Substitutions at residues 300 and 389 of the VP2 capsid protein serve as the minimal determinant of attenuation for canine parvovirus vaccine strain 9985-46.

Identifying molecular determinants of virulence attenuation in live attenuated canine parvovirus (CPV) vaccines is important for assuring their safety. To this end, we identified mutations in the attenuated CPV 9985-46 vaccine strain that arose during serial passage in Crandell-Rees feline kidney cells by comparison with the wild-type counterpart, as well as minimal determinants of the loss of virulence. Four amino acid substitutions (N93K, G300V, T389N and V562L) in VP2 of strain 9985-46 significantly restricted infection in canine A72 cells. Using an infectious molecular clone system, we constructed isogenic CPVs of the parental virulent 9985 strain carrying single or double mutations. We observed that only a single amino acid substitution in VP2, G300V or T389N, attenuated the virulent parental virus. Combinations of these mutations further attenuated CPV to a level comparable to that of 9985-46. Strains with G300V/T389N substitutions did not induce clinical symptoms in experimentally infected pups, and their ability to infect canine cells was highly restricted. We found that another G300V/V562L double mutation decreased affinity of the virus for canine cells, although its pathogenicity to dogs was maintained. These results indicate that mutation of residue 300, which plays a critical role in host tropism, is not sufficient for viral attenuation in vivo, and that attenuation of 9985-46 strain is defined by at least two mutations in residues 300 and 389 of the VP2 capsid protein. This finding is relevant for quality control of the vaccine and provides insight into the rational design of second-generation live attenuated vaccine candidates.

Efficacy of a new inactivated Chlamydophila felis vaccine in experimentally-infected cats.

A new inactivated and adjuvanted Chlamydophila felis vaccine was developed and its efficacy in cats was compared with that of commercially available inactivated and live vaccines. Two commercial vaccines conferred insufficient immunity on inoculated cats, as evaluated by antibody production and a challenge experiment, whereas cats administered the newly generated vaccine produced high-titre antibodies and acquired sufficient immunity. The cats immunised with the new vaccine revealed no or only mild clinical signs, and no chlamydiae were recovered from their tissue samples after exposure to a virulent C felis. However, they shed chlamydiae in their nasal and conjunctival secretions after challenge, as did those immunised with the commercial vaccines and the non-vaccinated controls. The newly developed vaccine caused no adverse reaction in the inoculated cats. These findings suggest that the new vaccine prepared here may be promising for practical use in controlling C felis infection in cats.

Immunological and genetic characterization of feline caliciviruses used in the development of a new trivalent inactivated vaccine in Japan.

Although vaccination against feline calicivirus (FCV) infection is widespread in Japan, FCV-associated diseases are still a significant problem in cats. Thus, we developed a new trivalent inactivated vaccine, Kyoto Biken Feline-CPR, consisting of three FCV strains; one was the production strain of our previous vaccine, and the others were screened from 60 field isolates obtained between 1998 and 2000 based on cross-neutralization tests. In this report, the three FCV strains used for development of the new vaccine were antigenically and genetically characterized. The three strains were antigenically quite different, as revealed by cross-neutralization tests. Alignment of deduced amino acid sequences of capsid regions A to E revealed that there were marked differences between the strains in both the N- and C ends of region E. Antisera against the three vaccine strains, our new vaccine and 2 commercial vaccines were then evaluated for neutralization with 58 field isolates collected between 2003 and 2006. Rat antisera against the three vaccine strains and a mixture of the 3 strains neutralized 49, 37, 42 and 55 isolates, respectively. Cat antiserum against the new vaccine neutralized 50 (86.2%) isolates, whereas the numbers neutralized by cat antisera against 2 commercial vaccines were 37 (63.8%) and 25 (43.1%). In conclusion, the immunological and genetic properties of the 3 vaccine strains investigated varied widely, and the Kyoto Biken Feline-CPR vaccine may have more potential to meet the antigenic diversity of FCVs spreading throughout Japan.

[Diagnosis of ocular sarcoidosis by diagnostic criteria for systemic sarcoidosis].

PURPOSE: To evaluate the diagnostic criteria for systemic sarcoidosis in diagnosis of ocular sarcoidosis. SUBJECTS AND METHODS: Subjects were 105 ocular sarcoidosis suspects and 37 patients with other uveitis. We diagnosed ocular sarcoidosis suspects using the diagnostic criteria for systemic sarcoidosis proposed by the Japanese Committee for Diffuse Lung Diseases. The criteria included histological and clinical diagnosis, and the clinical diagnosis required 5 systemic tests: 1) tuberculin skin test, 2) serum gamma-globulin(gamma-gl), 3) serum angiotensin converting enzyme(ACE), 4) serum lysozyme, and 5) 67Ga scintigraphy. Three positive findings including either 1) or 3) fulfilled the clinical diagnosis. RESULTS: Sixty-two patients were histologically and/or clinically diagnosed, and 43 patients remained undiagnosed. The histological and clinical diagnosis did not produce the same diagnostic yields. The sensitivity of ACE and gamma-gl was low. The percentage of patients showing increased lymphocytosis and/or CD 4/8 in bronchoalveolar lavage was similarly high in the diagnosed and undiagnosed, suggesting the presence of definitive ocular sarcoidosis in the undiagnosed. CONCLUSIONS: The diagnostic criteria for systemic sarcoidosis yielded false negative results in diagnosing ocular sarcoidosis. The selection and combination of systemic tests for clinical diagnosis should be further studied.

Characterization of a slow-migrating component of the rabies virus matrix protein strongly associated with the viral glycoprotein.

We investigated multiple forms of rabies virus matrix (M) protein. Under non-reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23- and 24-kDa) of M protein, an M antigen-positive slow-migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54-kDa and monomer components in the virion were about 20-30% and 70-80% of the whole M protein, respectively, while the content of the 54-kDa component was smaller (about 10-20% of the total M protein) in the cell than in the virion. The 54-kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% nonionic detergents by which most monomer components were solubilized. The 54-kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3-9-16), which recognized a linear epitope located at the N-terminal of the M protein. The mAb #3-9-16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54-kDa component of the M protein. Monomers and the 54-kDa polypeptide migrated to the same isoelectric point (pI) in twodimensional (2-D) gel electrophoresis, implicating that the 54-kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen-positive 54-kDa polypeptide is a homodimer of M protein, taking an N-terminal-exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface.

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.