Animal models of Rift Valley fever virus infection

Emerging and naturally occurring infectious diseases from bacterial and viral sources are constantly threatening humans and livestock. Recently, a variety of infectious diseases have emerged into previously disease-free areas, resulting in new epidemics. Consequently, governmental agencies and researchers in the area of biomedical research have started designing ways to prevent their further spread. Ongoing research activities are focused on developing therapeutic and prophylactic interventions against these emerging infections. Development and evaluation of vaccines, diagnostics and treatments often depend on the development of appropriate animal models to determine the efficacy of new therapeutic agents. In addition, animal models are necessary to understand the basic pathobiology of infection. In this minireview, the current animal models used for one of these emerging infectious diseases, Rift Valley fever virus (RVFV), and the specifics of infection and pathology associated with each model are discussed.     

Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins

The genomic M RNA segment of Rift Valley fever virus is transcribed to produce a single mRNA with multiple translation initiation sites. The products of translation are an N-terminal nested series of polyproteins. These polyproteins enter the secretory system of the host cell and are proteolytically processed to yield the mature virion glycoproteins, Gn and Gc, and two non-structural glycoproteins. By means of pulse-chase immune precipitation experiments we identify the Gn and Gc precursor molecules and also show that signal peptidase cleavage is required for mature Gn and Gc production. We also demonstrate that a hydrophobic domain at the N-terminus of Gn acts as a signal peptide only in the context of the polyprotein precursors that initiate at the second, fourth or fifth AUGs. In addition, we document that formation of Gn/Gc heteromeric complexes occur rapidly (<5 min) and can occur prior to signal peptidase processing of Gn, suggesting that this complex forms in the endoplasmic reticulum. Interestingly, Gc can form a complex with a glycoprotein that has been considered nonstructural, a discovery that has implications for both the topology and potential packaging of this glycoprotein.     

Rift Valley fever virus lacking NSm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease

Rift Valley fever virus is a significant human and veterinary pathogen responsible for explosive outbreaks throughout Africa and the Arabian Peninsula. Severe acute disease in humans includes rapid onset hepatic disease and hemorrhagic fever or delayed onset encephalitis. A highly efficient reverse genetics system was developed which allowed generation of recombinant RVF viruses to assess the role of NSm protein in virulence in a rat model in which wild-type RVF virus strain ZH501 (wt-ZH501) results in 100% lethal hepatic disease 2-3 days post infection. While extensive genomic analysis indicates conservation of the NSm coding capability of diverse RVF viruses, and viruses deficient in NSs proteins are completely attenuated in vivo, comparison of wt-ZH501, a reverse genetics generated wt-ZH501 virus (R-ZH501), and R-ZH501 virus lacking the NSm proteins (R-DeltaNSm-ZH501) demonstrated that the NSm proteins were nonessential for in vivo virulence and lethality. Surprisingly, while 44% of R-DeltaNSm-ZH501 infected animals quickly developed lethal hepatic disease similar to wt- and R-ZH501, 17% developed delayed onset neurologic disease (lethargy, head tremors, and ataxia) at 13 days post infection. Such infections may provide the basis for study of both RVF acute hepatic disease and delayed onset encephalitic disease in humans.     

Morphology and development of Rift Valley fever virus in Vero cell cultures

Rift Valley fever virus (RVFV) grown in vero cell cultures has a completed replication cycle within 13 hours. The first signs are the appearance of intranuclear fibrillar rods, followed by aggregations of precursor viral material in host cell cytoplasm and viral nucleocapsids budding into vacuoles associated with the Golgi apparati. Mature particles, liberated by the disintegration of vero cells, contained ribosomelike structures within the nucleocapsid, which was surrounded by a typical unit membrane through which were inserted some 350-375 surface spikes whose inner ends were incorporated into the nucleocapsid structure. In the negatively stained material, the overall diameter of the virion was 90-110 nm; the spikes were 10-18 nm in length and 5 nm in diameter.     

Rift valley fever virus infection in man in Nigeria

Recently, Rift Valley fever virus (RVFV) disease has assumed public health significance as associated with human fatalities on a significant scale both in the North, Central and South of Africa. In West Africa, little information is available about RVFV disease. However, in Nigeria, four strains of the virus have been isolated from sheep, culicoides, and mosquitoes; also, RVFV antibodies were demonstrated in some domestic animal sera. The West African dwarf sheep, which is indigenous to Nigeria, is highly suspectible to experimental RVFV infection. No information is available about RVFV disease in man. Human blood and serum samples collected from different parts of Nigeria were examined for RVFV and its specific antibodies using mouse inoculation technique, complement fixation (CF), and neutralization (N) tests. In addition, a limited number of hospital records of suspected RVFV cases were examined. No RVFV was isolated from 19,026 samples examined for virus content. Of the 2,223 sera tested, 146 or 6.6% were positive for RVFV antibodies by the CF test. There was a good correlation between the CF and N tests, in RVFV serodiagnosis. One hundred forty-four of the 146 CF-positive sera were positive in N tests; in addition, all of the 50 CF-negative sera were negative in N tests for RVFV antibodies. From the limited hospital records examined, it appears that RVFV infection in Nigeria presents as an influenza-like illness. On one occasion, there was ocular complication following a febrile illness, associated with the development of specific RVFV antibodies. With less than 10% of the surveyed population immune to RVFV, the potentials for a severe eqizootic of RVFV disease in Nigeria appear great. Further studies on such factors as climate and vector population, as well as the reservoir and host range of RVFV, are in progress.     

Single-particle cryo-electron microscopy of Rift Valley fever virus

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T = 12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.     

Punta Toro virus (Bunyaviridae, Phlebovirus) infection in mice: Strain differences in pathogenesis and host interferon response

The Adames strain of Punta Toro virus (PTV-A, Bunyaviridae, Phlebovirus) causes an acute lethal disease in hamsters and mice. The Balliet strain of the virus (PTV-B) is generally considered to be avirulent. The difference in hamster susceptibility is likely due to the ability of PTV-A to suppress interferon (IFN)-β similarly to that described for Rift Valley fever virus. Here we investigated strain differences in PTV pathogenesis and the IFN response in mice. Although PTV-B infection in mice did not induce systemic IFN-β release, primary macrophages produced dramatically higher levels when exposed to the virus in culture. The importance of IFN in resistance to PTV infection was borne out in studies employing STAT-1 knock-out mice. Also, a number of genes specific to IFN response pathways were upregulated in PTV-B-infected macrophages. Our findings provide new insights into the type I IFN response during PTV infection in the mouse model of phleboviral disease.     

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10^6.5 plaque forming units/ml, or up to 10¹⁰ viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.     

Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments

Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries. A number of single-gene target amplification assays have been developed for the rapid detection of RVF viral RNA. This paper describes the development of an improved amplification assay that includes two confirmatory target RNA segments (L and M) and a third target gene, NSs, which is deleted in the Clone 13 commercial vaccine and other candidate vaccines. The assay also contains an exogenous RNA control added during the PCR setup for detection of amplification inhibitors. The assay was evaluated initially with samples from experimentally infected animals, after which clinical veterinary and human samples from endemic countries were tested for further evaluation. The assay has a sensitivity range of 66.7–100% and a specificity of 92.0–100% depending on the comparison. The assay has an overall sensitivity of 92.5%, specificity of 95% and a positive predictive value of 98.7%. The single-tube assay provides confirmation of the presence of RVFV RNA for improved confidence in diagnostic results and a “differentiate infected from vaccinated animals” (DIVA) – compatible marker for RVFV NSs – deleted vaccines, which is useful for RVF endemic countries, but especially important in non-endemic countries.     

A replication-incompetent Rift Valley fever vaccine: Chimeric virus-like particles protect mice and rats against lethal challenge

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases.Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G_N and G_C, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates.Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.     

The pathogenesis of Rift Valley fever virus in the mouse model

Detailed studies describing the pathogenesis of Rift Valley fever (RVF) virus (RVFV) in the mouse model are lacking. A fully characterized small animal model of RVF is needed to evaluate potential vaccines and therapeutics. In this study, we characterized the pathogenesis of RVFV throughout the disease course in mice. Infection produced high-titer viremia and demonstrated RVFV tropism for a variety of tissue and individual cell types. Overwhelming infection of hepatocytes, accompanied by apoptosis, was a major consequence of infection. The majority of mice died or were euthanatized between days 3 and 6 postinfection with severe hepatitis. The remaining mice effectively cleared virus from the liver and blood, but exhibited neuroinvasion and developed panencephalitis. In addition, we characterized a number of other virological, clinicopathological, and histopathological features of RVFV infection in mice. The mouse model therefore mimics both the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF.     

Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus

The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and global changes in climate, travel and trade have increased the risk of RVF spreading to new regions and have emphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks. Highly sensitive viral detection systems like PCR have a limited use during outbreaks because of the short duration of viraemia, whereas antibodies like specific IgM which are serological indicators of acute infection, can be detected for up to 50 days after infection. Using the highly conserved and immunogenic recombinant nucleoprotein of RVF virus in an IgM capture ELISA, the risk of laboratory infection associated with traditional serological methods is avoided. The use of pre-coated/pre-blocked ELISA plates and the conjugation of the recombinant nucleoprotein with horseradish peroxidase simplified and shortened the assay procedure. Results showed the assay to be highly reproducible with a lower detection limit equal to that of a commercial competition ELISA. By receiver operating characteristic (ROC) curve analysis the area under curve (AUC) index was determined as 1.0 and the diagnostic sensitivity and specificity at a PP cut-off value of 4.1 as 100% and 99.78% respectively. The results of this study demonstrated that the IgM capture ELISA is a safe, reliable and highly accurate diagnostic tool which can be used on its own or in parallel with other methods for the early diagnosis of RVF virus infection and also for monitoring of immune responses in vaccinated domestic ruminants.     

Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA

This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation (R² = 0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection.     

Rift Valley fever virus inhibits a pro-inflammatory response in experimentally infected human monocyte derived macrophages and a pro-inflammatory cytokine response may be associated with patient survival during natural infection

Rift Valley fever virus (RVFV) causes significant morbidity and mortality in humans and livestock throughout Africa and the Middle East. The clinical disease ranges from mild febrile illness, to hepatitis, retinitis, encephalitis and fatal hemorrhagic fever. RVFV NSs protein has previously been shown to interfere in vitro with the interferon response, and RVFV lacking the NSs protein is attenuated in several animal models. Monocytes and macrophages are key players in the innate immune response via expression of various cytokines and chemokines. Here we demonstrate that wild-type RVFV infection of human monocyte-derived macrophages leads to a productive infection and inhibition of the innate immune response via decreased expression of IFN-α2, IFN-β and TNF-α. Using a recombinant virus lacking the NSs protein, we show that this effect is mediated by the viral NSs protein. Finally, analysis of RVF patient samples demonstrated an association between a pro-inflammatory cytokine response and patient survival.     

Emerging Infections of CNS: Avian Influenza A Virus,Rift Valley Fever Virus and Human Parechovirus

History is replete with emergent pandemic infections that have decimated the human population. Given the shear mass of humans that now crowd the earth, there is every reason to suspect history will repeat itself. We describe three RNA viruses that have recently emerged in the human population to mediate severe neurological disease. These new diseases are results of new mutations in the infectious agents or new exposure pathways to the agents or both. To appreciate their pathogenesis, we summarize the essential virology and immune response to each agent. Infection is described in the context of known host defenses. Once the viruses evade immune defenses and enter central nervous system (CNS) cells, they rapidly co‐opt host RNA processing to a cataclysmic extent. It is not clear why the brain is particularly susceptible to RNA viruses; but perhaps because of its tremendous dependence on RNA processing for physiological functioning, classical mechanisms of host defense (eg, interferon disruption of viral replication) are diminished or not available. Effectiveness of immunity, immunization and pharmacological therapies is reviewed to contextualize the scope of the public health challenge. Unfortunately, vaccines that confer protection from systemic disease do not necessarily confer protection for the brain after exposure through unconventional routes.     

The CD2v protein enhances African swine fever virus replication in the tick vector, Ornithodoros erraticus

The NH/P68 non-haemadsorbing (non-HAD) African swine fever virus (ASFV) isolate contains frameshift mutations in the EP402R and adjacent EP153R genes. These encode, respectively, the protein (CD2v) that is required for the haemadsorption (HAD) of swine erythrocytes to ASFV-infected cells and a C-type lectin protein. Two recombinant HAD viruses were constructed in this parental strain. In one of these the intact EP153R gene sequence was restored. Although restoration of the HAD phenotype did not increase virus virulence in pigs, a significant increase was observed in the number of pigs which developed viraemia. These HAD recombinant viruses replicated to titres approximately 1000-fold higher than the parental non-HAD isolate when membrane fed to Ornithodoros erraticus ticks. Inoculation of the non-HAD isolate across the gut wall increased viral replication to levels comparable to that of the HAD recombinant viruses. These results demonstrate a novel role for the CD2v protein in virus replication in ticks.     

Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection

Although antibody-mediated immune mechanisms have been shown to be important in immunity to ASF, it remains unclear what role virus neutralizing antibodies play in the protective response. Virus neutralizing epitopes have been identified on three viral proteins, p30, p54, and p72. To evaluate the role(s) of these proteins in protective immunity, pigs were immunized with baculovirus-expressed p30, p54, p72, and p22 from the pathogenic African swine fever virus (ASFV) isolate Pr4. ASFV specific neutralizing antibodies were detected in test group animals. Following immunization, animals were challenged with 10(4) TCID(50) of Pr4 virus. In comparison to the control group, test group animals exhibited a 2-day delay to onset of clinical disease and reduced viremia levels at 2 days postinfection (DPI); however, by 4 DPI, there was no significant difference between the two groups and all animals in both groups died between 7 and 10 DPI. These results indicate that neutralizing antibodies to these ASFV proteins are not sufficient for antibody-mediated protection.     

血清乙肝病毒外膜大蛋白检测及其与病毒复制的关系

目的探讨血清乙型肝炎病毒外膜大蛋白（LHBs）检测对于判定乙型肝炎病毒（HBV）复制的临床意义。方法分别采用ELISA法、时间分辨免疫荧光分析法和实时荧光定量PCR法检测340份慢性乙型肝炎患者血清中LHBs、Pre-S1蛋白、HBV-M和HBVDNA，并进行相关性分析。结果340份慢性乙型肝炎患者血清中LHBs水平与HBVDNA拷贝数变化相一致，两者呈正相关（r=0．899。P=0．038）；在不同模式的HBeAg血清中，LHBs与HBVDNA的阳性率差异均无统计学意义（P均〉0．05）；HBV DNA阳性血清中LHBs阳性率（83．15％）明显高于Pre-S1蛋白和HBeAg的阳性率（50．54％和54．48％），差异有统计学意义（P均〈0．05）。结论血清LHBs水平能反映HBV感染者体内HBV复制程度，其灵敏度高于Pre-S1蛋白和HBeAg，可作为判断HBV复制新的血清学指标。