Pathogenicity of the skalica strain (from the tick-borne encephalitis complex) for white mice

Outbred white mice of different age were inoculated by subcutaneous (s. c.) route with the Skalica strain of tick-borne encephalitis virus (TBE). In the CNS of 3-days-old mice diffuse necrotizing encephalitis, abundant cytoplasmic fluorescence of viral antigen in nearly each neuron and high levels of virus (8.8 log LD50/mg tissue) were found. In 10-days-old mice, the extent of encephalitis and that of immunofluorescence in neurons were less widespread; the peak titre of the virus did not exceed 5.5 log LD50/mg brain tissue. In the CNS of 21-days-old mice the infectivity titre was either very low (1.5 log50/mg on day 3 post infection) or the virus was not detected at all. A few neurons revealed positive fluorescence of viral antigen in the basal ganglia in 1 out of 2 mice examined by day 3 post infection (p. i.). No virus was isolated from the CNS of 2-months-old mice observed for 53 days. In the CNS of 3 out of 10 juvenile mice examined histologically within 8 days post infection, minimal inflammatory changes were seen; foci of neurons showing positive immunofluorescence were not found. The failure to recover infectious virus from cultured brain tissue fragments coming from these mice confirmed the negative outcome of direct virus isolations. It is concluded that the Skalica strain was not pathogenic for juvenile mice when administered by s. c. route.     

Replication of tick-borne encephalitis (TBE) virus in ticks Dermacentor marginatus

In the laboratory experiments, the virophoric period in D. marginatus ticks lasted 61 to 81 d, the premoulting period (nymphs - adults) amounted to 17 to 18 d. The titres of individually examined females and males for the presence of TBE virus ranged from 10(1) to 10(5.5) ic mouse LD50/0.03 ml between the 14th and 29th day after hatching. In the laboratory experiments 77% of ticks were positive. In the field experiments, the virophoric period in D. marginatus ticks lasted 79 to 114 d, the premoulting period amounted to 34 to 38 d. The titres of individually examined adults for the presence of virus ranged from 10(1) to 10(5.5) ic mouse LD50/0.03 ml between the 13th and 80th day after hatching. In the field experiments 96.5% of ticks were positive.     

Viraemia in Clethrionomys glareolus -- a new ecological marker of tick-borne encephalitis virus.

Viraemia was studied in adult Clethrionomys glareolus subcutaneously infected with 12 strains of tick-borne encephalitis (TBE) virus isolated in western and eastern foci of TBE. Nine strains caused viraemia regularly or irregularly, reaching titres higher than the threshold level of TBE virus infectivity for its vectors, ixodid ticks (2.5 -- 4.5 log LD50/0,03 ml) and three strains caused lower levels of hiraemia (0.4 -- 1.5 log LD50/0.03 ml). The ability or inability of various TBE virus strains to cause viraemia in adult C. glareolus in titres higher than the threshold level of infectivity for tick vectors was considered as an ecological marker of TBE virus. This marker was designated Cg: Cg+ and Cg- for TBE virus strains inducing respectively higher and lower levels of viraemia than the threshold of infectivity.     

Streptomycin--an activator of persisting tick-borne encephalitis virus

The effect of streptomycin (C) on persistence of tick-borne encephalitis (TBE) virus in Syrian hamsters infected with 3 strains of the virus (41/65, Aina/1448, Vasilchenko ) intracerebrally or subcutaneously was studied. In the animals not given C the infectious virus could be detected in the brain for 8-14 days but not later although their organs (mostly brains and spleens) contained the hemagglutinating antigen and viral antigen detectable by immunofluorescence. Intramuscularly C was given twice daily for 13-35 days in a daily dose of 200 mg/kg. The C-treated hamsters yielded 7 virulent TBE virus strains: 3 from the brain, 3 from the spleen, and one from the blood. No virus could be isolated from the liver, kidneys, or lungs despite the use of various methods for isolation including tissue explantation. The activating effect of C was observed against the background of 4-fold decrease in the titre of complement-fixing and antihemagglutinating antibodies. C exerted its activating effect both at early (70 days) and late (9 months) stages of TBE virus persistence. The activating effect of C appears to be due to its immunosuppressive properties and neurotoxic action on the CNS.     

Long-term persistence of cell-mediated and humoral immune response in specifically resistant mice given live attenuated langat (E5 "14") virus.

In a long-term experiment, the features of humoral and cell-mediated immunity (CMI) were investigated in mice given intravenously (i.v.) a single dose of live attenuated Langat (E5 "14") virus of the tick-borne encephalitis (TBE) complex. The inapparent infection induced in mice specific resistance against challenge with virulent TBE virus, CMI was assayed by the capillary method of murine splenic lymphocyte migrarion inhibition (LMI) in the presence of partially purified TBE vurus. Significant LMI values were observed as early as 4 days after inoculation (p.i.), persisting for at least 15 months. Specific virus neutralizing (VN) antibodies were detected in the pooled serum of mice on the 4th day p.i. by a VN test potentiated with monospecific anti-mouse IgG serum. For the whole experimental period, significant antibody levels were found in the immunized mice which also displayed a complete resistance against 1000 s.c. LD50 of the virulent virus. Organ cultures of brain and spleen from these mice did not yield infections virus.     

Monoclonal antibodies to tick-borne encephalitis (TBE) virus: their use for differentiation of the TBE complex viruses.

Monoclonal antibodies (MoAbs) to Central European tick-borne encephalitis virus (strain Hypr) were used for differentiation of eight viruses of the TBE complex by indirect immunofluorescence. MoAb 11/B3 (in Western blot recognizing 52 and 70 kD polypeptides) reacted with five out of the eight TBE complex viruses, MoAb 13/E5 (anti-52 kD protein) reacted with the western or eastern subtype of TBE virus only, while MoAb 12/G4 (anti-70 kD protein) distinguished the western subtype of TBE virus from the rest of the TBE complex. These three MoAbs were able to differentiate the virulent strain Hypr from attenuated strains Skalica and Hy-HK-18-"3". MoAb 2/10C (anti-56 and 70 kD proteins) which reacted with all viruses of the TBE complex, recognized both virulent and attenuated strains of TBE virus.     

Persistence of tick-borne encephalitis virus IV. Virus localization after intracerebral inoculation

Tick-borne encephalitis (TBE) virus was isolated from the brains and spinal cords, blood, livers, lymph nodes and kidneys from Macaca rhesus monkeys showing acute and subacute fatal encephalitis. In subacute encephalitis, virus titres in the CNS were lower than in acute disease (3.0--6.2 against 3.8--8.3 log LD50/ml). TBE virus localization in chronic encephalitis was largely the same as in acute and subacute disease. In monkeys with a chronic course and stable paralysis of the upper extremity, infectious TBE virus was isolated on day 383 from subcortical ganglia and spinal cord. In lymph nodes and spleen, it could be detected only by a combination of methods (co-cultivation in association with fluorescent antibody technique and complement-fixation test, explantation of organ fragments) more sensitive than is the inoculation of mice with organ homogenates. TBE virus was detected by the same methods on day 90 in the CNS and internal organs of a monkey with chronic encephalitis in the stage of remission.     

Genetic characterisation of a tick-borne encephalitis virus isolated from the brain of a naturally exposed monkey (Macaca sylvanus)

In a recently published case study, we have described a clinical case of severe tick-borne encephalitis (TBE) in a monkey (Macaca sylvanus) after natural exposure (tick bite) in a TBE endemic area in Germany. Using histological, immunohistochemical, serological methods, and RT-PCR, the TBE virus infection was confirmed. Here, we describe the isolation of a TBE virus from the brain tissue in Vero-B4 cell cultures and the sequencing of the complete genome of that isolate. The isolated TBE virus strain (named ‘Salem’) is closely related to the Kumlinge strain or strain Neudoerfl, the prototype of the Central European TBE virus subtype. However, a total of 268 nt changes were found in comparison with TBE virus strain Neudoerfl resulting in 28 amino acid changes, none of which affecting any of the known or supposed functional regions of the viral genome. Further investigation of the distribution of viral antigen in brain tissue and characterisation of the host's inflammatory reactions by immunohistology revealed similarities between the course of the TBE virus infection in the macaque and acute to peracute cases of TBE in humans.     

Infection and replication of avian influenza H5N1 virus in an infected human

The highly pathogenic avian influenza H5N1 viruses usually cause severe diseases and high mortality in infected humans. However, the tissue tropism and underlying pathogenesis of H5N1 virus infection in humans have not been clearly elucidated yet. In this study, an autopsy was conducted to better understand H5N1 virus distributions in tissues of infected humans, and whether H5N1 virus can replicate in extrapulmonary tissues. We found that the lungs had the higher viral load than the spleen, whereas no detectable viruses in tissues of heart, liver, kidney, large intestine, small intestine, or brain. Specifically, the viral load was higher in the left lung (7.1 log10 copies per ml) in relation to the right lung (5.7 log10 copies per ml), resulting in more severe pathological damage in the left lung, and lung tissues contained both positive- and negative-stranded viral RNA. However, there existed a low level of H5N1 viruses in the spleen (3.8 log10 copies per ml), with the absence of positive-stranded viral RNA. Our results indicate that replication of H5N1 viruses mainly occurs in the lungs, and the degree of lung damage is highly correlated with the viral load in the lungs. The low-load viruses in the spleen might be introduced through blood circulation or other ways. Jing-Jiao Zhou and Dan-Yun Fang equally contributed to this work.     

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk,non-risk and former risk areas of TBE,and in ticks removed from humans in Germany

Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a former risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from volunteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized.     

Variations of four Aedes aegypti mosquito strains in their susceptibility to and transmissibility of Tahyna virus

Aedes aegypti mosquitoes originating from different colonies were infected with Tahyna virus by sucking on viraemic newborn mice (peak titre 10(2) X 5 mouse i.c. LD50/0.01 ml). The mosquitoes of laboratory strain "London" proved most susceptible (infection rate 74.2%, transmission rate 60%), while mosquitoes of the laboratory strain "Basel" and strain "Bangkok" (Southeast Asia) were less susceptible (infection rate 23.7 and 17.6%, respectively, transmission rate 6.0 and 10.0%, respectively). Mosquitoes of "Ifakara" strain (East Africa) were found resistant to Tahyna virus infection.     

Transmission of Kyasanur Forest disease virus by Rhipicephalus haemaphysaloides ticks.

Larvae of Rhipicephalus haemaphysaloides were infected with Kaysanur Forest disease (KFD) virus by feeding on viraemic rodents and reared into next generation larvae. Fed larvae, nymphs, unfed adults, fed adult males, and females after oviposition were found infected, while the larvae were found free from infection. Nymphs and adults transmitted the infection by bite to rodents and rabbits respectively. The virus was also passed through a second rodent-tick cycle. Adult ticks showed a titre of 3.1 to 4.5 dex mouse LD50/0.03 ml, and the virus was also detected 245 days after infection.     

Human antibody response to immunization with 17D yellow fever and inactivated TBE vaccine

The antibody response against flaviviruses tick-borne encephalitis (TBE), Kyasanur Forest disease (KFD), Murray Valley encephalitis (MVE), West Nile fever (WNF), Japanese B encephalitis (JE), dengue 2 (DEN-2), and yellow fever (YF) was studied in humans after administration of an inactivated TBE virus vaccine. Individuals were either prevaccinated with 17D yellow fever (experimental group) or without any previous exposure to flaviviruses (control group). The appearance of serum titres of homologous and heterologous haemagglutination inhibition (HI) antibodies, heterotypic DEN-2 neutralizing antibodies, and TBE enzyme-linked immunosorbent assay (ELISA) antibodies were examined. Individuals prevaccinated with the 17D yellow fever developed an antibody pattern that contrasted with that of the control group. This pattern was characterized as follows: (1) Predominantly anti-TBE IgG antibodies appeared earlier and in higher titres than in the control group, (2) heterologous HI antibodies cross-reacting with the WN flavivirus subgroup preceded the appearance of homologous HI antibodies, (3) a broad spectrum HI response was observed against all flaviviruses tested, and (4) low titre heterotypic DEN-2 neutralizing antibodies were formed in about half of the cases. These observations are discussed in the context of cross-reactivity, cross-protection and virus infection enhancement.     

Influence of type 2 T cell responses on the severity of encephalitis associated with influenza virus infection

The role of type 2 T cell responses on the severity of post-infectious encephalitis was investigated in a mouse model of influenza virus infection. When mice were infected intracerebrally with 3.0 LD(50) of A/NWS33 strain of influenza virus, they all showed clinical signs of encephalitis, and 90% of them died within 10 days of the infection. However, the post-infectious encephalitis was not demonstrated in mice exposed to 0.5 LD50 of the same virus. The mortality rates of mice infected with 0.5 LD(50) of the virus were increased to levels observed in mice exposed to 3.0 LD(50) of influenza virus infection, after the administration of a mixture of interleukin (IL)-4 and IL-10 (2 ng/mouse each; immediately, 1 and 2 days after the infection). In contrast, mortality rates of mice exposed to 3.0 LD(50) of influenza virus were substantially decreased when these mice were treated with a mixture of monoclonal antibodies directed against IL-4 and IL-10. A predominance of type 2 T cell responses was demonstrated in splenic T cells of mice infected with 3.0 LD(50) of influenza virus, although these responses were minimal in mice infected with 0.5 LD(50) of the virus. After the treatment with the mixture of type 2 cytokines, an increase in the type 2 T cell responses in mice exposed to 0.5 LD(50) of the virus was shown. These results indicate that type 2 T cell responses associated with the viral infection play an important role in the severity of post-infectious encephalitis induced in mice by the intracerebral infection of influenza A virus.     

Physicochemical properties of Teschen disease virus RNA

The specific infectivity of virion RNA of teschen disease virus in a sensitive PP cell culture was 4-5 lg TCD50/ml per 1 microgram RNA. When virion RNA was inoculated into cell cultures insusceptible to the native virus, the virus replicated to a titre of 2.0-3.5 lg TCD50/ml. The molecular weight of virion RNA determined by two independent methods was 2.7 x 10(6) daltons. Tm calculated from the curve of virion RNA melting temperature was 57 degrees C. The double-stranded replicative form of RNA recovered from virus-infected PP cells was shown to have sucrose gradient sedimentation coefficient of 20 S. The specific infectivity was 2-3 lg TCD50/ml per 1 microgram of RNA.     

Effect of rearing at non-permissive temperature on silkworm larvae infected with temperature-sensitive lef-8 mutant of bombyx mori nucleopolyhedrovirus.

A temperature-sensitive (ts) mutant of bombyx mori nucleopolyhedrovirus (BmNPV), ts-S1, contains a mutation in a putative RNA polymerase gene, which is involved in late viral gene expression. When 4th-instar silkworm larvae were infected with 1.0 x 10(5) TCID50 of ts-S1 per larva and reared at 33 degrees C, the titre of budded virus (BV) and number of occlusion bodies (OBs) in the haemolymph of the infected larvae were very low in the early stage but markedly increased in the late stage of infection. In contrast, a rapid increase in both BV titre and OB number was detected in the haemolymph of infected larvae reared at 25 degrees C. LD50 values of ts-S1 and wild type BmNPV (wtBmNPV) for 4th-instar larvae were 2.41 and 0.96 TCID50 per larva at 25 degrees C, and > 1.0 x 10(6) and 1.70 TCID50 per larva at 33 degrees C, respectively. These results indicate that the virulence of ts-S1 for the larvae reared at 33 degrees C was markedly reduced. To examine further the reduction of ts-S1 virulence at the non-permissive temperature of 33 degrees C, silkworm larvae were infected with ts-S1 at the multiplicity of 1.0 x 10(2) - 1.0 x 10(6) TCID50 per larva, reared for various time at 33 degrees C and then shifted to 25 degrees C. Longer rearing periods at 33 degrees C resulted in better survival rates indicating that the reduction of virulence of ts-S1 was proportional to cumulative rearing time at 33 degrees C. When large virus inocula were used, a growth alteration of larvae was preferentially induced. However, when small virus inocula were used, the appearance of abortive infection due to the non-permissive temperature became more evident.     

Restricted replication of the E5"14" clone of Langat TP21 virus (from the tick-borne encephalitis complex) in CNS of subadult mice

Subadult ICR mice were infected with the low virulent Langat virus TP21 E5 strain clone "14" belonging to the tick-borne encephalitis (TBE) complex by subcutaneous (s.c.) and intracerebral (i.c.) routes. From 5 to 6 days post infection (p.i.), no virus was detected in cultured brain fragments of mice, which received 10(6) ic LD50 into interscapular area. Acute lethal encephalitis with lesions confined to the vicinity of the inoculation area (parietal cortex, basal ganglia, thalamus) has developed in all mice, which received greater than or equal to 3 PFU of the virus by i.c. route. However, no virus was recovered from the cultured fragments of brain stem and cerebellum of these animals, although direct isolation attempts were regularly positive from brain cortex and basal ganglia. Survivors, which did not succumb to i.c. administration of approximately equal to 1 ic LD50 (0.3 PFU) of the attenuated Langat strain were autopsied between 53-74 days p.i. Attempts to isolate the virus from cultured fragments of brain cortex and basal ganglia remained negative despite of the presence of focal residual histological lesions in g. hippocampi in 15% of of animals examined.     

Target-Organ Treatment of Neurotropic Virus Disease with Interferon Inducers

Interferon inducers were used against vaccinial encephalitis to study the target-organ treatment of neurotropic disease and to correlate interferon levels and the antiviral state following such treatment. A 45-mug amount of statolon, 30 mug of polyribinosinic-polyribocytidylic acid complex (poly I.poly C), or 0.0154 HA unit of Sendai virus given intracerebrally protected 100% of mice challenged the next day with 1,000 median lethal doses (LD(50)) of vaccinia virus. Significant protection against 1,000 LD(50) of vaccinia virus persisted for 1, 4, or 3 weeks after poly I.poly C, statolon, or Sendai virus (154 HA units), respectively. These doses of poly I.poly C and statolon were also used to study postinfection treatment. Mice challenged with 1, 10, 100, or 1,000 LD(50) were treated intracerebrally with poly I.poly C or statolon 24 or 48 hr later. Significant increases in survival time were seen in mice challenged with 1 to 100 LD(50) of vaccinia virus and treated 24 hr later. At challenges of 10 or 100 LD(50), statolon was more effective than poly I.poly C in increasing survival times. When treatment was delayed until 48 hr after infection, significant increases in survival time occurred only when the challenges were in the range of 1 to 10 LD(50), with poly I.poly C and statolon being equally effective. Interferon was measured by Finter's dye-uptake method, with L-929 cells and Semliki Forest virus. Poly I.poly C, statolon, or Sendai virus, given intracerebrally to mice, produced serum interferon peaks of 5,120 units/ml at 2 hr, 2,560 units/ml at 12 hr, or 320 units/ml at 18 hr, respectively. Corresponding brain interferon peaks were 640 units/g at 2 hr, 640 units/g at 4 to 24 hr, and 960 units/g at 72 hr.