detection probe (TBV TM: 6FAM-ACAGCTTAGGAGAACAAGAGCTGGGGAXT-PH) designed using Primer Express software (Applied Biosystems, Weiterstadt, Germany) and synthesized by TIB-MOLBIOL (Berlin, Germany) based on the AHFV sequences described by Charrel et al. [10]. The 5′-nuclease probe was labelled with 6-carboxyfluorescein at the 5′ end and with 6-carboxy-N,N,N′,N′-tetramethylrhodamine at the 3′ end. The 3′ end of each probe was phosphorylated to prevent elongation during PCR as previously described [11,12]. The QuantiFast Probe RT-PCR Kit (Qiagen) was used to amplify the AHFV genome for complete genome sequencing. The master mix consisted of 0.8 μM from each primer, 0.2 μM from the probe, 1 × PCR master mix buffer 1 µl of bovine serum albumin, 0.2 µl of QuantiFast RT Mix, 5 µl of the sample, and the final volume was brought up to 20 µl using RNase-free water. The PCR was performed using a Light Cycler (Roche, UK) with the following program: 10 min at 50° for cDNA synthesis, then 5 min for initial denaturation at 95°, followed by 45 cycles at 95° for 10 s, 57° for 30 s, 72° for 30 s, and a final extension step at 72° for 10 min.

Virus Isolation

The virus was inoculated in an LLC-MK2 monkey cell line as previously described [13]. Seven days after inoculation, 5 of the 7 samples exhibited a complete cytopathic effect. The supernatant medium was collected and clarified by low-speed centrifugation and tested using AHFV RT-PCR assay. Only one AHFV RNA-positive tissue culture sample was selected on the basis of the lowest Ct value in real-time RT-PCR. This isolate was designated as the AHFV/997/NJ/09/SA strain.

Primer Designing

Full-length coding sequences of AHFV strain 1176 were retrieved from the GenBank database. The primers' sequences were designed based on the AHFV strain 1176 polyprotein gene (AF331718). A total of 39 synthetic oligonucleotide primers were used to amplify and complete the whole sequence of the virus. The primer pairs were used to produce overlapping products of approximately 1.5 kb in length (table 1).

Table 1

List of primers used to sequence AHFV, AHFV/997/NJ/09/SA strain

List of primers used to sequence AHFV, AHFV/997/NJ/09/SA strain
List of primers used to sequence AHFV, AHFV/997/NJ/09/SA strain

PCR Amplification and Complete Genome Sequencing

Synthesis of cDNA and PCR of the target sequence was carried out using the Qiagen One-Step RT-PCR Kit. The genome of AHFV was amplified from the 5′ and 3′ ends of the virus by using a set of primers divided into 7 overlapping parts, named from A to G. Each part was amplified using the kit protocol parameters as follows: 5 × Qiagen One-Step RT-PCR Buffer, 400 μM dNTP mix, 2.0 μl Qiagen One-Step RT-PCR Enzyme Mix, 0.6 μM of each primer, 6 μl of the template, and the final volume was brought up to 50 μl with distilled water. Single-step RT-PCR cycling condition to amplify the fragments was 30 min at 50°, then 35 cycles of denaturation at 94° for 1 min, annealing (1 min at 65°), and extension (2 min at 72°, 10 min for the last cycle). The PCR product was purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions.

Cycle sequencing was performed, using 4-6 primers to cover each PCR product, according to the instructions of the manufacturer of the commercial kit (BigDye® Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems) using the Sanger dideoxy sequencing method. The sequencing reaction mixture contained 4 μl of premix, and 2 μl of ABI 5 × sequencing buffer, 3.2 μM of sequencing primer, and 1 μl of PCR product in a final volume of 20 μl. The amplification primers were used for sequencing by an initial denaturation at 96° for 1 min, followed by 25 cycles (10 s each) of denaturation at 96°, annealing (5 s at 50°), and extension (4 min at 60°). The sequencing products were purified using the ethanol/EDTA precipitation method and analyzed with the ABI Prism 3100 (Avant Genetic Analyzer, Applied Biosystems) according to the manufacturer's instructions.

Sequence and Phylogenetic Analysis

The retrieved sequences were aligned and the resulting complete nucleotide sequence was initially searched for similarity using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) [14]. The sequences that showed a higher score and similarity were selected for further analysis. Multiple sequence alignments were performed by ClustalW program (http://www.ebi.ac.uk/clustalw) using nucleotides sequences of AHFV/997/NJ/09/SA and 18 previously isolated AHFV strains, 1 strain of DENV, 3 strains of KFDV, 4 strains of Langat virus, 5 strains of OHFV, and 5 strains of TBEV from GenBank (table 2). A phylogenetic tree was constructed using the MEGA5 program from the aligned nucleotide sequences with neighbor joining and maximum parsimony methods using maximum composite likelihood for the DNA substitution test [15].

Table 2

Characteristics of AHFV strain AHFV/997/NJ/09/SA isolated from humans in Najran, Saudi Arabia, 18 previously isolated AHFV strains, 1 DENV strain of dengue virus, 3 KFDV strains, 4 Langat virus strains, 5 strains OHFV, and 5 TBEV strains used in the phylogenetic analysis tree

Characteristics of AHFV strain AHFV/997/NJ/09/SA isolated from humans in Najran, Saudi Arabia, 18 previously isolated AHFV strains, 1 DENV strain of dengue virus, 3 KFDV strains, 4 Langat virus strains, 5 strains OHFV, and 5 TBEV strains used in the phylogenetic analysis tree
Characteristics of AHFV strain AHFV/997/NJ/09/SA isolated from humans in Najran, Saudi Arabia, 18 previously isolated AHFV strains, 1 DENV strain of dengue virus, 3 KFDV strains, 4 Langat virus strains, 5 strains OHFV, and 5 TBEV strains used in the phylogenetic analysis tree

Complete Genome Sequence and Phylogenetic Analysis

Based on genome sequence analysis for structural and nonstructural genes, the genetic characterization of the AHFV/997/NJ/09/SA strain was compared with the selected AHFV, DENV, KFDV, Langat virus, OHFV, and TBEV isolates. The percent sequence identity matrix for structural and nonstructural genes is summarized in table 3.

Table 3

Percent nucleotide identity matrix of AHFV, strain AHFV/997/NJ/09/SA, with 18 previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains

Percent nucleotide identity matrix of AHFV, strain AHFV/997/NJ/09/SA, with 18 previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains
Percent nucleotide identity matrix of AHFV, strain AHFV/997/NJ/09/SA, with 18 previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains

The complete genome of the AHFV/997/NJ/09/SA strain was found to contain 10,546 nucleotides coding for 3,416 amino acids (table 2). The previously reported AHFV strains were composed of 10,685-10,749 nucleotides (table 2). Complete genome sequence analysis showed genetic variations in the form of deletions and insertions of nucleotides in the 5′ and 3′ ends of the untranslated region (UTR) as well as in other parts of the structural and nonstructural (NS) protein genes. When full genome nucleotide sequences of the AHFV/997/NJ/09/SA strain (10,546 nucleotides) were compared with the AHFV NC004355 strain (10,685 nucleotides), a total of 150 nucleotide deletions were identified at 5′ UTR-1-97, 3′ UTR-10635-10685, 1473-C (in the envelope gene), and 9709-G (in the NS5 gene) positions in the AHFV NC004355 strain. When sequences of the AHFV/997/NJ/09/SA strain were compared with the AHFV JF 416949 strain (10,749 nucleotides), a total of 214 nucleotide deletions were identified at 5′ UTR-1-88, 3′UTR-10626-10749, 1464-C (in the envelope gene), and 9700-G (in the NS5 gene) nucleotide positions in the AHFV JF 416949 strain. Eleven nucleotide insertions were identified in the AHFV/997/NJ/09/SA strain at various positions of the viral genome, namely the envelope gene (1409-T, 1940-C, and 1965-G), the NS4 gene (6601-T), and the NS5 gene (8087-C, 8149-T, 10027-C, 10028-A, 10029-A, 10030-T, and 10071-G).

The genetic characteristics of the AHFV/997/NJ/09/SA strain are listed in table 4. Comparison of the full-length sequence of the AHFV/997/NJ/09/SA strain with the other AHFV isolates, DENV, KFDV, Langat virus, OHFV, and TBEV isolates showed that the highest similarity (99.0%) was with AHFV isolate JF416957 and the lowest (58.6%) was with the DENV-1 isolate. Not much nucleotide sequence homology was observed among the Langat virus, OHFV, and TBEV isolates either in the full-length genome or in the structural and nonstructural genes. Phylogenetic analysis of these isolates showed that the AHFV/997/NJ/09/SA strain formed a close cluster with the AHFV strains previously isolated in Saudi Arabia (fig. 1).

Table 4

Genetic characteristics of AHFV strain AHFV/997/NJ/09/SA

Genetic characteristics of AHFV strain AHFV/997/NJ/09/SA
Genetic characteristics of AHFV strain AHFV/997/NJ/09/SA
Fig. 1

Phylogenetic tree of AHFV strain AHFV/997/NJ/09/SA (in bold), 18 previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains.Each isolate is indicated by its GenBank accession No.

Fig. 1

Phylogenetic tree of AHFV strain AHFV/997/NJ/09/SA (in bold), 18 previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains.Each isolate is indicated by its GenBank accession No.

Close modal

Envelope Protein

The retrieved and assembled sequences of the AHFV/997/NJ/09/SA strain were used for percent identity matrix and phylogenetic analysis with the selected AHFV, DENV, KFDV, Langat virus, OHFV, and TBEV isolates (table 3). The AHFV/997/NJ/09/SA strain formed a close cluster with Saudi Arabian AHFV isolates while KFDV, TBEV, OHFV, and Langat virus isolates formed a separate cluster (fig. 2). Comparison of nucleotide sequences showed that the AHFV/997/NJ/09/SA strain had the highest similarity (99.4%) with the AHFV JF416953 isolate followed by 91.6-91.7% similarities with the KFDV isolates (JF416958, JF416959, and JF416960). The lowest similarity (47.8%) was found with the DENV-1 isolate (JQ287667).

Fig. 2

Phylogenetic tree of the AHFV strain AHFV/997/NJ/09/SA (in bold), 18 other previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains based on the envelope protein gene. Each isolate is indicated by its GenBank accession No.

Fig. 2

Phylogenetic tree of the AHFV strain AHFV/997/NJ/09/SA (in bold), 18 other previously isolated AHFV strains, 1 DENV strain, 3 KFDV strains, 4 Langat virus strains, 5 OHFV strains, and 5 TBEV strains based on the envelope protein gene. Each isolate is indicated by its GenBank accession No.

Close modal

Core Protein

The highest (99.4%) similarity was observed with two AHFV isolates (J416953 and JF416957). A lower similarity (91.4%) was observed with the KFDV isolates (JF416958, JF416959, and JF416960). Significant variation was observed with two AHFV isolates (AF331718 and NC004355), which showed only 82.6% similarity with the AHFV/997/NJ/09/SA strain.

Premembrane

The highest nucleotide similarity (98.9%) was observed with the AHFV isolate (AJ416953), while the KFDV isolates (JF416958, JF416959, and JF416960) showed only 90.8% similarity.

Nonstructural Protein Gene Analysis

NS1 Gene

The highest similarity (99.8%) was observed with one AHFV isolate reported from Saudi Arabia (JF416957), while all three KFDV isolates (JF416958, JF416959, and JF416960) showed similarity ranging from 91.6 to 91.7%.

NS2a and 2b Gene

The highest nucleotide similarity (99.7%) was observed with two isolates of AHFV (JF416961 and JF416962). The KFDV isolates (JF416958, JF416959, and JF416960) showed 91.8-92.1% similarity. The NS2b gene sequence showed high similarity (99.7%) with three AHFV isolates (NC004355, JF416953, and JF 416957) and 94.4% similarity with the KFDV isolates (JF416958, JF416959, and JF416960).

NS3 Gene

The NS3 gene sequence showed high similarity (99.4%) with four AHFV isolates (NC004355, JF416952, JF416953, and JF416957) and less homology (93.1%) with KFDV isolates.

NS4a and 4b Genes

The NS4a gene sequence showed high homology (97.5%) with two AHFV isolates (JF416952 and JF416953) and less homology (89.2-89.5%) with KFDV isolates (JF416958, JF416959, and JF416960). Interestingly, only 85.1-85.8% homology was observed with two AHFV isolates (AF331718 and NC004355).

The NS4b sequence showed high homology (98.4-98.3%) with four AHFV isolates (JF416949, JF416954, JF416955, and JF416957) and 91.6-91.9% homology with the KFDV isolates (JF416958, JF416959, and JF416960).

NS5 Gene

The NS5 gene sequence showed high homology (99.3-99.4%) with five isolates of AHFV (JF416957, JF41696, 1 JF416962, NC004355, and JF416950) and 92.7-92.8% homology with the KFDV isolates (JF416958, JF416959, and JF416960).

Untranslated Region

The UTR of the AHFV/997/NJ/09/SA strain was found to have 269 nucleotides. The nucleotide sequence showed homology ranging from 63.0 to 67.0% with the selected AHFV and KFDV isolates, whereas TBEV, OHFV, and Langat virus showed 37-38% homology.

The complete genome sequence of the AHFV/997/NJ/09/SA strain was determined with various overlapping PCR primers. It had a total 10,546 nucleotides coding for 3,416 amino acids. When compared to the AHFV NC004355 and AHFV JF 416949 strains, a total of 139-203 nucleotides were missing from the AHFV/997/NJ/09/SA strain primarily in the UTR, likely due to passage of the virus in cell culture. Previous studies reported such deletions and even insertions and/or duplications of nucleotides of viruses when passaged in cultures [16,17,18,19,20,21]. Several studies have shown that nucleotide deletion/insertion/duplication occur in the 3′ UTR (formerly known as a noncoding region or NCR) among various members of flaviviruses and even among strains of the same viral species [16,17], as well as in the 5′ UTR of hepatitis A virus [18] and the Newcastle disease virus genome [19]. In another study it was observed that size variation between yellow fever virus strains was due to duplications and/or deletions of repeated nucleotide sequence elements that occurred in the 3′ UTR by passaging the virus in cell culture [20]. Another study showed that Brazilian yellow fever virus genomes ranged in size from 10,795 to 11,008 nucleotides. The variability in size was primarily due to high variability in the 3′ UTR with minor mutations occurring also in the 5′ UTR [21]. In our study, we observed that the AHFV/997/NJ/09/SA strain also had genetic variability due to deletion and insertions of nucleotides not only in the 5′ and 3′ UTRs, but also in other locations in the viral genome. Our analysis results showed that nucleotide deletion/insertion may occur in the UTR as well as in the structural and nonstructural protein genes.

Comparison of the complete nucleotide sequence of the AHFV/997/NJ/09/SA strain with other related flaviviruses have shown that the AHFV/997/NJ/09/SA strain is similar to the other AHFV isolates previously reported from Saudi Arabia [10,22]. The retrieved sequences of the full-length genome and the envelope protein gene of the AHFV/997/NJ/09/SA strain were analyzed and used to construct the phylogenetic tree with selected flaviviruses. The phylogenetic relationship showed that the AHFV/997/NJ/09/SA strain and all previously reported AHFV isolates formed a close cluster, while the KFDV, TBEV, OHFV, and Langat virus isolates formed a separate cluster. Phylogenetic tree analysis based on the full-length genome showed that AHFV/997/NJ/09/SA had identical homology with four of the previously reported AHFV isolates (JF416961, JF416962, JF416953, and JF 416957) and close homology with the other AHFV isolates, whereas the envelope gene sequences showed that AHFV/997/NJ/09/SA had identical homology with three of the previously reported AHFV isolates (JF416961, JF416962, and AF331718) and close homology with the other AHFV isolates. AHFV could be distinguished from KFDV in the hypervariable regions of the envelope protein where AHFV codes for the AHE sequence, which is distinct from the AQE motifs of KFDV [23]. Further analysis of the amino acid motif showed that AHFV is more closely related to KFDV. Our data concur with previously published findings that suggest AHFV and KFDV had a deeper evolutionary history than suggested by previous partial genome analysis [22]. It is estimated that the divergence of AHFV and KFDV occurred almost 700 years ago [22].

Because of the close phylogenetic similarity between AHFV and KFDV, ticks are believed to play an important role in the transmission cycle of AHFV [10]. This is further supported by the PCR-based detection of a virus closely related to AHFV from an Ornithodoros tick in Jeddah, and O. savignyi and Hyalomma dromedarii ticks in Najran, Saudi Arabia [22,24,25]. However, clinicoepidemiological studies indicate that ticks do not seem to play an important role in transmission from animals to humans even though the role of ticks as reservoirs of the virus in its ecologic niche and as vectors transmitting the virus between animals and perhaps also from animals to humans is possible[2,3,4]. Current epidemiological data suggest a clear association of human infection with livestock animals, particularly sheep, goats, and camels, despite the absence of any manifestations of illness in such animals [3]. Based on the current epidemiological data, direct contact with these animals or handling of their fresh raw meat is strongly suspected as a primary mode of transmission [2,3]. Additionally, mosquitoes also seem to be important vectors in the transmission of the virus from animals to humans [2,3]. The recent report of successful propagation of AHFV in mosquito cells lends further support to the speculated mosquito-borne mode of transmission [26].

In conclusion, the current study determined the complete genome sequences of the AHFV/997/NJ/09/SA strain. This virus showed high homology with the previously reported AHFV strains from Saudi Arabia. The previously reported AHFV strains are composed of 10,685-10,749 nucleotides, while the AHFV/997/NJ/09/SA strain is composed of 10,546 nucleotides. The most important variations were observed in the core protein and NS4a gene sequences of two AHFV isolates (AF331718 and NC004355). It is possible that the AHFV/997/NJ/09/SA strain has recently emerged as a variant of the previously isolated strain.

The authors thank Sheikh Mohammed Hussein Al-Amoudi for funding this research and the Scientific Chair for Viral Hemorrhagic Fever at King Abdulaziz University, Jeddah, Saudi Arabia.

The authors declare no conflicts of interest. This study was supported through the Scientific Chair of Sheikh Mohammad Hussein Al-Amoudi for Viral Hemorrhagic Fever, King Abdulaziz University, Jeddah, Saudi Arabia. The sponsor, Sheikh Mohammad Hussein Al-Amoudi, had no involvement in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.

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