Brian H. Bird

Rift Valley fever virus (Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention

Rift Valley fever (RVF) virus is an arbovirus in the Bunyaviridae family that, from phylogenetic analysis, appears to have first emerged in the mid-19th century and was only identified at the begininning of the 1930s in the Rift Valley region of Kenya. Despite being an arbovirus with a relatively simple but temporally and geographically stable genome, this zoonotic virus has already demonstrated a real capacity for emerging in new territories, as exemplified by the outbreaks in Egypt (1977), Western Africa (1988) and the Arabian Peninsula (2000), or for re-emerging after long periods of silence as observed very recently in Kenya and South Africa. The presence of competent vectors in countries previously free of RVF, the high viral titres in viraemic animals and the global changes in climate, travel and trade all contribute to make this virus a threat that must not be neglected as the consequences of RVF are dramatic, both for human and animal health. In this review, we present the latest advances in RVF virus research. In spite of this renewed interest, aspects of the epidemiology of RVF virus are still not fully understood and safe, effective vaccines are still not freely available for protecting humans and livestock against the dramatic consequences of this virus.

Rift Valley fever virus

Vet Med Today: Zoonosis Update 883 R Valley fever virus is a mosquito-borne pathogen of livestock and humans that historically has been responsible for widespread and devastating outbreaks of severe disease throughout Africa and, more recently, the Arabian Peninsula. The virus was first isolated and RVF disease was initially characterized following the sudden deaths (over a 4-week period) of approximately 4,700 lambs and ewes on a single farm along the shores of Lake Naivasha in the Great Rift Valley of Kenya in 1931. Since that time, RVF virus has caused numerous economically devastating epizootics that were characterized by sweeping abortion storms and mortality ratios of approximately 100% among neonatal animals and of 10% to 20% among adult ruminant livestock (especially sheep and cattle). Infections in humans are typically associated with selflimiting febrile illnesses. However, in 1% to 2% of affected individuals, RVF infections can progress to more severe disease including fulminant hepatitis, encephalitis, retinitis, blindness, or a hemorrhagic syndrome; among severely affected persons who are hospitalized, the case fatality ratio is approximately 10% to 20%. Rift Valley fever epizootics and epidemics can rapidly overwhelm the capacities of the public health and veterinary medical communities to provide rapid diagnostic testing and adequate medical care for affected humans and other animals, which can number in the tens if not hundreds of thousands. Veterinarians, other health personnel, farmers, and abattoir workers also are at high risk of infection from direct contact with infected animals and patients; indeed, many historical outbreaks of RVF disease in Africa were initially detected because of illnesses among veterinarians and their assistants after they performed necropsies on infected animals. In 2008, several veterinarians, staff, and veterinary students at a South African veterinary college were infected after handling and performing necropsies Rift Valley fever virus

Marburg virus infection detected in a common African bat.

Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80–90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat (Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.

Complete Genome Analysis of 33 Ecologically and Biologically Diverse Rift Valley Fever Virus Strains Reveals Widespread Virus Movement and Low Genetic Diversity due to Recent Common Ancestry

ABSTRACT Rift Valley fever (RVF) virus is a mosquito-borne RNA virus responsible for large explosive outbreaks of acute febrile disease in humans and livestock in Africa with significant mortality and economic impact. The successful high-throughput generation of the complete genome sequence was achieved for 33 diverse RVF virus strains collected from throughout Africa and Saudi Arabia from 1944 to 2000, including strains differing in pathogenicity in disease models. While several distinct virus genetic lineages were determined, which approximately correlate with geographic origin, multiple exceptions indicative of long-distance virus movement have been found. Virus strains isolated within an epidemic (e.g., Mauritania, 1987, or Egypt, 1977 to 1978) exhibit little diversity, while those in enzootic settings (e.g., 1970s Zimbabwe) can be highly diverse. In addition, the large Saudi Arabian RVF outbreak in 2000 appears to have involved virus introduction from East Africa, based on the close ancestral relationship of a 1998 East African virus. Virus genetic diversity was low (∼5%) and primarily involved accumulation of mutations at an average of 2.9 × 10−4 substitutions/site/year, although some evidence of RNA segment reassortment was found. Bayesian analysis of current RVF virus genetic diversity places the most recent common ancestor of these viruses in the late 1800s, the colonial period in Africa, a time of dramatic changes in agricultural practices and introduction of nonindigenous livestock breeds. In addition to insights into the evolution and ecology of RVF virus, these genomic data also provide a foundation for the design of molecular detection assays and prototype vaccines useful in combating this important disease.

Rift Valley Fever Virus Lacking the NSs and NSm Genes Is Highly Attenuated, Confers Protective Immunity from Virulent Virus Challenge, and Allows for Differential Identification of Infected and Vaccinated Animals

ABSTRACT Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ∼1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 × 104 PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, ∼1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.

Batai and Ngari Viruses: M Segment Reassortment and Association with Severe Febrile Disease Outbreaks in East Africa

ABSTRACT Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.

Inhibition of IRF-3 Activation by VP35 Is Critical for the High Level of Virulence of Ebola Virus

ABSTRACT Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

Multiple Virus Lineages Sharing Recent Common Ancestry Were Associated with a Large Rift Valley Fever Outbreak among Livestock in Kenya during 2006-2007

ABSTRACT Rift Valley fever (RVF) virus historically has caused widespread and extensive outbreaks of severe human and livestock disease throughout Africa, Madagascar, and the Arabian Peninsula. Following unusually heavy rainfall during the late autumn of 2006, reports of human and animal illness consistent with RVF virus infection emerged across semiarid regions of the Garissa District of northeastern Kenya and southern Somalia. Following initial RVF virus laboratory confirmation, a high-throughput RVF diagnostic facility was established at the Kenyan Central Veterinary Laboratories in Kabete, Kenya, to support the real-time identification of infected livestock and to facilitate outbreak response and control activities. A total of 3,250 specimens from a variety of animal species, including domesticated livestock (cattle, sheep, goats, and camels) and wildlife collected from a total of 55 of 71 Kenyan administrative districts, were tested by molecular and serologic assays. Evidence of RVF infection was found in 9.2% of animals tested and across 23 districts of Kenya, reflecting the large number of affected livestock and the geographic extent of the outbreak. The complete S, M, and/or L genome segment sequence was obtained from a total of 31 RVF virus specimens spanning the entire known outbreak period (December-May) and geographic areas affected by RVF virus activity. Extensive genomic analyses demonstrated the concurrent circulation of multiple virus lineages, gene segment reassortment, and the common ancestry of the 2006/2007 outbreak viruses with those from the 1997-1998 east African RVF outbreak. Evidence of recent increases in genomic diversity and effective population size 2 to 4 years prior to the 2006-2007 outbreak also was found, indicating ongoing RVF virus activity and evolution during the interepizootic/epidemic period. These findings have implications for further studies of basic RVF virus ecology and the design of future surveillance/diagnostic activities, and they highlight the critical need for safe and effective vaccines and antiviral compounds to combat this significant veterinary and public health threat.

Highly Sensitive and Broadly Reactive Quantitative Reverse Transcription-PCR Assay for High-Throughput Detection of Rift Valley Fever Virus

ABSTRACT Rift Valley fever (RVF) virus is a mosquito-borne virus associated with large-scale epizootics/epidemics throughout Africa and the Arabian peninsula. Virus infection can result in economically disastrous “abortion storms” and high newborn mortality in livestock. Human infections result in a flu-like illness, with 1 to 2% of patients developing severe complications, including encephalitis or hemorrhagic fever with high fatality rates. There is a critical need for a highly sensitive and specific molecular diagnostic assay capable of detecting the natural genetic spectrum of RVF viruses. We report here the establishment of a pan-RVF virus quantitative real-time reverse transcription-PCR assay with high analytical sensitivity (∼5 RNA copies of in vitro-transcribed RNA/reaction or ∼0.1 PFU of infectious virus/reaction) and efficiency (standard curve slope = −3.66). Based on the alignments of the complete genome sequences of 40 ecologically and biologically diverse virus isolates collected over 56 years (1944 to 2000), the primer and probe annealing sites targeted in this assay are known to be located in highly conserved genomic regions. The performance of this assay relative to serologic assays is illustrated by testing of known RVF case materials obtained during the Saudi Arabia outbreak in 2000. Furthermore, analysis of acute-phase blood samples collected from human patients (25 nonfatal, 8 fatal) during that outbreak revealed that patient viremia at time of presentation at hospital may be a useful prognostic tool in determining patient outcome.

Rift Valley Fever Virus Vaccine Lacking the NSs and NSm Genes Is Safe, Nonteratogenic, and Confers Protection from Viremia, Pyrexia, and Abortion following Challenge in Adult and Pregnant Sheep

ABSTRACT Rift Valley fever virus (RVFV) is a mosquito-borne human and veterinary pathogen causing large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Safe and effective vaccines are critically needed, especially those that can be used in a targeted one-health approach to prevent both livestock and human disease. We report here on the safety, immunogenicity, and efficacy of the ΔNSs-ΔNSm recombinant RVFV (rRVFV) vaccine (which lacks the NSs and NSm virulence factors) in a total of 41 sheep, including 29 timed-pregnant ewes. This vaccine was proven safe and immunogenic for adult animals at doses ranging from 1.0 × 103 to 1.0 × 105 PFU administered subcutaneously (s.c.). Pregnant animals were vaccinated with 1.0 × 104 PFU s.c. at day 42 of gestation, when fetal sensitivity to RVFV vaccine-induced teratogenesis is highest. No febrile reactions, clinical illness, or pregnancy loss was observed following vaccination. Vaccination resulted in a rapid increase in anti-RVFV IgM (day 4) and IgG (day 7) titers. No seroconversion occurred in cohoused control animals. A subset of 20 ewes progressed to full-term delivery after vaccination. All lambs were born without musculoskeletal, neurological, or histological birth defects. Vaccine efficacy was assessed in 9 pregnant animals challenged at day 122 of gestation with virulent RVFV (1.0 × 106 PFU intravenously). Following challenge, 100% (9/9) of the animals were protected, progressed to full term, and delivered healthy lambs. As expected, all 3 sham-vaccinated controls experienced viremia, fetal death, and abortion postchallenge. These results demonstrate that the ΔNSs-ΔNSm rRVFV vaccine is safe and nonteratogenic and confers high-level protection in sheep.