Dose Of Rabies Virus Research Articles

Experimental infection of Artibeus intermedius with a vampire bat rabies virus.

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n=35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n=3), stomach (n=1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.

A novel rabies vaccine based on the chimpanzee adenoviral vector AdC68

Objective To investigate the genetic stability, immunogenicity and protective efficacy of AdC68-rab.gp, a novel rabies vaccine based on the replication-defective chimpanzee adenoviral vector AdC68-ept. Methods The recombinant adenovirus AdC68-rab.gp expressing the glycoprotein of rabies virus ERA strain was constructed. Genomes of the AdC68-rab.gp of different generations were extracted and analyzed. HEK293 and Huh7 cells were infected with the AdC68-rab.gp of different generations. ICR mice were immunized with the AdC68-rab.gp and blood samples were collected 4 weeks or 6 months after immunization. Rapid fluorescent focus inhibition test (RFFIT) was performed to detect the neutralizing antibody against rabies virus in mice serum samples. ICR mice were challenged with lethal dose of rabies virus 4 weeks after the immunization with AdC68-rab.gp to evaluate the protective efficacy of AdC68-rab.gp. Results The genome of AdC68-rab.gp was stable after 15 passages, which was identical to that of the 5th and 1st generations. High levels of neutralizing antibody against rabies virus in serum samples were detected in mice immunized with AdC68-rab.gp and maintained for a long period of time. Immunization mice with one dose of AdC68-rab.gp could protect all mice from the lethal dose challenge of rabies virus. Conclusion The novel AdC68-rab.gp was characterized by good genetic stability and ideal protective efficacy. The adenoviral vector based vaccine could be further developed as a potential candidate for the substitute of current rabies vaccine. Key words: Chimpanzee adenoviral vector; Novel rabies vaccine; Immune response

A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

Donor-Derived Infections With Central Nervous System Pathogens After Solid Organ Transplantation

During the past decade, numerous instances have been reportedofdonor-derived infectionamongrecipientsof solidorgan transplants with pathogens associated with central nervous system (CNS) infections.1-7 Many cases have received significant attention both among the transplant community and the laymedia.Notablepathogenshave includedWestNile virus (WNV), Balamuthia mandrillaris, lymphocytic choriomeningitis virus, and rabies virus. In most cases, donorderived infection was suspected only after symptoms developed in recipients and further testing of archived specimens identified a donor source. In some cases, lack of recipient exposure to the pathogenprompted investigation for donor origin of the infection. Recipient outcomes were often poor; in part because even after identification of infection, effective treatment was not available. In this issueof JAMA, Vora andcolleagues8describe a case of donor-derived rabies in a recipient of a kidney transplant. The investigationwas prompted by the death from rabies of a recipient 18 months after transplant in the absence of exposures.Adetailed inquiry ledbyCenters forDiseaseControl and Prevention investigators confirmeddonor origin of the recipient’s infectionwith a raccoon rabies virus. The report contains a number of unexpected clinical features. Despite the immunosuppressed state of all 4 organ recipients, the affected patient remained asymptomatic for 18months after transplantation, and the 3 other patients did not develop rabies. The authors speculate that either a low dose of rabies virus or differential behavioral of the raccoon rabies variant explains these clinical features. Although the epidemiology and pathogenesis considerations are interesting, the most notable aspect of this report surrounds the implications for improving the safety of solid organ transplantation. Combined with previous cases,1-7 this report highlights a pattern of donor-derived infection caused by unidentified (at the time of procurement) donor infection with pathogens associated with CNS infection. Reducing the riskofdonor-derived infection requiresunderstanding thecurrent system of screening potential donors. Policy governing solid organ transplantation is established by the Organ Procurement and Transplantation Network (OPTN). Assessment of deceased donors for the risk of transmissible disease relies on amultipronged approach. The organ procurement organization (OPO)managing the process obtains amedical and social history from individuals familiar with the donor. Information that might reasonably be expected to affect potential recipients (eg, a history of recent intravenous drug usewould increase risk of acute viral hepatitis or human immunodeficiencyvirus [HIV] infection thatmightnotbedetectedon routine serologic donor screening tests) must be communicated to transplant programs.9 Limitations include the availability of friends and family and their familiaritywith thedonor’s exposures and behaviors. In addition, specific laboratory testingmust beperformedonall deceaseddonors. Required tests include serological testing forHIV, hepatitis C virus, hepatitis B virus, syphilis, cytomegalovirus, and Epstein-Barr virus as well as blood and urine cultures. At the time of a potential recipient being offered an organ for transplantation, the accepting transplant center must, in a time-pressured environment, integrate all this information, including the medical urgency of the potential recipient, andmake a decision regarding whether to accept the organs. Accepting centers necessarily rely on the clinical impression of treating physicians. For situations in which an increased riskof transmissible infection is present, specific informed consent must be obtained from the potential recipient.10 As an added safeguard, OPOs are required to report additional information that becomesavailable after transplant (eg, autopsy results) that might affect recipients. Furthermore, transplant centers must report any recipient events suggestive of donor-derived infection, thereby allowing notification of other centers that received organs from the same donor.For example, if a recipientwithnootherknownexposures is newly diagnosedwith hepatitis C after transplantation, the event should be reported to the OPTN. In the case described by Vora and colleagues,8 suspected donor-derived rabieswas reported to theOPTN, allowing rapidnotificationof the transplant centers caring for other recipients andadministrationof postexposure rabiesprophylaxis.AnOPTNcommittee, theDisease Transmission Advisory Committee (DTAC), reviews all such reports anddecideswhether theevent resulted indonorderived disease. Data are aggregated andused to guide policy development and educational efforts within the transplant community. Under the current system, episodes of unexpected donor-derived infection are rare. During 2008-2011, 113 622 patients received solid organ transplants from 57 488 donors.Donor-deriveddisease (infectionandmalignancy)developed in139 recipients (0.1%) resulting in29deaths (0.03%).11 A relevant comparison to make is that, during the same period, 26 852peoplediedwaiting for anorgan transplantation.12 Despite these safeguards, reports of CNS infection transmissionmandate consideration ofwhatmight be done to furRelated article page 398 Opinion

Glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice

Induction of apoptosis by rabies virus (RV) has been reported to be associated with the expression of the glycoprotein (G), but inversely correlated with pathogenicity. To further delineate the association between the expression of the G and the induction of apoptosis, recombinant RVs with replacement of only the G gene were used to infect mice by the intracerebral route. Recombinant viruses expressing the G from attenuated viruses expressed higher level of the G and induced more apoptosis in mice than recombinant RV expressing the G from wild-type (wt) or pathogenic RV, demonstrating that it is the G gene that determines the level of G expression and, consequently, the induction of apoptosis. Likewise, recombinant viruses expressing the G from wt or pathogenic RV are more pathogenic in mice than those expressing G from attenuated RV, confirming the inverse correlation between RV pathogenicity and the induction of apoptosis. To investigate the mechanism by which induction of apoptosis attenuates viral pathogenicity, mice were infected with wt or attenuated RV by the intramuscular route. It was found that low doses of attenuated RV induced apoptosis in the spinal cord and failed to spread to the brain or produce neurological disease. On the other hand, apoptosis was not observed in the spinal cord of mice infected with the same doses of wt RV and the virus spread to various parts of the brain and induced fatal neurologic disease. These results suggest that glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system (CNS) of mice.