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Bas-Congo Virus
Chapter 2
Bas-Congo Virus: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever Gilda Grard1, Joseph Fair2, Charles Chiu3,4,5 and Eric Leroy1,6 1
Unite´ des Maladies Virales Emergentes, Centre International de Recherches Me´dicales de Franceville, Franceville, Gabon, 2Metabiota, Incorporated, San Francisco, CA, USA, 3 Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA, USA, 4Department of Laboratory Medicine, University of California, San Francisco, CA, USA, 5UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA, UMR MIVEGEC (IRD 224 - CNRS 5290 - UM1 - UM2), 6 Institut de Recherche pour le De´veloppement, Montpellier, France
CLINICAL PRESENTATION A 32-year-old male nurse from Mangala village in the Bas-Congo province of Democratic Republic of Congo (DRC) fell ill on June 13, 2009. Symptoms included epistaxis, ocular and oral hemorrhages, hematemesis, and bloody diarrhea. Two days later, the patient developed fever .39 C, anorexia, headache, fatigue, and abdominal pain. He was transferred that same day to the regional general hospital of Boma, approximately 30 km from Mangala village, where a serum sample was taken for analysis. Supportive care was then provided, including fluid resuscitation, blood transfusion, and empiric antibiotics. Initial laboratory tests were negative for malaria, tuberculosis, dengue, and bacterial sepsis. The patient recovered spontaneously from the episode of presumptive acute hemorrhagic fever.
EPIDEMIOLOGICAL CONTEXT Within the past 3 weeks prior to becoming ill with hemorrhagic fever, the nurse had cared directly for two teenagers who had presented to the Mangala village health center with hemorrhagic symptoms. The first patient was a 15-year-old boy seen on May 25, 2009 with malaise, epistaxis, conjunctival injection, gingival bleeding, hematemesis, and diarrhea with blood. Emerging Infectious Diseases. DOI: http://dx.doi.org/10.1016/B978-0-12-416975-3.00002-9 © 2014 Elsevier Inc. All rights reserved.
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Although no fever was documented, he died rapidly within 2 days after onset of symptoms. The second patient was a 13-year-old girl who attended the same public school, although there was no history of any direct contact between them. She presented on June 5, 2009 with headache, fever .39 C, abdominal pain, epistaxis, conjunctival injection, mouth bleeding, hematemesis, and diarrhea with blood. Despite receiving symptomatic treatment for fever and quinine for malaria, she died within 3 days after onset of symptoms. All three cases lived approximately 50 meters from one another in the same neighborhood in Mangala village. Household contacts for each of the three patients were closely monitored for 21 days, the duration required upon suspicion of viral hemorrhagic fever, and no other cases were observed. Notably, although DRC is an endemic country for Ebola and Marburg viral hemorrhagic fever, no previous outbreaks had ever been reported in the BasCongo province.
LABORATORY INVESTIGATION FOR VIRAL HEMORRHAGIC FEVER (VHF) DIAGNOSIS The only available serum sample from the surviving 32-year-old nurse was received by the Centre International de Recherches Me´dicales de Franceville (CIRMF) on June 29, 2009 for viral hemorrhagic fever laboratory testing. The patient sample tested negative for Ebola virus, Marburg virus, CrimeanCongo hemorrhagic fever virus, yellow fever virus, Rift Valley fever virus, and dengue virus.
DISCOVERY OF BAS-CONGO VIRUS, A NOVEL RHABDOVIRUS, IN PATIENT SERUM Extracted RNA from the patient serum sample was then subjected to metagenomic investigation, leading to the detection of viral genome sequences from a previously unrecognized novel rhabdovirus, subsequently named BasCongo virus (BASV). Likely due to the break in cold chain encountered during sample collection in DRC, the virus failed to grow on cell cultures and in suckling mice brain, but in-depth genetic analysis by next-generation sequencing and de novo genome assembly allowed full genomic characterization of the virus, phylogenetic confirmation of its membership in the Rhabdoviridae family, and development of a virus neutralization serological assay for BASV using a “pseudotyped” construct. Using the serological test, specific BASV-neutralizing antibodies were detected in serum from the surviving nurse and from an asymptomatic close contact, another nurse who took care of him upon onset of hemorrhagic symptoms in Mangala village and assisted in transporting him to the hospital in Boma. These three cases and associated research investigations were previously published in 2012.1
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1. WHY THIS CASE WAS SIGNIFICANTLY IMPORTANT AS AN EMERGING INFECTION Bas-Congo virus is a newly discovered rhabdovirus found in serum from a patient with acute hemorrhagic fever. Rhabdoviruses that are known to be pathogenic for humans have previously been associated with encephalitic syndromes (rabies virus and Chandipura virus)2,3 or influenza-like syndromes (vesicular stomatitis virus).4 This case represents the first time that a member of the family Rhabdoviriadae has been associated with acute hemorrhagic fever in humans.
2. WHAT IS THE CAUSATIVE AGENT? 2.1 BASV as the Etiologic Agent of an Acute Hemorrhagic Fever Syndrome Upon discovery of a new microorganism, establishing a causal and unambiguous relationship to the disease requires several lines of evidence. This is particularly true when the suspected agent is assigned to a group that has never been associated with the type of disease under investigation. This is the case for the association of BASV with hemorrhagic fever syndrome, as was also the case with Sin Nombre hantavirus and acute pulmonary syndrome during the 1993 Four Corners outbreak in the United States.5,6 Evidence of causality can be obtained by detecting the virus in geographically and temporally linked clusters of sick individuals with similar clinical presentations, and/or inoculation of the virus (either naturally cultured or artificially generated by reverse genetics) in a healthy animal model to induce the observed pathology. To date, the lines of evidence supporting BASV as the etiologic agent of the hemorrhagic fever cases described here include: 1. BASV was the only credible pathogen found in the serum of the acutely ill patient 2. The viral copies per ml were 1.09 3 106 RNA copies/ml, titers similar to those observed in survivors of Ebola virus infection and correlated with disease severity 3. The close epidemiological clustering of the three patients who lived in the same neighborhood and presented with similar symptoms over a 3-week time frame 4. Clearance of viral RNA and detection of specific neutralizing antibodies in the serum of the surviving patient after convalescence.
2.2 BASV Taxonomy The Rhabdoviridae family (order Mononegavirales) is associated with an extremely diverse host range and is currently divided into eight genera.7,8 The genera Cytorhabdovirus and Nucleorhabdovirus are associated with
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plant infections, the genera Perhabdovirus and Novirhabdovirus with fish infections, and the genera Sigmavirus with insect infections. The genus Lyssavirus is associated with infections of bats and carnivores and includes the highly pathogenic rabies viruses. Finally, the genera Ephemerovirus, Tibrovirus, and Vesiculovirus form together, with about 120 unclassified viruses, the dimarhabdovirus supergroup (dipteran-mammal-associated rhabdovirus), the genus Vesiculovirus including human pathogens and viruses responsible for hemorrhagic disease in fish. Phylogenetic (Figure 2.1) and whole genome analysis of BASV identifies it is a member of the dimarhabdovirus supergroup, most closely related to the genera Ephemerovirus and Tibrovirus.1 Viruses from these genera are usually associated with arthropod and cattle infections but have not previously been known to infect humans. Additionally, the phylogenetic branching pattern and the high amino acid divergence (70%) of BASV suggest it represents a distinct viral group that has yet to be taxonomically classified.
2.3 Virus Description Rhabdoviruses are enveloped viruses with single-stranded negative-sense RNA genomes. Virions are bullet shaped, from 100 to 430 nm long and 45 to 100 nm wide.7,9 The glycoprotein G is anchored in the lipid bilayer and is responsible for virus entry into cells (Figure 2.2A). Viral RNA is encapsulated with the nucleocapsid protein (N). This ribonucleocapsid is associated with the viral polymerase (L) through interaction with the viral phosphoprotein (P). The ribonucleocapsid is packaged with matrix protein (M), which gives the virion its characteristic form.9 The near-complete BASV genome sequence is 11,892 nucleotides long (GenBank accession number JX297815). The genome organization of BASV displays eight open reading frames (ORFs) in the following order (Figure 2.2B): 30 - nucleoprotein (N), phosphoprotein (P), matrix protein (M), U1 protein (U1), U2 protein (U2), glycoprotein (G), U3 protein (U3), and RNA-dependent RNA polymerase (L). The N-P-M-G-L genome organization is common to rhabdoviruses while the insertion of U1, U2, and U3 genes is shared only with members of the genus Tibrovirus. Functions of the proteins U1 and U2 are not known, although U3 protein is hypothesized to be a candidate viroporin.10
3. WHAT IS THE FREQUENCY OF THE DISEASE? To date, BASV has only been found in the Bas-Congo province of Democratic Republic of Congo, Central Africa (Figure 2.3). Infection was confirmed for one surviving patient, who presented with hemorrhagic fever symptoms, and suspected for two other closely epidemiologically linked patients, who died rapidly from fulminant hemorrhagic fever before samples could be collected. Infection by BASV was retrospectively confirmed in
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FIGURE 2.1 Phylogenetic analysis of the L proteins of rabdovirus. Bayesian tree topologies were assessed with MrBayes V.32 software (20,000 sampled trees; 5000 trees discarded as burn-in). Original figure was published by Grard et al.1 under the creative commons license (CC-BY, available at: http://creativecommons.org/licenses/by/3.0/) and slightly modified for taxonomic updates. Virus abbreviations are listed as follows, in alphabetical order: ABLV, Australian bat lyssavirus; ARAV, Aravan virus; BEFV, bovine ephemeral fever virus; BYSMV, barley yellow striate mosaic virus (BYSMV); CHPV, Chandipura virus; CPV, Coastal Plains virus; COCV, cocal virus; DURV, Durham virus; DUVV, Duvenhage virus; EBLV1, European bat lyssavirus 1; EBLV2, European bat lyssavirus 2; EVEX, eel virus European X virus; FLAV, Flanders virus; HIRRV, Hirame rhabdovirus; IHNV, infectious hematopoietic necrosis virus; IRKV, Irkut virus; ISFV, Isfahan virus; KHUV, Khujand virus; LBV, Lagos bat virus; LNYV, lettuce necrotic yellow virus; MARAV, Maraba virus; MMV, maize mosaic virus; MOKV, Mokala virus; MOUV, Moussa virus; NCMV, northern cereal mosaic virus; NGAV, Ngaingan virus; OVRV, Oak Vale rhabdovirus; PFRV, pike fry rhabdovirus; RABV, rabies virus; RYSV, rice yellow stunt rhabdovirus; SIGMAV, sigma virus; SCRV, Siniperca chuatsi rhabdovirus; SHRV, snakehead virus; SMRV, Scophthalmus maximus rhadovirus; SVCV, spring viremia of carp virus; SYNV, Sonchus yellow net virus; TIBV, Tibrogargan virus; TUPV, Tupaia virus; TVCV, tomato vein clearing virus; VHSV, viral hemorrhagic septicemia virus; VSIV, vesicular stomatitis virus, Indiana; VSNJV, vesicular stomatitis virus, New Jersey; WCBV, West Caucasian bat virus; WONGV, Wongabel virus.
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FIGURE 2.2 Panel A. Schematic representation of the rhabdovirus virion structure. Panel B. Schematic representation of the Bas-Congo virus genome organization.
another asymptomatic contact, a nurse who cared directly for the surviving patient, by seroneutralization studies. The proportion of asymptomatic vs. symptomatic infections with severe or possibly mild clinical manifestations is not known. A preliminary epidemiological study that included (1) molecular screening of 50 serum samples from patients presenting with hemorrhagic fever syndrome in DRC and (2) serological screening of 50 plasma samples from blood donors in the Kasai-Orientale province of DRC failed to detect additional cases of BASV infection. These results suggest that human infection with BASV is infrequent or that the virus has only emerged very recently. Larger epidemiological studies and surveillance of active cases of unknown hemorrhagic fever are needed to establish the frequency and impact of BASV-associated disease.
4. HOW IS THE VIRUS TRANSMITTED? To date, the source of infection and the possible ways of transmission have not been established. Waterborne or airborne transmission appears unlikely
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FIGURE 2.3 Map of viral hemorrhagic fever outbreaks in Africa. Yellow fever and dengue fever have a wide geographic distribution throughout sub-Saharan Africa, as well as Rift Valley fever which extends to the south of Africa and are not shown. Mangala village, located in Democratic Republic of the Congo, is represented by a red star. Original figure was published by Grard et al.1 under the creative commons license (CC-BY, available at: http://creativecommons.org/licenses/by/3.0/) and slightly modified.
due to the small number of reported cases. The phylogenetic position of BASV in the dimarhabdovirus supergroup is consistent with a possible arthropod-borne transmission. Additionally, the epidemiological data also suggest potential human-to-human transmission. Indeed, the surviving nurse directly cared for the first two patients who presented with hemorrhagic fever symptoms, and he in turn was taken care of by the serologically confirmed asymptomatic contact. However, none of the household contacts became ill, suggesting that human-to-human transmission may be limited. This putative pattern of transmission for BASV is also seen with CrimeanCongo hemorrhagic fever virus, which is an arboviral VHF agent associated with additional cases of nosocomial human-to-human transmission.11
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5. WHAT ARE THE CLINICAL MANIFESTATIONS? Clinical symptoms shared by all three cases were an abrupt onset of disease, with hemorrhagic manifestations not limited to gastrointestinal sites but also affecting mucosa: epistaxis (nose bleeding), ocular hemorrhage or conjunctival injection (eye bleeding), oral hemorrhage (mouth bleeding), hemorrhagic vomiting, and hemorrhagic diarrhea. Associated unspecific symptoms included one or more of the following: fever, malaise, headache, abdominal pain, fatigue, and anorexia. Notably, fever may be concomitant with early symptoms or appear several days later, potentially explaining why one patient died without documented fever. As revealed by the retrospective serologic study, asymptomatic infection from BASV may occur. In addition, BASV-associated clinical presentations with subclinical, mild, or unrelated symptoms, such as rashes, arthralgias, hepatitis, or neurologic disorders, cannot be excluded at the present time. Many hemorrhagic fever viruses, such as yellow fever12, are associated with a variety of associated clinical manifestations ranging from mild to severe.
6. HOW DO YOU DIAGNOSE? 6.1 Molecular Diagnosis Diagnosis of severe viral hemorrhagic fevers classically includes detection of viral RNA or antigens in serum or blood samples taken during the acute phase of the disease. BASV was first detected by random metagenomic analysis from RNA extracted from a serum sample taken during the acute phase of the disease. Conventional reverse transcription (RT)-PCR and real-time RT-PCR systems were subsequently implemented in the lab for viral RNA detection in human sera and are currently used for diagnostic investigations of suspected VHF cases.
6.2 Viral Isolation Virus isolation is usually the gold standard technique for diagnosis of viral infection. However, as the time of virus cultivation is several days long, this approach is not suitable for emergency detection of highly pathogenic VHF viruses, which require immediate outbreak response and management. With BASV, attempts were made to grow the virus on several cell lines with unsuccessful results: Vero (green monkey kidney), LLC-MK2 (rhesus monkey kidney), BHK (baby hamster kidney cells), CCL-106 (rabbit kidney), and C6/36 (Aedes albopictus mosquito). Since intracerebral inoculation of suckling mice also failed in growing the virus, we assume that the failure in virus cultivation resulted from viral inactivation due to breakdown in cold chain. A recent study of a viral pseudotype carrying the immunogenic BASV glycoprotein in a vesicular stomatitis virus (VSV, genus Vesiculovirus) backbone demonstrated the susceptibility of numerous cell lines including Vero
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and C6/36 to BASV infection in vitro.13 In the absence of samples from additional cases of BASV, recovery of infectious virus may be possible by reverse genetics, as previously performed for rabies virus and VSV.14,15
6.3 Serologic Diagnosis Serological testing for BASV during the acute phase of the disease is not recommended. To date, the kinetics of production of IgM and IgG antibodies relative to onset of symptoms and disease course are not known. In addition, we cannot exclude a limited or impaired humoral immune response in patients with fatal outcome as previously reported for Ebola hemorrhagic fever.16 A seroneutralization assay for BASV using a pseudotyped viral construct is available for epidemiological serosurveys of BASV prevalence13 but is currently not useful for diagnosis.
7. HOW DO YOU DIFFERENTIATE THIS DISEASE FROM SIMILAR ENTITIES? To date, BASV has only been detected in Central Africa where numerous viral hemorrhagic fevers are encountered (Figure 2.3). In this context, other viral etiologies to consider are Ebola and Marburg viruses, Crimean-Congo hemorrhagic fever virus, yellow fever virus, dengue viruses, and Rift Valley fever virus. Lassa and Lujo viruses should also be considered despite currently occurring in a different geographic range. However, the early development of hemorrhagic signs (within the first 2 days of symptoms) seen in BASV infection appears unusual with respect to other VHF agents. Ebola, Marburg, Crimean-Congo, and Lassa hemorrhagic fevers typically begin with a non-specific prodrome that may be confused with the other infections listed in Table 2.1, and hemorrhagic manifestations generally appear by day 3 to 5 after symptom onset.11,1719 In addition to viral hemorrhagic fevers, many other infections are associated with acute febrile disease in tropical and subtropical regions (Table 2.1). The two most frequent infections to exclude are malaria and typhoid fever.20 Gastrointestinal infections by bacteria such as Shigella, Campylobacter, and Salmonella can also be accompanied by bleeding, and are part of the differential diagnosis. Other causes of acute febrile illness frequently encountered in Central Africa include typhus, plague, relapsing fever, leptospirosis, anthrax, and viral hepatitis.19
8. WHAT IS THE THERAPEUTIC APPROACH? To date the therapeutic approach is limited to supportive care.
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TABLE 2.1 BASV Infection Differential Diagnosis Viral infections Hemorrhagic fever viruses Family Filoviridae
Zaire ebolavirus Bundibugyo ebolavirus Tai Forest ebolavirus Sudan ebolavirus Marburg marburgvirus
Family Flaviviridae
Dengue virus Yellow fever virus
Family Bunyaviridae
Crimean-Congo hemorrhagic fever virus Rift Valley fever virus
Family Arenaviridae
Lassa virus Lujo virus
Other viral infections Measles Hemorrhagic varicella Rubella Hemorrhagic smallpox Viral hepatitis Chikungunya fever Parasitic infections Malaria African trypanosomiasis Bacterial infections Gram-negative bacteria Gram-negative bacterial septicemia Shigellose Salmonellose Typhoid fever Septicemic plague Cholera Q fever Meningococcemia Typhus Murin typhus Leptospirose Siphilis Relapsing fever Leptospirosis Gram-positive bacteria Staphylococcal or streptococcal toxic shock syndrome Anthrax Leptospirosis
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9. WHAT ARE THE PREVENTIVE AND INFECTION CONTROL MEASURES? As the source of infection and way(s) of transmission are not clearly established, instructions to prevent and control the infection cannot be precisely defined. However, a minimum of rules should already be considered. Public health authorities must be informed whenever there is VHF suspicion and contact tracing should be engaged as soon as possible. Compliance with the standard guidelines for biosafety and hygiene (use of lab coat, gloves, sharps containers, hand and surface cleaning, etc.) during clinical examination and blood sampling is an absolute requirement to prevent a potential nosocomial transmission. An aerosol transmission seems unlikely but use of masks and face shields should be considered in light of the precautionary principle and would provide an additional protection in the event of accidental exposure to body fluids. The scientific data do not allow a definitive and obvious risk assessment, so that prevention and control measures are a sensitive point with strong ethical concerns. Going beyond the minimal set of rules proposed here, in considering stronger isolation measures for patient management for example, must be collectively discussed with additional medical experts, including those experienced in the management of VHF outbreaks in the field, to reach a consensus on solid and balanced recommendations.
REFERENCES 1. Grard G, Fair JN, Lee D, Slikas E, Steffen I, Muyembe JJ, et al. A novel rhabdovirus associated with acute hemorrhagic fever in central Africa. PLoS Pathog [serial online]. 2012 [cited 2012 Sep];8(9):e1002924. Available from: ,http://www.plospathogens.org.. 2. Gurav YK, Tandale BV, Jadi RS, Gunjikar RS, Tikute SS, Jamgaonkar AV, et al. Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007. Indian J Med Res 2010;132:3959. 3. Warrell MJ, Warrell DA. Rabies and other lyssavirus diseases. Lancet 2004;363:95969. 4. Rodriguez LL. Emergence and re-emergence of vesicular stomatitis in the United States. Virus Res 2002;85:2119. 5. Fredericks DN, Relman DA. Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 1996;9:1833. 6. Chiu C, Fair J, Leroy EM. Bas-Congo virus: another deadly virus? Future Microbiol 2013;8:13941. 7. Kuzmin IV, Novella IS, Dietzgen RG, Padhi A, Rupprecht CE. The rhabdoviruses: biodiversity, phylogenetics, and evolution. Infect Genet Evol 2009;9:54153. 8. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Virus taxonomy: classification and nomenclature of viruses: ninth report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press; 2012. 9. Lyles DS, Rupprecht CE. Rhabdoviridae. In: Knipe DM, Howley PM, editors. Fields virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2007. p. 1363408.
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10. Gubala A, Davis S, Weir R, Melville L, Cowled C, Boyle D. Tibrogargan and Coastal Plains rhabdoviruses: genomic characterization, evolution of novel genes and seroprevalence in Australian livestock. J Gen Virol 2011;92:216070. 11. Ergonul O. Crimean-Congo haemorrhagic fever. Lancet Infect Dis 2006;6:20314. 12. Monath TP, Barrett AD. Pathogenesis and pathophysiology of yellow fever. Adv Virus Res 2003;60:34395. 13. Steffen I, Liss NM, Schneider BS, Fair JN, Chiu CY, Simmons G. Characterization of the Bas-Congo virus glycoprotein and its function in pseudotyped viruses. J Virol 2013;87:955868. 14. Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. EMBO J 1994;13:4195203. 15. Stanifer ML, Cureton DK, Whelan SP. A recombinant vesicular stomatitis virus bearing a lethal mutation in the glycoprotein gene uncovers a second site suppressor that restores fusion. J Virol 2011;85:810515. 16. Baize S, Leroy EM, Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debre P, et al. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med 1999;5:4236. 17. Yun NE, Walker DH. Pathogenesis of lassa fever. Viruses 2012;4:203148. 18. Bwaka MA, Bonnet MJ, Calain P, Colebunders R, De Roo A, Guimard Y, et al. Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo: clinical observations in 103 patients. J Infect Dis 1999;179(Suppl. 1):S17. 19. Sanchez A, Geisbert TW, Feldmann H. Filoviridae: Marburg and Ebola viruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2007. p. 140948. 20. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet 2011;377:84962.
Bas-Congo Virus: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever Gilda Grard1, Joseph Fair2, Charles Chiu3,4,5 and Eric Leroy1,6 1
Unite´ des Maladies Virales Emergentes, Centre International de Recherches Me´dicales de Franceville, Franceville, Gabon, 2Metabiota, Incorporated, San Francisco, CA, USA, 3 Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA, USA, 4Department of Laboratory Medicine, University of California, San Francisco, CA, USA, 5UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA, UMR MIVEGEC (IRD 224 - CNRS 5290 - UM1 - UM2), 6 Institut de Recherche pour le De´veloppement, Montpellier, France
CLINICAL PRESENTATION A 32-year-old male nurse from Mangala village in the Bas-Congo province of Democratic Republic of Congo (DRC) fell ill on June 13, 2009. Symptoms included epistaxis, ocular and oral hemorrhages, hematemesis, and bloody diarrhea. Two days later, the patient developed fever .39 C, anorexia, headache, fatigue, and abdominal pain. He was transferred that same day to the regional general hospital of Boma, approximately 30 km from Mangala village, where a serum sample was taken for analysis. Supportive care was then provided, including fluid resuscitation, blood transfusion, and empiric antibiotics. Initial laboratory tests were negative for malaria, tuberculosis, dengue, and bacterial sepsis. The patient recovered spontaneously from the episode of presumptive acute hemorrhagic fever.
EPIDEMIOLOGICAL CONTEXT Within the past 3 weeks prior to becoming ill with hemorrhagic fever, the nurse had cared directly for two teenagers who had presented to the Mangala village health center with hemorrhagic symptoms. The first patient was a 15-year-old boy seen on May 25, 2009 with malaise, epistaxis, conjunctival injection, gingival bleeding, hematemesis, and diarrhea with blood. Emerging Infectious Diseases. DOI: http://dx.doi.org/10.1016/B978-0-12-416975-3.00002-9 © 2014 Elsevier Inc. All rights reserved.
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Although no fever was documented, he died rapidly within 2 days after onset of symptoms. The second patient was a 13-year-old girl who attended the same public school, although there was no history of any direct contact between them. She presented on June 5, 2009 with headache, fever .39 C, abdominal pain, epistaxis, conjunctival injection, mouth bleeding, hematemesis, and diarrhea with blood. Despite receiving symptomatic treatment for fever and quinine for malaria, she died within 3 days after onset of symptoms. All three cases lived approximately 50 meters from one another in the same neighborhood in Mangala village. Household contacts for each of the three patients were closely monitored for 21 days, the duration required upon suspicion of viral hemorrhagic fever, and no other cases were observed. Notably, although DRC is an endemic country for Ebola and Marburg viral hemorrhagic fever, no previous outbreaks had ever been reported in the BasCongo province.
LABORATORY INVESTIGATION FOR VIRAL HEMORRHAGIC FEVER (VHF) DIAGNOSIS The only available serum sample from the surviving 32-year-old nurse was received by the Centre International de Recherches Me´dicales de Franceville (CIRMF) on June 29, 2009 for viral hemorrhagic fever laboratory testing. The patient sample tested negative for Ebola virus, Marburg virus, CrimeanCongo hemorrhagic fever virus, yellow fever virus, Rift Valley fever virus, and dengue virus.
DISCOVERY OF BAS-CONGO VIRUS, A NOVEL RHABDOVIRUS, IN PATIENT SERUM Extracted RNA from the patient serum sample was then subjected to metagenomic investigation, leading to the detection of viral genome sequences from a previously unrecognized novel rhabdovirus, subsequently named BasCongo virus (BASV). Likely due to the break in cold chain encountered during sample collection in DRC, the virus failed to grow on cell cultures and in suckling mice brain, but in-depth genetic analysis by next-generation sequencing and de novo genome assembly allowed full genomic characterization of the virus, phylogenetic confirmation of its membership in the Rhabdoviridae family, and development of a virus neutralization serological assay for BASV using a “pseudotyped” construct. Using the serological test, specific BASV-neutralizing antibodies were detected in serum from the surviving nurse and from an asymptomatic close contact, another nurse who took care of him upon onset of hemorrhagic symptoms in Mangala village and assisted in transporting him to the hospital in Boma. These three cases and associated research investigations were previously published in 2012.1
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1. WHY THIS CASE WAS SIGNIFICANTLY IMPORTANT AS AN EMERGING INFECTION Bas-Congo virus is a newly discovered rhabdovirus found in serum from a patient with acute hemorrhagic fever. Rhabdoviruses that are known to be pathogenic for humans have previously been associated with encephalitic syndromes (rabies virus and Chandipura virus)2,3 or influenza-like syndromes (vesicular stomatitis virus).4 This case represents the first time that a member of the family Rhabdoviriadae has been associated with acute hemorrhagic fever in humans.
2. WHAT IS THE CAUSATIVE AGENT? 2.1 BASV as the Etiologic Agent of an Acute Hemorrhagic Fever Syndrome Upon discovery of a new microorganism, establishing a causal and unambiguous relationship to the disease requires several lines of evidence. This is particularly true when the suspected agent is assigned to a group that has never been associated with the type of disease under investigation. This is the case for the association of BASV with hemorrhagic fever syndrome, as was also the case with Sin Nombre hantavirus and acute pulmonary syndrome during the 1993 Four Corners outbreak in the United States.5,6 Evidence of causality can be obtained by detecting the virus in geographically and temporally linked clusters of sick individuals with similar clinical presentations, and/or inoculation of the virus (either naturally cultured or artificially generated by reverse genetics) in a healthy animal model to induce the observed pathology. To date, the lines of evidence supporting BASV as the etiologic agent of the hemorrhagic fever cases described here include: 1. BASV was the only credible pathogen found in the serum of the acutely ill patient 2. The viral copies per ml were 1.09 3 106 RNA copies/ml, titers similar to those observed in survivors of Ebola virus infection and correlated with disease severity 3. The close epidemiological clustering of the three patients who lived in the same neighborhood and presented with similar symptoms over a 3-week time frame 4. Clearance of viral RNA and detection of specific neutralizing antibodies in the serum of the surviving patient after convalescence.
2.2 BASV Taxonomy The Rhabdoviridae family (order Mononegavirales) is associated with an extremely diverse host range and is currently divided into eight genera.7,8 The genera Cytorhabdovirus and Nucleorhabdovirus are associated with
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plant infections, the genera Perhabdovirus and Novirhabdovirus with fish infections, and the genera Sigmavirus with insect infections. The genus Lyssavirus is associated with infections of bats and carnivores and includes the highly pathogenic rabies viruses. Finally, the genera Ephemerovirus, Tibrovirus, and Vesiculovirus form together, with about 120 unclassified viruses, the dimarhabdovirus supergroup (dipteran-mammal-associated rhabdovirus), the genus Vesiculovirus including human pathogens and viruses responsible for hemorrhagic disease in fish. Phylogenetic (Figure 2.1) and whole genome analysis of BASV identifies it is a member of the dimarhabdovirus supergroup, most closely related to the genera Ephemerovirus and Tibrovirus.1 Viruses from these genera are usually associated with arthropod and cattle infections but have not previously been known to infect humans. Additionally, the phylogenetic branching pattern and the high amino acid divergence (70%) of BASV suggest it represents a distinct viral group that has yet to be taxonomically classified.
2.3 Virus Description Rhabdoviruses are enveloped viruses with single-stranded negative-sense RNA genomes. Virions are bullet shaped, from 100 to 430 nm long and 45 to 100 nm wide.7,9 The glycoprotein G is anchored in the lipid bilayer and is responsible for virus entry into cells (Figure 2.2A). Viral RNA is encapsulated with the nucleocapsid protein (N). This ribonucleocapsid is associated with the viral polymerase (L) through interaction with the viral phosphoprotein (P). The ribonucleocapsid is packaged with matrix protein (M), which gives the virion its characteristic form.9 The near-complete BASV genome sequence is 11,892 nucleotides long (GenBank accession number JX297815). The genome organization of BASV displays eight open reading frames (ORFs) in the following order (Figure 2.2B): 30 - nucleoprotein (N), phosphoprotein (P), matrix protein (M), U1 protein (U1), U2 protein (U2), glycoprotein (G), U3 protein (U3), and RNA-dependent RNA polymerase (L). The N-P-M-G-L genome organization is common to rhabdoviruses while the insertion of U1, U2, and U3 genes is shared only with members of the genus Tibrovirus. Functions of the proteins U1 and U2 are not known, although U3 protein is hypothesized to be a candidate viroporin.10
3. WHAT IS THE FREQUENCY OF THE DISEASE? To date, BASV has only been found in the Bas-Congo province of Democratic Republic of Congo, Central Africa (Figure 2.3). Infection was confirmed for one surviving patient, who presented with hemorrhagic fever symptoms, and suspected for two other closely epidemiologically linked patients, who died rapidly from fulminant hemorrhagic fever before samples could be collected. Infection by BASV was retrospectively confirmed in
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FIGURE 2.1 Phylogenetic analysis of the L proteins of rabdovirus. Bayesian tree topologies were assessed with MrBayes V.32 software (20,000 sampled trees; 5000 trees discarded as burn-in). Original figure was published by Grard et al.1 under the creative commons license (CC-BY, available at: http://creativecommons.org/licenses/by/3.0/) and slightly modified for taxonomic updates. Virus abbreviations are listed as follows, in alphabetical order: ABLV, Australian bat lyssavirus; ARAV, Aravan virus; BEFV, bovine ephemeral fever virus; BYSMV, barley yellow striate mosaic virus (BYSMV); CHPV, Chandipura virus; CPV, Coastal Plains virus; COCV, cocal virus; DURV, Durham virus; DUVV, Duvenhage virus; EBLV1, European bat lyssavirus 1; EBLV2, European bat lyssavirus 2; EVEX, eel virus European X virus; FLAV, Flanders virus; HIRRV, Hirame rhabdovirus; IHNV, infectious hematopoietic necrosis virus; IRKV, Irkut virus; ISFV, Isfahan virus; KHUV, Khujand virus; LBV, Lagos bat virus; LNYV, lettuce necrotic yellow virus; MARAV, Maraba virus; MMV, maize mosaic virus; MOKV, Mokala virus; MOUV, Moussa virus; NCMV, northern cereal mosaic virus; NGAV, Ngaingan virus; OVRV, Oak Vale rhabdovirus; PFRV, pike fry rhabdovirus; RABV, rabies virus; RYSV, rice yellow stunt rhabdovirus; SIGMAV, sigma virus; SCRV, Siniperca chuatsi rhabdovirus; SHRV, snakehead virus; SMRV, Scophthalmus maximus rhadovirus; SVCV, spring viremia of carp virus; SYNV, Sonchus yellow net virus; TIBV, Tibrogargan virus; TUPV, Tupaia virus; TVCV, tomato vein clearing virus; VHSV, viral hemorrhagic septicemia virus; VSIV, vesicular stomatitis virus, Indiana; VSNJV, vesicular stomatitis virus, New Jersey; WCBV, West Caucasian bat virus; WONGV, Wongabel virus.
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FIGURE 2.2 Panel A. Schematic representation of the rhabdovirus virion structure. Panel B. Schematic representation of the Bas-Congo virus genome organization.
another asymptomatic contact, a nurse who cared directly for the surviving patient, by seroneutralization studies. The proportion of asymptomatic vs. symptomatic infections with severe or possibly mild clinical manifestations is not known. A preliminary epidemiological study that included (1) molecular screening of 50 serum samples from patients presenting with hemorrhagic fever syndrome in DRC and (2) serological screening of 50 plasma samples from blood donors in the Kasai-Orientale province of DRC failed to detect additional cases of BASV infection. These results suggest that human infection with BASV is infrequent or that the virus has only emerged very recently. Larger epidemiological studies and surveillance of active cases of unknown hemorrhagic fever are needed to establish the frequency and impact of BASV-associated disease.
4. HOW IS THE VIRUS TRANSMITTED? To date, the source of infection and the possible ways of transmission have not been established. Waterborne or airborne transmission appears unlikely
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FIGURE 2.3 Map of viral hemorrhagic fever outbreaks in Africa. Yellow fever and dengue fever have a wide geographic distribution throughout sub-Saharan Africa, as well as Rift Valley fever which extends to the south of Africa and are not shown. Mangala village, located in Democratic Republic of the Congo, is represented by a red star. Original figure was published by Grard et al.1 under the creative commons license (CC-BY, available at: http://creativecommons.org/licenses/by/3.0/) and slightly modified.
due to the small number of reported cases. The phylogenetic position of BASV in the dimarhabdovirus supergroup is consistent with a possible arthropod-borne transmission. Additionally, the epidemiological data also suggest potential human-to-human transmission. Indeed, the surviving nurse directly cared for the first two patients who presented with hemorrhagic fever symptoms, and he in turn was taken care of by the serologically confirmed asymptomatic contact. However, none of the household contacts became ill, suggesting that human-to-human transmission may be limited. This putative pattern of transmission for BASV is also seen with CrimeanCongo hemorrhagic fever virus, which is an arboviral VHF agent associated with additional cases of nosocomial human-to-human transmission.11
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5. WHAT ARE THE CLINICAL MANIFESTATIONS? Clinical symptoms shared by all three cases were an abrupt onset of disease, with hemorrhagic manifestations not limited to gastrointestinal sites but also affecting mucosa: epistaxis (nose bleeding), ocular hemorrhage or conjunctival injection (eye bleeding), oral hemorrhage (mouth bleeding), hemorrhagic vomiting, and hemorrhagic diarrhea. Associated unspecific symptoms included one or more of the following: fever, malaise, headache, abdominal pain, fatigue, and anorexia. Notably, fever may be concomitant with early symptoms or appear several days later, potentially explaining why one patient died without documented fever. As revealed by the retrospective serologic study, asymptomatic infection from BASV may occur. In addition, BASV-associated clinical presentations with subclinical, mild, or unrelated symptoms, such as rashes, arthralgias, hepatitis, or neurologic disorders, cannot be excluded at the present time. Many hemorrhagic fever viruses, such as yellow fever12, are associated with a variety of associated clinical manifestations ranging from mild to severe.
6. HOW DO YOU DIAGNOSE? 6.1 Molecular Diagnosis Diagnosis of severe viral hemorrhagic fevers classically includes detection of viral RNA or antigens in serum or blood samples taken during the acute phase of the disease. BASV was first detected by random metagenomic analysis from RNA extracted from a serum sample taken during the acute phase of the disease. Conventional reverse transcription (RT)-PCR and real-time RT-PCR systems were subsequently implemented in the lab for viral RNA detection in human sera and are currently used for diagnostic investigations of suspected VHF cases.
6.2 Viral Isolation Virus isolation is usually the gold standard technique for diagnosis of viral infection. However, as the time of virus cultivation is several days long, this approach is not suitable for emergency detection of highly pathogenic VHF viruses, which require immediate outbreak response and management. With BASV, attempts were made to grow the virus on several cell lines with unsuccessful results: Vero (green monkey kidney), LLC-MK2 (rhesus monkey kidney), BHK (baby hamster kidney cells), CCL-106 (rabbit kidney), and C6/36 (Aedes albopictus mosquito). Since intracerebral inoculation of suckling mice also failed in growing the virus, we assume that the failure in virus cultivation resulted from viral inactivation due to breakdown in cold chain. A recent study of a viral pseudotype carrying the immunogenic BASV glycoprotein in a vesicular stomatitis virus (VSV, genus Vesiculovirus) backbone demonstrated the susceptibility of numerous cell lines including Vero
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and C6/36 to BASV infection in vitro.13 In the absence of samples from additional cases of BASV, recovery of infectious virus may be possible by reverse genetics, as previously performed for rabies virus and VSV.14,15
6.3 Serologic Diagnosis Serological testing for BASV during the acute phase of the disease is not recommended. To date, the kinetics of production of IgM and IgG antibodies relative to onset of symptoms and disease course are not known. In addition, we cannot exclude a limited or impaired humoral immune response in patients with fatal outcome as previously reported for Ebola hemorrhagic fever.16 A seroneutralization assay for BASV using a pseudotyped viral construct is available for epidemiological serosurveys of BASV prevalence13 but is currently not useful for diagnosis.
7. HOW DO YOU DIFFERENTIATE THIS DISEASE FROM SIMILAR ENTITIES? To date, BASV has only been detected in Central Africa where numerous viral hemorrhagic fevers are encountered (Figure 2.3). In this context, other viral etiologies to consider are Ebola and Marburg viruses, Crimean-Congo hemorrhagic fever virus, yellow fever virus, dengue viruses, and Rift Valley fever virus. Lassa and Lujo viruses should also be considered despite currently occurring in a different geographic range. However, the early development of hemorrhagic signs (within the first 2 days of symptoms) seen in BASV infection appears unusual with respect to other VHF agents. Ebola, Marburg, Crimean-Congo, and Lassa hemorrhagic fevers typically begin with a non-specific prodrome that may be confused with the other infections listed in Table 2.1, and hemorrhagic manifestations generally appear by day 3 to 5 after symptom onset.11,1719 In addition to viral hemorrhagic fevers, many other infections are associated with acute febrile disease in tropical and subtropical regions (Table 2.1). The two most frequent infections to exclude are malaria and typhoid fever.20 Gastrointestinal infections by bacteria such as Shigella, Campylobacter, and Salmonella can also be accompanied by bleeding, and are part of the differential diagnosis. Other causes of acute febrile illness frequently encountered in Central Africa include typhus, plague, relapsing fever, leptospirosis, anthrax, and viral hepatitis.19
8. WHAT IS THE THERAPEUTIC APPROACH? To date the therapeutic approach is limited to supportive care.
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TABLE 2.1 BASV Infection Differential Diagnosis Viral infections Hemorrhagic fever viruses Family Filoviridae
Zaire ebolavirus Bundibugyo ebolavirus Tai Forest ebolavirus Sudan ebolavirus Marburg marburgvirus
Family Flaviviridae
Dengue virus Yellow fever virus
Family Bunyaviridae
Crimean-Congo hemorrhagic fever virus Rift Valley fever virus
Family Arenaviridae
Lassa virus Lujo virus
Other viral infections Measles Hemorrhagic varicella Rubella Hemorrhagic smallpox Viral hepatitis Chikungunya fever Parasitic infections Malaria African trypanosomiasis Bacterial infections Gram-negative bacteria Gram-negative bacterial septicemia Shigellose Salmonellose Typhoid fever Septicemic plague Cholera Q fever Meningococcemia Typhus Murin typhus Leptospirose Siphilis Relapsing fever Leptospirosis Gram-positive bacteria Staphylococcal or streptococcal toxic shock syndrome Anthrax Leptospirosis
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9. WHAT ARE THE PREVENTIVE AND INFECTION CONTROL MEASURES? As the source of infection and way(s) of transmission are not clearly established, instructions to prevent and control the infection cannot be precisely defined. However, a minimum of rules should already be considered. Public health authorities must be informed whenever there is VHF suspicion and contact tracing should be engaged as soon as possible. Compliance with the standard guidelines for biosafety and hygiene (use of lab coat, gloves, sharps containers, hand and surface cleaning, etc.) during clinical examination and blood sampling is an absolute requirement to prevent a potential nosocomial transmission. An aerosol transmission seems unlikely but use of masks and face shields should be considered in light of the precautionary principle and would provide an additional protection in the event of accidental exposure to body fluids. The scientific data do not allow a definitive and obvious risk assessment, so that prevention and control measures are a sensitive point with strong ethical concerns. Going beyond the minimal set of rules proposed here, in considering stronger isolation measures for patient management for example, must be collectively discussed with additional medical experts, including those experienced in the management of VHF outbreaks in the field, to reach a consensus on solid and balanced recommendations.
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