Life-long shedding of Puumala hantavirus in wild bank voles (Myodes glareolus).

The knowledge of viral shedding patterns and viraemia in the reservoir host species is a key factor in assessing the human risk of zoonotic viruses. The shedding of hantaviruses (family Bunyaviridae) by their host rodents has widely been studied experimentally, but rarely in natural settings. Here we present the dynamics of Puumala hantavirus (PUUV) shedding and viraemia in naturally infected wild bank voles (Myodes glareolus). In a monthly capture-mark-recapture study, we analysed 18 bank voles for the presence and relative quantity of PUUV RNA in the excreta and blood from 2 months before up to 8 months after seroconversion. The proportion of animals shedding PUUV RNA in saliva, urine and faeces peaked during the first month after seroconversion, but continued throughout the study period with only a slight decline. The quantity of shed PUUV in reverse transcription quantitative PCR (RT-qPCR) positive excreta was constant over time. In blood, PUUV RNA was present for up to 7 months but both the probability of viraemia and the virus load declined with time. Our findings contradict the current view of a decline in virus shedding after the acute phase and a short viraemic period in hantavirus infection - an assumption widely adopted in current epidemiological models. We suggest the life-long shedding as a means of hantaviruses to survive over host population bottlenecks, and to disperse in fragmented habitats where local host and/or virus populations face temporary extinctions. Our results indicate that the kinetics of pathogens in wild hosts may differ considerably from those observed in laboratory settings.

Dark chocolate and reduced snack consumption in mildly hypertensive adults: an intervention study.

BACKGROUND: Several studies have shown that cocoa and cocoa-containing foods have the potential to lower blood pressure and improve endothelial function. Most of the studies reporting the beneficial effects of dark chocolate on blood pressure have been short (≤ 4 weeks). The aim of the present 8-wks (weeks) study was to assess the effects of regular consumption of dark chocolate during a reduced snack consumption intervention on blood pressure and other cardiovascular risk factors in mildly hypertensive individuals. DESIGN: This was a randomized, controlled, cross-over trial involving 22 adults (8 women, 14 men), aged 33-64 y, BMI 27.7 ± 3.7 kg/m(2) with mild hypertension. During the intervention period (8-wks) the participants reduced the intake of habitual snacks and replaced them with dark chocolate (49 g/day). In the control period, they only reduced the snacks without any added chocolate. Data (blood lipid profile, glucose, insulin, 24 h blood pressure) was collected in the beginning and end of both periods (intervention and control), and some variables also in the run-in and run-out periods (weight, body fat percentage, blood pressure, arterial stiffness index, diet and physical activity). RESULTS: Daily consumption of dark chocolate had no effects on 24 h blood pressure, resting blood pressure (mean ± SD, pre 142 ± 11.5/89 ± 8.4 mmHg vs. post 142 ± 14.2/88 ± 9.4 mmHg in systolic and diastolic blood pressure, respectively) or arterial stiffness (mean ± SD, pre 7.68 ± 0.88 vs. post 7.76 ± 0.89). Weight was reduced by 1.0 ± 2.2 kg during the control (reduced snack only) period, but was unchanged while eating chocolate (p < 0.027 between the treatments). CONCLUSION: The data collected in this study indicates that inclusion of dark chocolate daily in the diet had no significant effects on blood pressure or other cardiovascular risk factors during a reduced snack period. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02130141.

Surveillance of endemic foci of tick-borne encephalitis in Finland 1995-2013: evidence of emergence of new foci.

The geographical risk areas for tick-borne encephalitis (TBE) in Finland remained the same until the beginning of the 21st century, but a considerable geographical expansion has been observed in the past 10 years. In order to support public health measures, the present study describes the number of laboratory-confirmed TBE cases and laboratory tests conducted and the associated trends by hospital district, with a particular emphasis on the suspected geographical risk areas. An additional investigation was conducted on 1,957 clinical serum samples throughout the country taken from patients with neurological symptoms to screen for undiagnosed TBE cases. This study identified new TBE foci in Finland, reflecting the spread of the disease into new areas. Even in the most endemic municipalities, transmission of TBE to humans occurred in very specific and often small foci. The number of antibody tests for TBE virus more than doubled (an increase by 105%) between 2007 and 2013. Analysis of the number of tests also revealed areas in which the awareness of clinicians may be suboptimal at present. However, it appears that underdiagnosis of neuroinvasive TBE is not common.

Tick-borne encephalitis virus in wild rodents in winter, Finland, 2008-2009.

Rodents might maintain tick-borne encephalitis virus (TBEV) in nature through latent persistent infections. During 2 subsequent winters, 2008 and 2009, in Finland, we detected RNA of European and Siberian subtypes of TBEV in Microtus agrestis and Myodes glareolus voles, respectively. Persistence in rodent reservoirs may contribute to virus overwintering.

Recent establishment of tick-borne encephalitis foci with distinct viral lineages in the Helsinki area, Finland.

Number of tick-borne encephalitis (TBE) cases has increased and new foci have emerged in Finland during the last decade. We evaluated risk for locally acquired TBE in the capital region inhabited by 1.2 million people. We screened ticks and small mammals from probable places of TBE virus (TBEV) transmission and places without reported circulation. The TBEV positive samples were sequenced and subjected to phylogenetic analysis. Within the study period 2007-2017, there was a clear increase of both all TBE cases and locally acquired cases in the Helsinki area. The surveillance of ticks and small mammals for TBEV confirmed four distinct TBEV foci in the Helsinki area. All detected TBEV strains were of the European subtype. TBEV genome sequences indicated that distinct TBEV lineages circulate in each focus. Molecular clock analysis suggested that the virus lineages were introduced to these foci decades ago. In conclusion, TBE has emerged in the mainland of Helsinki area during the last decade, with at least four distinct virus lineages independently introduced into the region previously. Although the overall annual TBE incidence is below the threshold for recommending general vaccinations, the situation requires further surveillance to detect and prevent possible further emergence of local TBE clusters.

Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

Serological evidence of tick-borne encephalitis virus infection in moose and deer in Finland: sentinels for virus circulation.

BACKGROUND: The incidence of tick-borne encephalitis (TBE) in humans has increased in Finland, and the disease has emerged in new foci. These foci have been investigated to determine the circulating virus subtype, the tick host species and the ecological parameters, but countrywide epidemiological information on the distribution of TBEV has been limited. METHODS: In this study, we screened sera from hunter-harvested wild cervids for the presence of antibodies against tick-borne encephalitis virus (TBEV) with a hemagglutination inhibition test. The positive results were confirmed by a neutralisation assay. RESULTS: Nine (0.74%) of 1213 moose, one (0.74%) of 135 white-tailed deer, and none of the 17 roe deer were found seropositive for TBEV. A close geographical congruence between seropositive cervids and recently reported human TBE cases was observed: nine of the ten seropositive animals were from known endemic areas. CONCLUSIONS: Our results confirm the local circulation of TBEV in several known endemic areas. One seropositive moose had been shot in an area where human TBE cases have not been reported, suggesting a possible new focus. Moose appear to be a useful sentinel animal for the presence of TBEV in the taiga region.

Siberian subtype tick-borne encephalitis virus in Ixodes ricinus in a newly emerged focus, Finland.

The first tick-borne encephalitis (TBE) cases in Kotka, Finland appeared in 2010. Altogether ten human cases have been diagnosed by 2014. Four had long-lasting sequelae. We collected 195 Ixodes ricinus ticks, nine rodents, and eleven shrews from the archipelago of Kotka in 2011. Three Siberian subtype TBE virus (TBEV) strains were isolated from the ticks and three mammals were positive for TBEV antibodies. The archipelago of Kotka is a newly emerged TBE focus of Siberian subtype TBEV circulating notably in I. ricinus. The patients had on average longer hospitalization than reported for the European subtype infection.

Assessing the abundance, seasonal questing activity, and Borrelia and tick-borne encephalitis virus (TBEV) prevalence of Ixodes ricinus ticks in a Lyme borreliosis endemic area in Southwest Finland.

Studies have revealed that Ixodes ricinus (Acari: Ixodidae) have become more abundant and their geographical distribution extended northwards in some Nordic countries during the past few decades. However, ecological data of tick populations in Finland are sparse. In the current study, I. ricinus abundance, seasonal questing activity, and their Borrelia spp. and tick-borne encephalitis virus (TBEV) prevalence were evaluated in a Lyme borreliosis endemic area in Southwest Finland, Seili Island, where a previous study mapping tick densities was conducted 12 years earlier. A total of 1940 ticks were collected from five different biotopes by cloth dragging during May-September 2012. The overall tick density observed was 5.2 ticks/100m(2) for nymphs and adults. Seasonal questing activity of ticks differed between biotopes and life stages: bimodal occurrences were observed especially for nymphal and adult ticks in forested biotopes, while larvae in pastures exhibited mostly unimodal occurrence. Prevalence of Borrelia and TBEV in ticks was evaluated using conventional and real-time PCR. All samples were negative for TBEV. Borrelia prevalence was 25.0% for adults (n=44) and the minimum infection rate (MIR) 5.6% for pooled nymph samples (191 samples, 1-14 individuals per sample; 30/191 positive). No Borrelia were detected in pooled larval samples (63 samples, 1-139 individuals per sample). Five species of Borrelia were identified from the samples: B. afzelii, B. burgdorferi s.s., B. garinii, B. valaisiana and B. miyamotoi. In Finland, B. valaisiana and B. miyamotoi have previously been reported from the Åland Islands but not from the mainland or inner archipelago. The results of the present study suggest an increase in I. ricinus abundance on the island.

The three subtypes of tick-borne encephalitis virus induce encephalitis in a natural host, the bank vole (Myodes glareolus).

Tick-borne encephalitis virus (TBEV) infects bank voles (Myodes glareolus) in nature, but the relevance of rodents for TBEV transmission and maintenance is unclear. We infected colonized bank voles subcutaneously to study and compare the infection kinetics, acute infection, and potential viral persistence of the three known TBEV subtypes: European (TBEV-Eur), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). All strains representing the three subtypes were infective and highly neurotropic. They induced (meningo)encephalitis in some of the animals, however most of the cases did not present with apparent clinical symptoms. TBEV-RNA was cleared significantly slower from the brain as compared to other organs studied. Supporting our earlier findings in natural rodent populations, TBEV-RNA could be detected in the brain for up to 168 days post infection, but we could not demonstrate infectivity by cell culture isolation. Throughout all time points post infection, RNA of the TBEV-FE was detected significantly more often than RNA of the other two strains in all organs studied. TBEV-FE also induced prolonged viremia, indicating distinctive kinetics in rodents in comparison to the other two subtypes. This study shows that bank voles can develop a neuroinvasive TBEV infection with persistence of viral RNA in brain, and mount an anti-TBEV IgG response. The findings also provide further evidence that bank voles can serve as sentinels for TBEV endemicity.