High prevalence of Seoul hantavirus in a breeding colony of pet rats.

As part of further investigations into three linked haemorrhagic fever with renal syndrome (HFRS) cases in Wales and England, 21 rats from a breeding colony in Cherwell, and three rats from a household in Cheltenham were screened for hantavirus. Hantavirus RNA was detected in either the lungs and/or kidney of 17/21 (81%) of the Cherwell rats tested, higher than previously detected by blood testing alone (7/21, 33%), and in the kidneys of all three Cheltenham rats. The partial L gene sequences obtained from 10 of the Cherwell rats and the three Cheltenham rats were identical to each other and the previously reported UK Cherwell strain. Seoul hantavirus (SEOV) RNA was detected in the heart, kidney, lung, salivary gland and spleen (but not in the liver) of an individual rat from the Cherwell colony suspected of being the source of SEOV. Serum from 20/20 of the Cherwell rats and two associated HFRS cases had high levels of SEOV-specific antibodies (by virus neutralisation). The high prevalence of SEOV in both sites and the moderately severe disease in the pet rat owners suggest that SEOV in pet rats poses a greater public health risk than previously considered.

Expansion of spatial and host range of Puumala virus in Sweden: an increasing threat for humans?

Hantaviruses are globally distributed and cause severe human disease. Puumala hantavirus (PUUV) is the most common species in Northern Europe, and the only hantavirus confirmed to circulate in Sweden, restricted to the northern regions of the country. In this study, we aimed to further add to the natural ecology of PUUV in Sweden by investigating prevalence, and spatial and host species infection patterns. Specifically, we wanted to ascertain whether PUUV was present in the natural reservoir, the bank vole (Myodes glareolus) further south than Dalälven river, in south-central Sweden, and whether PUUV can be detected in other rodent species in addition to the natural reservoir. In total, 559 animals were collected at Grimsö (59°43'N; 15°28'E), Sala (59°55'N; 16°36'E) and Bogesund (59°24'N; 18°14'E) in south-central Sweden between May 2013 and November 2014. PUUV ELISA-reactive antibodies were found both in 2013 (22/295) and in 2014 (18/264), and nine samples were confirmed as PUUV-specific by focus reduction neutralization test. Most of the PUUV-specific samples were from the natural host, the bank vole, but also from other rodent hosts, indicating viral spill-over. Finally, we showed that PUUV is present in more highly populated central Sweden.

Seoul hantavirus in brown rats in the Netherlands: implications for physicians--Epidemiology, clinical aspects, treatment and diagnostics.

The recent discovery of Seoul hantavirus (SEOV) presence in wild rat populations in the Netherlands has direct implications for Dutch clinicians and hantavirus diagnostics. SEOV is amongst the Old World hantaviruses which cause haemorrhagic fever and renal syndrome (HFRS) in humans. HFRS is characterised by a classical triad of fever, acute kidney injury and haemorrhage, but can show different signs and symptoms in specific cases. SEOV is transmitted from infected rats to humans by inhalation of aerosolised excreta. When compared with the known circulating hantaviruses in the Netherlands, Puumala (PUUV) and Tula (TULV), SEOV causes a more severe form of HFRS. Data from cohort studies undertaken in China and Northern Europe show differences in signs and symptoms at onset of disease, (haemorrhagic) complications and mortality. Furthermore, routine diagnostics currently available for hantavirus diagnosis in the Netherlands are not optimised for SEOV detection. The clinical outcome of an SEOV and PUUV infection will greatly benefit from an early diagnosis which will reduce the costs of unnecessary tests and treatments as well. The discovery of SEOV circulation in the Netherlands follows recent findings of SEOV infections in both rodents and humans in England, Wales, France, Belgium and Sweden, indicating the emerging character of SEOV and a high importance of this hantavirus for Public Health in large areas of Europe. Here, we review the current knowledge on the clinical manifestation of SEOV versus PUUV infections in humans, the treatment of clinical cases and diagnostics.

The hanta hunting study: underdiagnosis of Puumala hantavirus infections in symptomatic non-travelling leptospirosis-suspected patients in the Netherlands, in 2010 and April to November 2011.

Leptospirosis and haemorrhagic fever with renal syndrome (HFRS) are hard to distinguish clinically since these two important rodent-borne zoonoses share hallmark symptoms such as renal failure and haemorrhage. Leptospirosis is caused by infection with a spirochete while HFRS is the result of an infection with certain hantaviruses. Both diseases are relatively rare in the Netherlands. Increased incidence of HFRS has been observed since 2007 in countries that border the Netherlands. Since a similar rise in incidence has not been registered in the Netherlands, we hypothesise that due to overlapping clinical manifestations, hantavirus infections may be confused with leptospirosis, leading to underdiagnosis. Therefore, we tested a cohort of non-travelling Dutch patients with symptoms compatible with leptospirosis, but with a negative diagnosis, during 2010 and from April to November 2011. Sera were screened with pan-hantavirus IgG and IgM enzyme-linked immunosorbent assays (ELISAs). Sera with IgM reactivity were tested by immunofluorescence assay (IFA). ELISA (IgM positive) and IFA results were confirmed using focus reduction neutralisation tests (FRNTs). We found hantavirus-specific IgG and/or IgM antibodies in 4.3% (11/255) of samples taken in 2010 and in 4.1% (6/146) of the samples during the 2011 period. After FRNT confirmation, seven patients were classed as having acute Puumala virus infections. A review of hantavirus diagnostic requests revealed that at least three of the seven confirmed acute cases as well as seven probable acute cases of hantavirus infection were missed in the Netherlands during the study period.

Stability of a formalin-inactivated Rift Valley fever vaccine: evaluation of a vaccination campaign for cattle in Mozambique.

In Africa and the Arabian Peninsula, outbreaks of Rift Valley fever (RVF) are characterized by abortions in gestating animals and high mortality rates among domestic ruminants. An immunization program using a formalin-inactivated vaccine was initiated in Mozambique in 2002 to control RVF in cattle. In this intervention, the vaccine must be transported for more than a week within the country before it can be administered to the animals, and it is practically impossible to maintain low storage temperatures during that time. Here, we evaluated the influence of transportation conditions on the efficacy of the vaccine. Sixty-three previously unvaccinated and RVF virus seronegative cattle were divided into four groups, which were given vaccine that had been stored for 1 week at 4°C (n=9, group A), at 25°C (n=8, group B), or alternating between 4 and 25°C (n=8, group C), or under the temperature conditions ordinarily occurring during transportation within Mozambique (n=38, group D). The antibody responses induced were monitored for 6-9 months and in some animals up to 21 months. Two immunizations (3 weeks apart) with the formalin-inactivated vaccine induced a long-lasting neutralizing antibody response that was still detectable up to 21 months later. The antibody titers in the animals did not differ significantly between the temperature-assigned vaccine groups A, B, and C, whereas they were significantly higher in group D. These results show that the formalin-inactivated RVF virus vaccine is stable, and, importantly, it is not adversely affected by the variation in temperature that ordinarily occurs during transport within Mozambique.

Identification of Anaplasma phagocytophilum in tick populations in Estonia, the European part of Russia and Belarus.

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.

Dengue virus infection is the most frequent arthropod-borne infection affecting humans in the world. Our understanding of the pathophysiological events leading to mild or severe outcomes of the disease remains limited by the fact that viral target cells in the human body are poorly characterized. One of the most sensitive strategies for detecting cells supporting active replication of this positive-strand RNA virus is the search for the replicative intermediate, an antigenome of negative polarity, by RT-PCR. However, a phenomenon described as 'false priming' of the reverse transcriptase (RT) prevents strand-specific detection. The results of the current study showed that this event corresponds to cDNA synthesis that is independent of any primer addition. This property was general to all RNAs tested and was not associated with small free nucleic acids, such as tRNAs and microRNAs. Rather, it corresponded to initiation of cDNA synthesis from the 3' end of the RNA template, and a model is proposed in which the template RNA snaps back upon itself and creates a transient RNA primer suitable for the RT. Such a property would explain why many assays proposed for detection of a replicative intermediate are not specific, and may help in the development of a molecular biology protocol that could allow replication studies of RNA viruses of human interest, such as dengue virus, hepatitis C virus and enteroviruses.

The European network of Biosafety-Level-4 laboratories: enhancing European preparedness for new health threats.

Emerging and re-emerging infections and possible bioterrorism acts will continue to challenge both the medical community and civilian populations worldwide, urging health authorities to respond rapidly and effectively. Established in 2005, the European Community (EC)-funded European Network of Biosafety-Level-4 laboratories (Euronet-P4), which brings together the laboratories in Porton Down, London, Hamburg, Marburg, Solna, Lyon and Rome, seeks to increase international collaboration in the areas of high containment laboratory biosafety and viral diagnostic capability, to strengthen Europe's capacity to respond to an infectious disease emergency, and to offer assistance to countries not equipped with such costly facilities. Network partners have agreed on a common strategy to fill the gaps identified in the field of risk group-4 agents' laboratory diagnosis, namely the lack of standardization and of reference samples. The network has received a further 3-year funding, to offer assistance to external laboratories, and to start the planning of field activities.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium.

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

Gene segment reassortment between American and Asian lineages of avian influenza virus from waterfowl in the Beringia area.

Since prehistoric times, the Bering Strait area (Beringia) has served as an avenue of dispersal between the Old and the New Worlds. On a field expedition to this area, we collected fecal samples from dabbling ducks, geese, shorebirds, and gulls on the Chukchi Peninsula, Siberia, and Pt. Barrow, Alaska, and characterized the subtypes of avian influenza virus present in them. Four of 202 samples (2%) from Alaska were positive for influenza A virus RNA in two independent polymerase chain reaction (PCR)-based screening assays, while all shorebird samples from the Chukchi Peninsula were negative. Subtypes H3N8 and H6N1 were recorded once, while subtype H8N4 was found in two samples. Full-length sequences were obtained from the three unique isolates, and phylogenetic analysis with representative sequences for the Eurasian and North American lineages of influenza A virus showed that one HA gene clustered with the Eurasian rather than the North American lineage. However, the closest relative to this sequence was a North American isolate from Delaware described in 2002, indicating that a H6 spillover from Asia has established itself in North America.

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Exogenous nitric oxide inhibits Crimean Congo hemorrhagic fever virus.

Crimean Congo hemorrhagic fever virus (CCHFV) is a geographically widespread pathogen that causes severe hemorrhagic fever with high mortality. Even though one of the main objectives focuses on the progress of antiviral agents, the research on CCHFV is strongly hampered due to its BSL-4 classification. Nitric oxide (NO), a mediator with broad biological effects, has been shown to possess inhibitory properties against various pathogens. The molecule constitutes a component of the innate immunity and serves to assist in the early immunological events where it contributes to clearance of microorganisms. In this study, we investigated the inhibitory properties of exogenous NO on CCHFV. We found that NO had a significant antiviral activity against CCHFV replication. By using the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) we were able to show up to 99% reduction in virion progeny yield. In contrast, 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, had no significant antiviral activity against CCHFV. Furthermore the expression of viral proteins; the nucleocapsid protein and the glycoprotein, were clearly reduced with increasing concentrations of SNAP. We have also shown that the amount of total vRNA in SNAP-treated cells was reduced by about 50% compared to the controls.

Delayed viremia and antibody responses in Puumala hantavirus challenged passively immunized cynomolgus macaques.

No specific therapy is currently available against hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome. In order to study if passive immunization could inhibit hantavirus infection and/or symptoms, we inoculated two cynomolgus macaques with neutralizing monoclonal antibodies and subsequently challenged them with wild-type Puumala virus (PUUV), recently shown to induce typical signs of milder HFRS in cynomolgus macaques. Although viral load and antibody titers did not differ substantially as compared to the two control monkeys, a delayed onset of viremia and seroconversion was observed in the immunized monkeys. Interestingly, one of the immunized monkeys showed no symptoms, nor elevated of levels of IL-6, IL-10, and TNF-alpha, while the other developed severe symptoms and elevated levels of those cytokines, believed to be involved in PUUV-pathogenesis.

Tick-borne virus diseases of human interest in Europe.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.

Rapid field test for detection of hantavirus antibodies in rodents.

Puumala virus (PUUV) is the causative agent of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome. PUUV is transmitted to humans via aerosolized excreta of the infected bank vole (Clethrionomys glareolus). Current methods for screening of the PUUV prevalence among bank vole populations are laborious, combining sampling in the field and subsequent analyses in the laboratory. In order to facilitate animal testing, a new serological immunochromatographic rapid test was developed. The test uses PUUV nucleocapsid protein as antigen, and it detects anti-PUUV IgG antibodies in rodents. With fresh and undiluted bank-vole blood samples (n = 105) the efficacy of the test was 100%, and with frozen and diluted samples (n = 78) the efficacy was 91%. The test was also shown to detect related hantavirus infections in Norway lemmings and sibling voles (n = 31) with 99% efficacy. The test provides an applicable tool for studying PUUV and related hantavirus infections in arvicoline rodents.

Distribution of hantavirus foci in Belgium.

During 1999 and 2000, we performed rodent captures on 15 sites all over Belgium to evaluate the presence of hantaviruses in local rodent populations. Viral antibody and RNA detection was performed by ELISA/focus reduction neutralisation test and RT-PCR, respectively. We found hantavirus-positive rodents on 13 out of 15 trapping sites and 3 rodent species were found positive for hantavirus infection. Apart from Puumala virus that was carried by Clethrionomys glareolus, 2 additional rodent species, Microtus arvalis and Apodemus sylvaticus, were found antibody- and/or RNA-positive.