Differential pathogenesis of intracerebral and intramuscular inoculation of street rabies virus and CVS-11 strains in a mouse model.

OBJECTIVES: The mechanisms of rabies evasion and immunological interactions with the host defense have not been completely elucidated. Here, we evaluated the dynamic changes in the number of astrocytes, microglial and neuronal cells in the brain following intramuscular (IM) and intracerebral (IC) inoculations of street rabies virus (SRV). MATERIALS AND METHODS: The SRV isolated from a jackal and CVS-11 were used to establish infection in NMRI-female mice. The number of astrocytes (by expression of GFAP), microglial (by Iba1), and neuronal cells (by MAP-2) in the brain following IM and IC inoculations of SRV were evaluated by immunohistochemistry and H & E staining 7 to 30 days post-infection. RESULTS: Increased numbers of astrocytes and microglial cells in dead mice infected by SRV via both IC and IM routes were recorded. The number of neuronal cells in surviving mice was decreased only in IC-infected mice, while in the dead group, this number was decreased by both routes.The risk of death in SRV-infected mice was approximately 3 times higher than in the CVS-11 group. In IC-inoculated mice, viral dilution was the only influential factor in mortality, while the type of strain demonstrated a significant impact on the mortality rate in IM inoculations. CONCLUSION: Our results suggested that microglial cells and their inflammatory cytokines may not contribute to the neuroprotection and recovery in surviving mice following intracerebral inoculation of SRV. An unexpected decrease in MAP2 expression via intramuscular inoculation indicates the imbalance in the integrity and stability of neuronal cytoskeleton which aggravates rabies infection.

Role of autophagy in nerve cell apoptosis in mice infected with street rabies virus.

Rabies is an important zoonotic disease in Iran. Autophagy is a process that maintains homeostasis and can be used as an innate defense mechanism against viruses. Apoptosis is the process of programmed cell death induced by physiological and pathological conditions. The crosstalk of autophagy and apoptosis plays a key role in rabies virus infection. In the current study, NMRI mice intra-cranially received 3-Methyl Adenine (3-MA), rapamycin, street rabies virus (SRABV) and drugs plus SRABV. SRABV and Map1lc3, Beclin-1, Atg5 gene expression were assayed by real-time PCR. Immunohistochemistry was carried out via LC3 protein staining as an autophagy marker, and apoptotic cell death was measured using a TUNEL assay. Map1lc3, Beclin-1 and Atg5 genes expression was significantly increased in drug-plus-SRBV-treated tissues compared to control at 24 hpi. Map1lc3 and Atg5 gene expression showed a slight change in the drugs-plus-virus group compared with the control at 72 hpi. The presence of LC3 in the tissues of the group treated with rapamycin plus SRBV confirmed induction of autophagy, but it was not present in the tissues treated with 3-MA plus SRBV. Our data revealed that apoptosis was induced only in the groups receiving the SRBV or rapamycin or both at 24 hpi. Apoptosis was observed after 72 hours, when the drugs' effect had disappeared in all but the autophagy inhibitor group. Understanding the interaction of SRABV with autophagy pathway genes and its effect on host cell apoptosis may open a new horizon for human intervention and allow a deeper understanding of rabies infections.

A phylogenetic study of new rabies virus strains in different regions of Iran.

Rabies is the most critical zoonotic disease in Iran, which imposes many extra costs on health care system in each country. The present study aimed to determine the molecular characteristics of the wild circulating strains of the rabies virus (RABV) collected in Iran during 2015-2017. Rabies-suspected samples were collected from different regions of Iran and identified for RABV antigen confirmation using fluorescent antibody tests. Polymerase chain reaction (PCR) was performed on positive samples and gene sequencing was done on rabies nucleoprotein and glycoprotein genes to determine the rabies molecular characteristics. Accordingly, nine street RABVes were isolated. Then, N (802 bp) and G (735 bp) genes were amplified with specific primers using PCR. The sequence of nine strains was determined and compared with another 50 close to them, and the phylogenetic tree was plotted using neighbor-joining method by Mega 7 software. The molecular characteristic results indicated that all new strains belong to RABV wild species. As a result, the most prevalent strains of RABV in northwest, west, center, and south of Iran were identified. The present study may provide a better insight into the identification of all RABV strains, and understanding the evolutionary nature of RABV and how its hosts change in the world over the centuries.

Ante-mortem Diagnosis of Human Rabies Cases Using SYBR Green Real-Time PCR.

BACKGROUND: Rabies, as the most important zoonotic disease, is transmitted through a bite or scratch by an infected domestic or wild carnivores and bats or contact of open wound with infected saliva. The fluorescent antibody test (FAT) is the "gold standard" diagnostic method for suspected brain samples. For close monitoring of unknown encephalitis, rabies surveillance, and also the limitations for post-mortem diagnosis of rabies in human and performing fast prophylactic measures for other individuals in contact with rabid patients, ante-mortem diagnosis based on molecular methods such as real-time polymerase chain reaction (PCR) seems to be more reliable. In this study, we detected 2 positive rabid cases using SYBR Green real-time PCR for the first time in Iran. METHODS: In this study, 3 saliva samples at intervals up to 6 hours were collected from any of the nine suspected patients with nonspecific symptoms between March 2016 and March 2017. Total RNA extraction, cDNA synthesis and real-time PCR were performed along with confirmed negative and positive controls. Then, we tracked the patients for follow-up and understanding of their status. On brain samples of patients who died, FAT and MIT (mouse inoculation test) were performed to obtain definitive results. RESULTS: In this study, the patients were 4 females and 5 males, between 8 and 80 years old from different geographical areas of Iran. The ante-mortem saliva samples of 2 out of nine patients who died were positive by SYBR Green real-time PCR. Positive results of FAT test on these samples confirmed the presence of rabies virus infection in their brains and also the ante-mortem diagnosis results. CONCLUSION: The results of this study suggest that SYBR Green real-time PCR technique on saliva sample can be used as an applicable method for ante-mortem diagnosis of rabies to avoid infection of other people such as the treating medical staff or family members of the patient.

Evaluation of G2 Citric Acid-Based Dendrimer as an Adjuvant in Veterinary Rabies Vaccine.

For induction of an appropriate immune response, especially in the case of an inactivated vaccine, the use of an adjuvant is crucial. In this study, adjuvanticity effect of G2 dendrimer in veterinary rabies vaccine has been investigated. A nonlinear globular G2 dendrimer comprising citric acid and polyethylene glycol 600 (PEG-600) was synthesized and the toxicity was studied in vitro on the J774A.1 cell line. The adjuvanticity effect of the dendrimer was then investigated on rabies virus in NMRI mice as a model. Different concentrations of dendrimer were used to determine the best formulation for the survival of the mice after virus challenge. The rise of neutralizing antibody was also checked by rapid fluorescent focus inhibition test (RFFIT). The relative potency of the prepared formulation was finally calculated using standard NIH test and the results were compared (and discussed) with the commercially available rabies vaccine. The accuracy of dendrimer synthesis was confirmed using Fourier transform infrared (FT-IR), size, and zeta potential analysis. The in vitro toxicity assay revealed that no significant toxic effect is observed in cells when data are compared with the control group. The in vivo assay showed that a higher survival rate in the mice received a special formulation due to adjuvanticity effect of dendrimer, which is also confirmed by RFFIT. However, the relative potency of that formulation does not give expected results when compared with the alum-containing rabies vaccine. In the current investigation, the adjuvanticity effect of G2 dendrimer was demonstrated for the first time in rising of neutralizing antibodies against rabies virus. Our data confirm that nanoparticles can enhance immune responses in an appropriate manner. Moreover, engineered nanoparticles will enable us to develop novel potent multivalent adjuvants in vaccine technology.

Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine.

BACKGROUND: Green synthesis of nanoparticles by plant extracts plays a significant role in different applications. Recently, several studies were conducted on the use of nanoparticles as adjuvant. The main aim of this study was to evaluate green synthesized silver nanoparticles (AgNPs) as adjuvant in rabies veterinary vaccine and compare the results with the existing commercially available alum adjuvant. MATERIALS AND METHODS: In the current study, AgNPs were prepared by the reduction of aqueous silver nitrate by leaf extract of Eucalyptus procera. The formation of AgNPs was confirmed by ultraviolet (UV)-visible spectrophotometer, scanning electron microscopy, dynamic light scattering, and X-ray diffraction analysis. Then, different amounts of AgNPs (200 µg, 400 µg, 600 µg, and 800 µg) were added to 1 mL of inactivated rabies virus. The loaded vaccines (0.5 mL) were injected intraperitoneally into six Naval Medical Research Institute mice in each group on days 1 and 7. On the 15th day, the mice were intracerebrally challenged with 0.03 mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD50]), and after the latency period of rabies disease in mice (5 days), the mice were monitored for 21 days. Neutralizing antibodies against rabies virus were also investigated using the rapid fluorescent focus inhibition test method. The National Institutes of Health test was performed to determine the potency of optimum concentration of AgNPs as adjuvant. In vitro toxicity of AgNPs was assessed in L929 cell line using MTT assay. In addition, in vivo toxicity of AgNPs and AgNPs-loaded vaccine was investigated according to the European Pharmacopeia 8.0. RESULTS: AgNPs were successfully synthesized, and the identity was confirmed by UV-visible spectrophotometry and X-ray diffraction analysis. The prepared AgNPs were spherical in shape, with an average size of 60 nm and a negative zeta potential of -14 mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15 mg/kg and 20 mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15 mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10 mg/mL was toxic, but AgNPs were not toxic. The in vivo toxicity also elucidated the safety of AgNPs and AgNPs-loaded vaccine in mice and dogs, respectively. CONCLUSION: In the current study, for the first time, the adjuvanticity effect of green synthesized AgNPs on veterinary rabies vaccine potency with no in vivo toxicity was elucidated according to the European Pharmacopeia 8.0.

Phylogenetic Comparison of Influenza Virus Isolates from Three Medical Centers in Tehran with the Vaccine Strains during 2008-2009.

BACKGROUND: Influenza virus is a major infectious pathogen of the respiratory system causing a high degree of morbidity and mortality annually. The worldwide vaccines are decided and produced annually by World Health Organization and licensed companies based on the samples collected from all over the world. The aim of this study was to determine phylogenecity and heterogenecity of the circulating influenza isolates during 2008-2009 outbreaks in Tehran, compare them with the vaccine strains that were recommended by WHO for the same period. METHODS: Nasopharyngeal swabs (n=142) were collected from patients with influenza and influenza-like illness. Typing and subtyping of the isolates were performed using multiplex RT-PCR and phylogenetic analysis was carried out for hemagglutinin genes of the isolates. RESULTS: Fifty out of 142 samples were positive for influenza A virus, and no influenza B virus was detected. Phylogenetic analyses revealed that the A/H1N1 isolates were related closely to A/Brisbane/59/2007, and the A/H3N2 isolates were close to A/Brisbane/10/2007 vaccine strains. CONCLUSION: The findings of the present study demonstrate that the A/H1N1 was the predominant subtype of human influenza virus among the patients studied in Tehran during 2008-2009 winter seasons. In addition, some amino acid variation was found in Tehran/2008/H1N1 isolates from the 2008-2009 vaccine strain, but the H3N2 isolates showed higher genetic resemblance to the vaccine strain.

Molecular and phylogenetic analysis of human influenza virus among Iranian patients in Shiraz, Iran.

Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation namely A/H1N1, A/H3N2, and B. The objective of this study was to determine the human influenza virus genotypes in Shiraz, the capital of the Fars province of Iran. Three hundred patients suspected with human influenza virus infection were enrolled in this survey (2004-2005). The throat samples were cultured and titrated by hemagglutination (HA) assay. Typing and subtyping were performed by an in-house developed multiplex RT-PCR. Moreover, the phylogenetic analysis was carried out for HA gene. A total of 24 samples were found to be positive for human influenza virus infection, 17 H1N1 and 7 H3N2. These results were in agreement with the HI assay. The phylogenetic analysis results revealed that the Iranian H1N1 isolated were close to the A/New Caledonia/20/99 vaccine strain genetically and the Iranian H3N2 isolates were also related closely to the Fujian/411/021 and California/7/2004 vaccine strains. However, a slight genetic drift was found in these isolates. In conclusion, it was demonstrated that both influenza A subtypes A/H1N1 and A/H3N2 were dominant among Iranian patients in Shiraz during the 2004/5 winter season.