Characterization of microminipig as a laboratory animal for safety pharmacology study by analyzing fluvoxamine-induced cardiovascular and dermatological adverse reactions.

Fluvoxamine is a selective serotonin-reuptake inhibitor, of which IC50 values for serotonin- and noradrenaline-uptake process were reported to be 3.8 and 620 nmol/L, respectively, also known to directly inhibit cardiac Na+, Ca2+, and K+ channels. We characterized microminipig as a laboratory animal by analyzing fluvoxamine-induced cardiovascular and dermatological responses under halothane anesthesia. Fluvoxamine maleate was infused in doses of 0.1, 1, and 10 mg/kg over 10 min with a pause of 20 min (n = 4). The peak plasma concentrations were 35, 320, and 1906 ng/mL, of which free plasma concentrations were estimated as 20, 187, and 1108 nmol/L, respectively. The low and middle doses did not alter any cardiovascular variable. The high dose increased heart rate and mean blood pressure, prolonged QRS width, but shortened QT interval, whereas no significant change was detected in PR interval or QTcF. Moreover, it induced systemic erythema on the skin. Pretreatment of H1/5-HT2A antagonist cyproheptadine hydrochloride sesquihydrate in a dose of 0.3 mg/kg significantly attenuated the fluvoxamine-induced pressor response; but tended to further enhance sinus automaticity, atrioventricular nodal conduction; and ventricular repolarization in addition to intraventricular conduction delay; whereas it markedly suppressed onset of systemic erythema (n = 4). In microminipigs, cardiovascular adverse effects of the high dose may be manifested as a sum of its inhibitory action on the cardiac ionic channels and its stimulatory effects on serotonergic and adrenergic systems, whereas dermatologic reaction can be induced primarily through H1/5-HT2A receptor-dependent mechanism. Thus, microminipigs may be used for analyzing such multifarious adverse events of clinical serotonergic pharmacotherapy.

Isolation and characterization of hantaviruses in Far East Russia and etiology of hemorrhagic fever with renal syndrome in the region.

Hemorrhagic fever with renal syndrome (HFRS) is a serious public health issue in Far East Russia. Two different hantaviruses were isolated from rodents captured in the Khabarovsk region: Amur virus (AMRV; Khekhtsir/AP209/2005 strain from Apodemus peninsulae) and Hantaan virus (HTNV; Galkino/AA57/2002 strain from A. agrarius). Genetic analysis of the new isolates revealed that the M and L segments were apparently different between AMRV and HTNV, but S segments of the two viruses were closer. The antigenicities of AMRV, HTNV, and Seoul virus (SEOV) were differentiated by cross-neutralization. Serological differential diagnoses of 67 HFRS patients in the Prymorsky and Khabarovsk regions of Far East Russia were conducted using a neutralization test. The results revealed that the major cause of HFRS varied with location in Far East Russia: SEOV for Vladivostok city in the Prymorsky region, AMRV in rural areas of the Primorsky region, and probably HTNV for the Khabarovsk region.

Puumala virus infection in Syrian hamsters (Mesocricetus auratus) resembling hantavirus infection in natural rodent hosts.

The mechanism of hantavirus persistent infection in natural hosts is poorly understood due to a lack of laboratory animal models. Herein, we report that Syrian hamsters (Mesocricetus auratus) infected with Puumala virus (PUUV) at 4 weeks old show persistent infection without clinical symptoms for more than 2 months. IgG and IgM antibodies against the viral nucleocapsid protein and neutralizing antibody were first detectable at 14 days postinoculation (dpi) and maintained through 70 dpi. Viral RNA was first detected from 3 dpi in lungs and blood clots, and was detected in all tissues tested at 7 dpi. The viral RNA persisted for at least 70 days in the lungs, kidney, spleen, heart, and brain. The highest level of RNA copies was observed at 14 dpi in the lungs. Slight inflammatory reactions were observed in the lungs, adrenal glands, and brain. Immunohistochemical analysis revealed that PUUV antigen persisted until 56 dpi in the kidneys and adrenal glands. Infected hamsters showed no body weight loss or clinical signs. These results indicate that PUUV infection in hamsters is quite similar to the hantavirus infection of natural host rodents.

An efficient in vivo method for the isolation of Puumala virus in Syrian hamsters and the characterization of the isolates from Russia.

Puumala virus (PUUV) and other Arvicolinae-borne hantaviruses are difficult to cultivate in cell culture. To isolate these hantaviruses efficiently, hantavirus nucleocapsid protein (NP)-positive but seronegative wild rodents were selected by NP-detection ELISA. Three of 68 Myodes glareolus captured in Samara, Russia, were NP-positive and seronegative. Syrian hamsters were inoculated with lung homogenates from NP-positive rodents for virus propagation. Virus isolation in vitro was carried out by inoculation of lung homogenates of NP-positive hamsters to Vero E6 cell monolayers. Two PUUV strains (Samara49/CG/2005 and Samara94/CG/2005) from M. glareolus were isolated in Vero E6 cells. Nucleotide and amino acid sequence identities of the S segment of these isolates to those of PUUV F-s808 from a fatal HFRS patient in Samara region were 96.7-99.3% and 99.3-100.0%, respectively. Morphologic features of Vero E6 cells infected with PUUV strain Samara49/CG/2005 were quite similar to those of Hantaan virus-infected cells. Isolation of Hokkaido virus from Myodes rufocanus captured in Hokkaido, Japan, was also performed. Hokkaido virus NP and RNA were recovered and maintained in hamsters. These results suggest that inoculation of Syrian hamsters with rodent samples is an efficient method for the isolation and maintenance of PUUV and other Arvicolinae-borne hantaviruses.

Epidemiological study of hantavirus infection in the Samara Region of European Russia.

European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.