Immunohistochemical detection of virus antigen in the nasal planum of rabid dogs

The rabies virus is one of the most neurotropic of all viruses infecting mammals. During the terminal phases of infection, the virus spreads to peripheral tissues, including the skin. The external skin of the nose, called the nasal planum, is a sensory organ where numerous nerve bundles and terminal nerves are distributed. Therefore, the nasal planum is expected to serve as a postmortem diagnostic material. However, the distribution of rabies virus antigens in the nasal planum in rabid animals has not yet been studied. In this study, the nasal planum was obtained from 45 rabid dogs. In all rabid dogs, the viral antigen was detected in the peripheral nerve tissues, Merkel cells, and squamous cells. The viral antigen in the epidermis exhibited three patterns: first, a diffuse positive pattern from the basal layer to the squamous layer; second, a reticular positive pattern along the cell membrane in the squamous layer; and third, a basal layer pattern of the epidermis. In the dermis, viral antigens were detected more often in lamellated corpuscles just beneath the rete pegs. These results suggest that the nasal planum could serve as a useful alternative source for postmortem diagnosis in rabies endemic countries.     

Cellular response to rabies virus infection

The effects of rabies virus on host cells were studied and compared to those obtained with another rhabdovirus, vesicular stomatitis virus [J. Virol. 34, 777-781 (1980)]. We show here: (1) that rabies infection has no effect on cell morphology, while infection with vesicular stomatitis virus caused cell retraction. Thus, only vesicular stomatitis virus induced a depolymerization of the microfilaments; and (2) that microtubules and microfilaments do not play a major role in rabies virus production, as it is suggested by results obtained with several effectors (colcemid, colchicine and cytochalasin-B) which directly or indirectly affect cytoskeleton organization. The same properties were observed with directly or indirectly affect cytoskeleton organization. The same properties were observed with vesicular stomatitis virus. Furthermore, the use of cytochalasin-B shows that an inhibition of glycosylation of the virion spike protein occurs only in rabies infected cells. As vesicular stomatitis viral glycosylation is normal in cytochalasin-B treated cells, results obtained indicate that two types of interactions can occur between a virion and the host-cell depending on the rhabdovirus type.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.     

Effect of inhibitors of cytoplasmic structures and functions on rabies virus infection in vitro

The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.     

Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.     

Infection of Bergmann glia in the cerebellum of a skunk experimentally infected with street rabies virus.

Rabies virus is a highly neuronotropic virus and glial cell infection is not prominent in the central nervous system (CNS). Paraffin-embedded tissues from the cerebella of skunks experimentally infected with either a skunk salivary gland isolate of street rabies virus or the challenge virus standard (CVS) strain of fixed rabies virus were examined with immunoperoxidase staining for rabies virus antigen by using an anti-rabies virus nucleocapsid protein monoclonal antibody. A skunk infected with street rabies virus showed prominent infection of Bergmann glia. Although infected Purkinje cells were observed, they usually demonstrated a relatively small amount of antigen in their perikarya. A CVS-infected skunk showed many intensely labeled Purkinje cells and a relatively small number of infected Bergmann glia. These findings indicate that although rabies virus is a highly neuronotropic virus, street rabies virus strains do not always demonstrate strict neuronotropism in the central nervous system.     

应用免疫胶体金标记技术对狂犬病病毒形态发生学的抗原定位研究

利用包埋后免疫胶体金标记技术，通过透射电子显微镜，对感染ＢＨＫ２１细胞的鹿狂犬病病毒进行了形态发生的抗原定位。结果：感染细胞的细胞质内有５种包涵体，其中４种被免疫胶体金特异性标记，这些病毒抗原是多成分的，另一种未被免疫胶体金标记的包涵体可能是病毒核酸；感染细胞的细胞质内大量堆积的病毒前体物质包涵体，其中可能有病毒装配后多余的成分，表明病毒各组分并不是按比例需要而合成的。     

In vitro effect of isoprinosine on rabies virus

The in vitro effect of isoprinosine, an antiviral compound, was evaluated on ERA and VA319 rabies viral strains. Isoprinosine diminished the number of immunofluorescent cells, viral titers, and structures involved in rabies virus replication. Mechanism of antiviral action was probably mediated by modification of the polyribosome conformation and function.     

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.     

狂犬病病毒Vero ERA株G蛋白333位点精氨酸突变株的构建

狂犬病病毒糖蛋白（G）在病毒的致病性中起着主要作用,其第333位点精氨酸（Arg）是决定病毒神经细胞侵嗜性的重要分子基础之一。为研制更加安全有效的狂犬病减毒活疫苗,本试验采用负链RNA病毒反向遗传学方法,在体外将狂犬病病毒Vero细胞适应株ERA糖蛋白G333位点精氨酸突变为谷氨酸,构建减毒株ERAGΔ,减弱了其潜在的毒力返强的危险。救获的ERAGΔ突变株在Vero细胞上表现出与ERA株亲本病毒相似的生长动力学特性。本研究成功构建并拯救出了狂犬病病毒ERA减毒疫苗株,为研制开发狂犬病病毒无毒疫苗提供了候选株。     

Antigenic characterisation of virus isolates from vaccinated dogs dying of rabies

Four rabies virus isolates from dogs that succumbed to rabies infection in Nigeria within one year of anti-rabies vaccination were characterised by monoclonal antibodies (MAbs). The samples were screened for rabies and rabies-related viral antigens by the indirect fluorescent antibody test, performed with MAb 502-2, which recognises the nucleocapsid (NC) protein of all known Lyssaviruses and with MAb 422-5 which identifies African rabies-related viruses. All four canine virus isolates displayed positive fluorescence with MAb 502-2 and were negative with MAb 422-5. In the anti-NC MAb characterisation with a panel of 34 additional MAbs, all isolates displayed positive staining with 32 of the MAbs, were negative with MAb 102-27 and all displayed poor immunofluorescence with MAb 377-7. On the basis of reactivity with a panel of 40 anti-glycoprotein (G) MAbs the isolates were separated into four distinct viral subtypes. None of these canine isolates was identified as the common attenuated Flury LEP rabies strain used for domestic animal vaccination and none resembled other previously characterised rabies viruses from Nigeria.     

检测狂犬病病毒抗原的夹心间接Dot—ELISA的建立和应用研究

本研究建立了检测狂犬病病毒抗原的夹心间接斑点酶联免疫吸附试验(SI-Dot-ELISA).本法检出狂犬病病毒抗原的最低浓度为:0.01IU/mL的标准抗原,1:100000 (相当于20 LD_(50))的狂犬病病毒CVS株和鹿8202株的鼠脑悬液,1:400(相当于7906TCID_(50)/mL)的狂犬病病毒SAG株的细胞培养物.对多种健康动物的组织、健康细胞培养物以及犬瘟热病毒、犬腺病毒等检测均为阴性.用SI—Dot—ELISA、夹心间接ELISA(SI—ELISA)和小鼠脑内接种3种方法,检测了388份材料,检出的阳性份数分别为173、171和179,经统计学处理,3种方法在狂犬病病毒抗原的检测上,可以相互代替.甲醛灭活病毒不影响本方法的检测,而加入氢氧化铝胶后的病毒悬液则不能用本法检测.本法适用于狂犬病的诊断、流行病学调查、疫苗效价的测定和实验研究中病毒抗原的测定.     

Experimental rabies in skunks: Immune response and salivary gland infection

Groups of striped skunks (Mephitis mephitis) were inoculated intramuscularly with graded doses of street rabies virus. At various intervals after inoculation, saliva and sera were tested for rabies virus and neutralizing antibodies, respectively. Skunks that developed rabies were killed in terminal stages of the disease and the following examinations were made: titers of virus and antibody in submandibular salivary glands and brain, extent of immunofluorescence in submandibular salivary glands, and histologic examination of various tissues.

Skunks that received inocula containing 4 × 10⁴ to 4 × 10⁵ mouse intracerebral lethal dose₅₀ (MICLD₅₀) had detectable serum neutralizing antibodies by 7–12 days postinoculation; however, most of the skunks that received lower doses (40 to 4 × 10³ MICLD₅₀) did not have detectable serum neutralizing antibodies until clinical signs began. In the salivary glands, slight and extensive immunofluorescence corresponded to high and low titers of tissue neutralizing antibody. Also low viral titers were associated with high tissue neutralizing antibody titers. There was a close correlation between viral titers in right and left submandibular salivary glands.

The results suggest that the immune response can impede the process of infection of the salivary glands resulting in lack of antigen or low amounts of antigen in this tissue. This could occur through interference with centrifugal neural transport of virus and/or neutralization of virus during transfer from neural elements to epithelial cells. Lack of infectious virus or low viral titers in salivary glands containing antigen and high levels of tissue neutralizing antibodies can be caused partly by postmortem virus neutralization (during viral titration).     

Immunohistochemical and histopathological study of experimental rabies infection in mice.

An immunohistochemical and histopathological study using the ABC technique was carried out to examine time-sequential virus spread in the central nervous system (CNS) of mice after inoculation with the CVS strain of fixed rabies virus by different routes; intracerebral (ic), intraocular (io), intranasal (in), intramuscular (im) and subcutaneous (sc). Only the ic and io inoculations caused fatal infections, so that detailed analysis was conducted on mice inoculated by these two routes. In ic-inoculated mice, viral antigens were detected mainly in neurons in the cerebral cortex and in the pyramidal cells and granular cells of the hippocampus. After io inoculation, viral antigen was first detected in the trigeminal nerve ganglia, following which it spreads to the cerebral cortex and cerebellum. In the hippocampus only a few cells were viral antigen-positive at the early stage after io inoculation. There were no inflammatory lesions or Negri bodies in the CNS of mice infected by either route. This suggests that clinical signs such as ataxia or depression leading to death may be due to the direct effect of the virus on the functions of neural cells, but not to inflammatory reactions. The ABC method will be useful for the early diagnosis of suspected patients or animals to have the disease when conventional histopathological and immunofluorescent antibody techniques can not detect lesions or viral antigens.     

The efficacy of a new single post-exposure treatment of rabies in mice without vaccination.

Local application of rabies immune serum and isoprinosine, an immunomodulator with antiviral activity was effective in mice infected with a sylvatic rabies virus. In this way, a single medical or veterinary treatment is only required, which is particularly important for developing but also for developed countries. The importance of using a post-exposure potency test to monitor rabies vaccines is emphasized. The same principle could be applied to other emerging viral infections of humans (for example, human immunodeficiency virus infection) and animals, for which no effective vaccines are available at this moment.     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

A pathological study of the salivary glands of rabid dogs in the Philippines

Rabies is a zoonotic disease caused by the rabies virus. While the salivary glands are important as exit and propagation sites for the rabies virus, the mechanisms of rabies excretion remain unclear. Here, we investigated the histopathology of the salivary glands of rabid dogs and analyzed the mechanism of excretion into the oral cavity. Mandibular and parotid glands of 22 rabid dogs and three control dogs were used. Mild to moderate non-suppurative sialadenitis was observed in the mandibular glands of 19 of the 22 dogs, characterized by loss of acinar epithelium and infiltration by lymphoplasmacytic cells. Viral antigens were detected in the mucous acinar epithelium, ganglion neurons and myoepithelium. Acinar epithelium and lymphocytes were positive for anti-caspase-3 antibodies and TUNEL staining. In contrast, no notable findings were observed in the ductal epithelial cells and serous demilune. In the parotid gland, the acinar cells, myoepithelium and ductal epithelium all tested negative. These findings confirmed the path through which the rabies virus descends along the facial nerve after proliferation in the brain to reach the ganglion neurons of the mandibular gland, subsequently traveling to the acinar epithelium via the salivary gland myoepithelium. Furthermore, the observation that nerve endings passing through the myoepithelium were absent from the ductal system suggested that viral proliferation and cytotoxicity could not occur there, ensuring that secretions containing the virus are efficiently excreted into the oral cavity.