Increased pathogenicity of rabies virus due to modification of a non-coding region.

Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV.

Rabies vaccination at a virus-inoculated site as an alternative option to rabies immunoglobulin.

Combined active and passive immunization has been established to be an optimal strategy for rabies post-exposure prophylaxis (PEP). Prompt administration of vaccine and rabies immunoglobulin (RIG) can reliably prevent the disease. However, RIG is unavailable and unaffordable in the majority of cases. On the basis of a model experiment using hamsters, we demonstrated that vaccine injection at the wound site in the same manner as administration of RIG provided protective efficacy that was not inferior to the current optimal PEP, a combination of vaccination and RIG. Further study is needed to determine whether it can replace the use of RIG.

Rabies neutralizing antibody after 2 intradermal doses on days 0 and 21 for pre-exposure prophylaxis.

Pre-exposure prophylaxis is recommended for people who will be exposed to rabies virus in the laboratory or who will contact with mammals. World Health Organization recommends 2 doses of a cell-culture rabies vaccine given 1 week apart, and a third booster dose given 2-3 weeks later. Neutralizing antibody response is virtually 100%, and the individual remains sensitized indefinitely. Intradermal pre-exposure regimen for rabies prophylaxis is more economical compared with the conventional intramuscular regimen in terms of vaccine volume. However, both regimens require three clinic visits. In order to reduce non-medical expenses such as transportation to the clinics and to increase compliance, the immunogenicity and safety of two-visit intradermal regimen for pre-exposure prophylaxis were studied. Fifty-five healthy subjects aged between 18 and 24 years were enrolled and divided into two groups. Group A (n=39) received 0.1 ml of purified Vero cell rabies vaccine (PVRV; Sanofi Pasteur, Lyon, France; Lot no. Z0996 with an antigenic value of 4.8 IU/0.5 ml vial) intradermally each at two sites on days 0 and 21. Group B (n=16) received 0.5 ml of PVRV intramuscularly on days 0, 7 and 21, as conventional intramuscular regimen for pre-exposure prophylaxis. Rabies neutralizing antibody (Nab) titers were measured on days 0, 35, 365 and 379 (14 days after simulated post-exposure booster vaccination). All subjects from two groups had Nab titers ≥0.5 IU/ml on day 35. In addition, the difference between geometric mean titers for group A (4.51 IU/ml; range of Nab titers 1.69-13.0 IU/ml) and group B (6.74 IU/ml; range of Nab titers 2.20-14.23 IU/ml) on day 35 was not statistically significant (p>0.05). One year after pre-exposure vaccination, all subjects in both groups received simulated post-exposure booster vaccination with 0.1 ml of PVRV ID on days 0 and 3 (day 365 and day 368 after pre-exposure vaccination). After simulated booster vaccinations with 0.1 ml PVRV ID on days 0 and 3, all subjects in groups A (GMT 14.38 IU/ml; range 2.99-308.44 IU/ml) and in group B (GMT 14.06 IU/ml; range 3.12-62.09 IU/ml) had rabies Nab titers ≥0.5 IU/ml on day 14 post-booster (p>0.05). Mild local adverse events such as pain at injection site, pruritus and erythema were observed. Our study indicated that 2-site intradermal pre-exposure regimen on days 0 and 21 with 0.1 ml of cell-culture rabies vaccine is safe and immunogenic as the conventional intramuscular regimen.

Molecular analysis of the mutational effects of Thai street rabies virus with increased virulence in mice after passages in the BHK cell line.

QS-BHK-P7, street rabies virus, after passages in the BHK cell line, had an in vitro phenotype that distinguished it from its parental virus. Both viruses caused lethal infection in mice by central nervous system inoculation; however, only QS-BHK-P7 killed mice by the intramuscular route. We found four mutations, S23R and H424P in ectodomain of the glycoprotein (G), I1711 V in the polymerase genes, and another at the non-coding region between the phosphoprotein and matrix protein genes of QS-BHK-P7. None of the mutations in the G gene occurred in previously reported pathogenic determinants. The roles of mutations in particular non-coding regions remain to be elucidated.

Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs.

Using the principle of immunochromatography, we previously developed a method called RAPINA (Rapid Neutralizing Antibody detection test) that can measure the level of rabies virus -neutralizing antibody (VNA) in serum samples [Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods 2009;161:58-62]. RAPINA is faster, simpler, and easier to perform compared with a virus-neutralizing test or enzyme-linked immunosorbent assay (ELISA). The improved version of RAPINA has greater positive and negative predictive values corresponding to a VNA level of 0.5 IU/mL, as recommended by the World Health Organization and the World Organization for Animal Health. To verify the efficacy of this improved method, serum samples were collected from humans and dogs before and after immunization against rabies and were tested in Japan, Sri Lanka, and Thailand. The results were compared between RAPINA and the true VNA levels measured by the Rapid Fluorescent Focus Inhibition Test (RFFIT). The improved RAPINA accurately predicted seropositivity for 182 of 183 seropositive human samples as assessed by RFFIT (99.5%) and for 138 of 140 RFFIT-negative human samples (98.6%). In dog serum samples, the positive and negative predictive values were 99.7% (345/355) and 95.6% (174/182), respectively. RAPINA was also used to estimate VNA levels in a semiquantitative manner by using serial dilution of serum samples. Our results show that RAPINA is an easy and rapid method for measuring VNA levels before and after immunization with the rabies vaccine and does not need a high skill level or sophisticated equipment. RAPINA can be used to monitor the success of preexposure prophylaxis in at-risk persons, vaccine coverage, and animal control. It can also be used in laboratories with modest facilities and where a large number of samples are screened.

Evaluation of a monoclonal antibody-based rapid immunochromatographic test for direct detection of rabies virus in the brain of humans and animals.

Rabies diagnosis uses a direct fluorescent antibody test (FAT) that is difficult, costly, and time-consuming, and requires trained personnel. We developed a rapid immunochromatographic test (RICT) for the diagnosis of rabies. The efficacy of the RICT was compared with that of the FAT. Brain samples were collected from humans, dogs, cats, and other animals in Sri Lanka (n = 248), Bhutan (n = 27), and Thailand (n = 228). The sensitivity (0.74-0.95), specificity (0.98-1.0), positive predictive value (0.98-1.0), negative predictive value (0.75-0.97), accuracy (0.91-0.98), and kappa measure of agreement (0.79-0.93) were all satisfactory for animal samples and samples preserved in 50% glycerol saline solution. Because the RICT showed high sensitivity but low specificity with human brain samples, it is unsuitable for confirming rabies in humans. No amino acid substitutions were found in the antibody attachment sites of the nucleoprotein gene with FAT-positive, RICT-negative samples. The RICT is reliable, user friendly, rapid, robust, and can be used in laboratories with a modest infrastructure.

One clinic visit for pre-exposure rabies vaccination (a preliminary one year study).

We performed an abbreviated prospective study of rabies pre-exposure (PREP) vaccination in 109 volunteers. Group 1, the control group, received the conventional 3 intradermal injections on days 0, 7 and 21. Group 2 received one rabies vaccine injection (0.1 ml intradermally) at 2 sites on a single day. Group 3 was given one full ampule intramuscularly. One year later, all 3 groups received booster injections (0.1 ml at 4 sites) intradermally at one time or 2 injections intramuscularly on days 0 and 3. All subjects achieved a vigorous anamnestic antibody response 7 days after the boosters. These data suggest that one time immunization of one full dose intramuscularly or 2 site injections of 0.1 intradermally on a single day are adequate to prime immune memory and obtain an accelerated immune response one year later.

Immunogenicity of Simulated PCECV Postexposure Booster Doses 1, 3, and 5 Years after 2-Dose and 3-Dose Primary Rabies Vaccination in Schoolchildren.

Objectives. To assess the immunogenicity of intradermal (ID) booster doses of Purified Chick Embryo Cell rabies vaccine (PCECV, Rabipur) administered to Thai schoolchildren one, three and five years after a primary ID pre-exposure (PrEP) vaccination series. Methods. In this follow-up study of a randomized, open-label, phase II clinical trial, two simulated post-exposure booster doses of PCECV were administered on days 0 and 3 intradermally to 703 healthy schoolchildren, one, three or five years after primary vaccination with either two or three ID doses of 0.1 mL PCECV. Blood was drawn immediately before and 7, 14 and 365 days after the first booster dose to determine rabies virus neutralizing antibody (RVNA) concentrations. Results. An anamnestic response of approximately 30-fold increase in RVNA concentrations was demonstrated within 14 days after booster. All children (100%) developed adequate RVNA concentrations above 0.5 IU/mL. No vaccine related serious adverse events were seen in any of the vaccinees. Conclusion. ID rabies PrEP with PCECV is safe and immunogenic in schoolchildren and the anamnestic response to a two booster dose vaccination series was found to be adequate one, three, and five years after a two- or three-dose primary PrEP vaccination series.

Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR.

Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.

Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus.

Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein-Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP-Flury, and Nishigahara strains and recognized a well-conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT50 activity against CVS, 3.7 × 10⁻7 M of the Kd value, and the enhancing effect of complement-dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post-exposure prophylaxis.

A preliminary study of chemo- and cytokine responses in rabies vaccine recipients of intradermal and intramuscular regimens.

Plasma from 10 patients who had received rabies vaccine either intradermally (ID) or intramuscularly (IM) was examined for 20 chemo- and cytokines. Plasma samples were withdrawn on days 0, 3 and 7 after vaccination. These chemo- and cytokines and sampling days were chosen based on data collected from a protein array analysis of 122 cytokines conducted on one recipient of vaccine administered IM and one recipient of vaccine administered ID. Although eotaxin, interleukin (IL)-5 in the ID and IL-1 beta in the IM group were the only chemo- and cytokines that reached statistical significance (p<0.05), the overall trends may suggest bias on Th1 or Th2 according to vaccination routes. IL-1 alpha, -2, and -6, hemofiltrate cysteine-cysteine chemokine (HCC-4), glucocorticoid induced tumor necrosis factor receptor (GITR), tumor necrosis factor (TNF) related apoptosis inducing ligand-receptor (TRAIL-R3) had some degree of elevation in the ID group. TNF-alpha, gamma-interferon, granulocytes/macrophages - colony stimulating factor (GM-CSF), transforming growth factor (TGF)-beta, lymphotactin and pulmonary and activation-regulated chemokine (PARC) were elevated, although not to a significant level, in the IM group. IL-12, interferon-inducible T cell alpha chemoattractrant (I-TAC) and sertoli cell factor (SCF) were not significantly elevated in both groups whereas IL-4 and -10 were unchanged. Further studies are required to determine whether the presence of specific chemokines, such as eotaxin, is responsible for the production of high levels of rabies virus neutralizing antibody after administration of the dose-sparing ID regimen.

Failure of rabies postexposure prophylaxis in patients presenting with unusual manifestations.

We report an atypical case of paralytic rabies presenting with trismus followed by limb weakness, areflexia, ophthalmoparesis, and bilateral ptosis. Atypical presentations and history of rabies postexposure prophylaxis led to delayed diagnosis. Nucleocapsid and glycoprotein genes of rabies viruses from the patient's and biting dog's brains were of identical sequences.

Postexposure rabies prophylaxis completed in 1 week: preliminary study.

BACKGROUND: Patients exposed to a rabid animal often travel long distances to receive postexposure prophylaxis (PEP), which requires 4 or 5 visits. Reducing the number of clinic visits would not only reduce costs for the patient but may also help increase compliance to receive complete PEP. We made an effort to develop PEP completed in 1 week. METHODS: We administered the 4-site intradermal injections of 0.1 mL of purified Vero cell rabies vaccine to the deltoids and thighs on days 0, 3, and 7, with and without equine rabies immunoglobulin (40 IU/kg). A control group received the World Health Organization-approved and widely used Thai Red Cross regimen (2-site intradermal injections on days 0, 3, and 7 and 1 injection on days 28 and 90) with equine rabies immunoglobulin. We then determined rabies neutralizing antibody (NAb) up to day 360. RESULTS: Geometric mean titers for subjects receiving the 4-site intradermal regimen, with or without equine rabies immunoglobulin, had significantly higher NAb values than did the control group on day 14 and 28 (P<.001). All subjects in all groups had a NAb value > or =0.5 IU/mL on days 14 and 28. The percentages of subjects who had a NAb value > or =0.5 IU/mL from days 0 through 360 were not significantly different among the 3 groups. CONCLUSIONS: After any PEP regimen, World Health Organization recommendations require a NAb value > or =0.5 IU/mL on days 14 and 28. The 1-week PEP regimen, therefore, appears promising. It increased immunogenicity over the 2-site intradermal schedule, and it is convenient and can be used in small clinics, because it consumes almost the entire supplied vaccine ampoule volume.

Inhibition of rabies virus replication by multiple artificial microRNAs.

The RNA interference (RNAi) technology has been recognized as a promising antiviral therapy for a few years. One of the potential limitations for applying this technology against wild type rabies virus is its high rate of genetic variation. Recently, an RNAi vector system that incorporated modified dsRNA within microRNA structure [or artificial miRNAs(amiRNAs)] has been described. This allowed expression of multiple amiRNAs of single or multiple targets from a single construct. In this study, we evaluated a benefit of using amiRNA vector against different rabies strains. We found that applying single targeting amiRNA against challenged rabies virus standard (CVS) rabies nucleocapsid (N) mRNA resulted in more than 90% reduction of viral genome in Neuro2A cells up to 72 h after infection. Multiple amiRNAs aiming at single or multiple NmRNA target(s) yielded comparable inhibitory results as with a single amiRNA against perfectly matched target. Although the level of each mature miRNA generated from multiple amiRNA construct was slightly reduced as assessed by stem-loop RT and real-time PCR techniques, its effectiveness remained unchanged even when an ineffective or scrambled amiRNA was also included in the transcript. Against highly pathogenic wild type virus, single amiRNA construct activity was reduced when mismatching with target sequence occurred at critical site whereas multiple targeting amiRNA construct remained highly effective. Our results suggest the benefit of using multiple targeting amiRNAs when confronting with wild type rabies virus, the sequence of which is not completely known.

Development and evaluation of a rapid neutralizing antibody test for rabies.

The level of virus-neutralizing antibody, which plays a crucial role in the prevention of rabies, is determined by rabies virus (RABV) neutralizing test, which are time- and cost-consuming. In order to determine the level of neutralizing antibody in vaccinees, an easy and reliable method is needed. Based on the principle of immunochromatography, we developed a RAPINA (RAPId Neutralizing Antibody) test to determine the presence of neutralizing antibody in serum. In the RAPINA test, if neutralizing antibody equivalent to 0.5IU/ml of serum sample are mixed with an optimal amount of inactivated RABV (iRABV) and are completely absorbed by the virus, none of the iRABV can bind with monoclonal antibody that recognizes the iRABV glycoprotein (G) on the test strip. A total of 115 human sera samples were tested. The sensitivity, specificity and accuracy of the RAPINA test compared with rapid fluorescent focus inhibition test (RFFIT) as a standard test, were 88.7, 91.9 and 90.4%, respectively. The RAPINA test is a simple, safe and rapid method, which can be a substitute for neutralizing tests that use live viruses, cultured cells and fluorescence microscopy. This test might be useful for screening a large number of sera.

A pilot study on intradermal vaccination of Japanese rabies vaccine for pre-exposure immunization.

Japanese rabies vaccine, a purified chick embryo cell vaccine manufactured by Kaketsuken (PCEC-K), is normally given subcutaneously; however, this requires a large amount of vaccine, and the pre-exposure vaccination regimen requires 6 months to complete. These factors often hamper appropriate vaccination. Therefore, we examined whether this vaccine could induce adequate level of viral neutralizing antibody (VNA) when vaccinated according to the World Health Organization (WHO) intradermal regimen. Our pilot study showed that this regimen resulted in all subjects developing adequate VNA levels. Intradermal route was effective not only for pre-exposure but also for booster vaccination. The intradermal PCEC-K regimen was found to be safe and effective in inducing adequate VNA levels with the use of a smaller quantity of vaccine and within a shorter period of time.

A simple and rapid immunochromatographic test kit for rabies diagnosis.

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine-pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.

Molecular epidemiology of rabies in Vietnam.

The present study was done to determine the molecular epidemiology of rabies virus (RV) in Vietnam. The nucleoprotein (N) and glycoprotein (G) genes of RVs were amplified from the brains of ten rabid dogs of Ho Chi Minh City, Vietnam. The nucleotide sequences of these genes were compared with those of other Asian strains to find the possible relationship among them. Phylogenetic analysis revealed that the Asian N gene segregated into three main branches, namely South-East Asia 1 (SEA 1), South-East Asia 2 (SEA 2) and Indian subcontinent (ISC) genotypes. The SEA 1 genotype comprised RVs from Malaysia, Vietnam and Thailand. The SEA 2 genotype contained strains from the Philippines, and the ISC genotype comprised strains from Sri Lanka and India. Phylogenetically G genes of RVs from Vietnam and Thailand were clustered together. Our study suggests that Vietnamese and Thai RVs are closely related and might have originated from a common ancestor.

Pre-exposure rabies vaccination using purified chick embryo cell rabies vaccine intradermally is immunogenic and safe.

OBJECTIVE: To demonstrate the safety and immunogenicity of intradermal rabies pre-exposure prophylaxis with purified chick embryo cell vaccine (PCECV) in schoolchildren age 5 to 8 years in Thailand. STUDY DESIGN: In a randomized, open-label, phase II clinical trial, 2 or 3 intradermal doses of 0.1 mL PCECV (Rabipur) were administered to 703 schoolchildren on days 0 and 28 or on days 0, 7, and 28. In 206 children, 2 simulated post-exposure booster doses were given 1 year after the primary vaccination series. Rabies virus- neutralizing antibody (RVNA) titers were determined by the rapid fluorescent focus inhibition test. RESULTS: In school-age children in Thailand, a pre-exposure immunization regimen of 3 intradermal doses of PCECV produced adequate immune responses. After primary vaccination, all subjects developed RVNA titers > or =0.5 IU/mL and demonstrated a rapid increase in RVNA titer after 2 simulated post-exposure booster immunizations 1 year after the primary vaccination series. No serious adverse drug reactions occurred. CONCLUSIONS: Rabies pre-exposure immunization with PCECV is safe and immunogenic, and its implementation could save the lives of many children in rabies-endemic areas.