Åke Lundkvist

Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia

Dobrava hantavirus (DOB) was isolated from the striped field mouse (Apodemus agrarius) trapped on Saaremaa Island, Estonia, and its genetic and antigenic characteristics were subsequently analysed. Phylogenetic analysis showed that the Estonian DOB strain, together with several wild strains carried by Apodemus agrarius, forms a well-supported lineage within the DOB clade. The topography of the trees calculated for the S, M and L nucleotide sequences of the Estonian DOB suggests a similar evolutionary history for all three genes of this virus and, therefore, the absence of heterologous reassortment in its evolution. A cross-neutralization comparison of the Estonian virus with the prototype DOB, isolated from a yellow-necked mouse (A. flavicollis) in Slovenia, revealed 2- to 4-fold differences in the end-point titres of rabbit and human antisera. When studied with a panel of 25 monoclonal antibodies (MAbs), the Estonian and Slovenian DOB isolates showed similar antigenic patterns that could be distinguished by two MAbs. Genetic comparison showed sequence differences in all three genome segments of the two DOB isolates, including an additional N-glycosylation site in the deduced sequence of the G2 protein from the Estonian virus. Whether any of these mutations relates to the different rodent hosts rather than to the distant geographical origin of the two isolates remains to be resolved. Taken together, our observations suggest that A. agrarius, which is known to harbour Hantaan virus in Asia, carries another hantavirus, DOB, in north-east Europe.

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.

How Did Zika Virus Emerge in the Pacific Islands and Latin America

ABSTRACT The unexpected emergence of Zika virus (ZIKV) in the Pacific Islands and Latin America and its association with congenital Zika virus syndrome (CZVS) (which includes microcephaly) and Guillain-Barré syndrome (GBS) have stimulated wide-ranging research. High densities of susceptible Aedes spp., immunologically naive human populations, global population growth with increased urbanization, and escalation of global transportation of humans and commercial goods carrying vectors and ZIKV undoubtedly enhanced the emergence of ZIKV. However, flavivirus mutations accumulate with time, increasing the likelihood that genetic viral differences are determinants of change in viral phenotype. Based on comparative ZIKV complete genome phylogenetic analyses and temporal estimates, we identify amino acid substitutions that may be associated with increased viral epidemicity, CZVS, and GBS. Reverse genetics, vector competence, and seroepidemiological studies will test our hypothesis that these amino acid substitutions are determinants of epidemic and neurotropic ZIKV emergence.

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.

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.

Hepatitis E Virus in Domestic Pigs, Wild Boars, Pig Farm Workers, and Hunters in Estonia

AbstractWhile hepatitis E is a growing health concern in Europe, epidemiological data on hepatitis E virus (HEV) in Estonia are scarce. Along with imported HEV infections, autochthonous cases are reported from European countries. Both domestic and wild animals can be a source of human cases of this zoonosis. Here, we investigated the presence of anti-HEV antibodies and HEV RNA in domestic pigs and wild boars, as well as in pig farm workers and hunters in Estonia. Anti-HEV antibodies were detected in 234/380 (61.6xa0%) of sera from domestic pigs and in all investigated herds, and in 81/471 (17.2xa0%) of meat juice samples from wild boars. HEV RNA was detected by real-time PCR in 103/449 (22.9xa0%) of fecal samples from younger domestic pigs and 13/81 (16.0xa0%) of anti-HEV-positive wild boar samples. Analysis of sera from 67 pig farm workers and 144 hunters revealed the presence of HEV-specific IgG in 13.4 and 4.2xa0% of the samples, respectively. No HEV RNA was detected in the human serum samples. Phylogenetic analyses of HEV sequences from domestic pigs and wild boars, based on a 245xa0bp fragment from the open reading frame 2 nshowed that all of them belonged to genotype 3. The present study demonstrates the presence of HEV in Estonian domestic pig and wild boar populations, as well as in humans who have direct regular contact with these animals. Our results suggest that HEV infections are present in Estonia and require attention.

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.

Serological Evidence of Human Hantavirus Infections in Indonesia

Here we describe the first evidence of hantavirus infection in humans in Indonesia. Hantaviruses belong to the family Bunyaviridae. Several serotypes have been described to cause disease in man. Hantaan (HNT), Seoul (SEO), Dobrava (DOB) and Puumala (PUU) hantaviruses cause severe to milder hemorrhagic fever with renal syndrome (HFRS) [1]. New World hantavirus serotypes (e.g. Sin Nombre [SNV],Andes) are associated with pulmonary syndrome (hantavirus pulmonary syndrome [HPS]) [1]. In Southeast Asia, evidence of hantavirus infections have been reported from Korea, the Philippines, Singapore and Thailand [2–5]. In Indonesia, a serological study indicated the presence of a Seoul-like virus in wild rats [6].So far no human cases of hantavirus infection have been reported from this country. Serum samples from 94 febrile patients initially suspected of a dengue virus infection were investigated for the presence of hantavirus-specific antibodies.All patients were residents of Yogyakarta or Semarang, Central Java, Indonesia and had presented as outpatients or had been admitted to hospital with symptoms of febrile illness between May 1995 and January 1996 during a dengue epidemic. Patients were divided into two age-groups; the first group included 69 patients in the age range 2–20 years (mean 13.9 years), the second group included 25 patients in the age range 21–47 years (mean 27.2 years). Dengue virus-specific serology in serum samples of these 94 patients was not indicative of recent infection with this virus.Differential diagnosis included measles, rubella, influenza and chikungunya viruses, as well as rickettsiosis and leptospirosis. Nevertheless, the etiology of their illness remained elusive. Serial serum samples of each patient were tested for the presence of hantavirus-specific IgM serum antibodies with a commercially available enzyme immunoassay (EIA) (Focus Technologies,Cypress,USA),using microplates coated with a mixture of SEO and SNV virus recombinant nucleocapsid proteins [7]. Positive EIA results were confirmed by immunofluorescence assay (using SEO, HNT and PUU serotypes) [8] and tested further for the presence of hantavirus-specific IgG serum antibodies by EIA [7], IFA [8] and immunoblotting using native hantavirus antigens, essentially as described earlier [9] (Table 1). In ten (11%) of the 94 patients, serology indicated a recent hantavirus infection.Five had hantavirus-specific IgM and IgG serum antibodies and five had hantavirus-specific IgM but no specific IgG serum antibodies. The ratio in the EIA for the positive samples ranged between 1.1 and 3.1 and between 1.6 and 4.6 for the IgM and IgG, respectively. In two patients only hantavirusspecific IgG serum antibodies were demonstrated, indicating a past infection. Seven sera were confirmed positive for hantavirus-specific IgG serum antibodies by immunoblotting.The serum samples of these patients reacted predominantly with the PUU serotype in the IFA (titers ranged between 64 and ≥ 256) and to a lesser extent with HNT and SEO serotypes (titers ranged between 16 and 128).The differences between IFA and ELISA pattern were probably caused by the fact that the antigen in the IFA is based on whole virus, whereas recombinant proteins were used in the ELISA.The scattered serological pattern in IFA and immunoblot might also indicate that an unknown hantavirus serotype is circulating in this region. Nine of the ten recently infected patients were under 20 years of age (mean 17.6 years), the youngest being 13 years old.This finding is in agreement with previous observations, showing the majority of hantavirus patients to be older than 15 years [10]. Previous epidemiological studies have demonstrated that the distribution of hantavirus infection in Europe is skewed towards the male population,whereas in Southeast Asia the distribution of hantavirus infections is almost equal among the sexes, which may be related to outdoor occupational activities [3,10]. In our study,eight of the patients were

Global population divergence and admixture of the brown rat (Rattus norvegicus)

Native to China and Mongolia, the brown rat (Rattus norvegicus) now enjoys a worldwide distribution. While black rats and the house mouse tracked the regional development of human agricultural settlements, brown rats did not appear in Europe until the 1500s, suggesting their range expansion was a response to relatively recent increases in global trade. We inferred the global phylogeography of brown rats using 32 k SNPs, and detected 13 evolutionary clusters within five expansion routes. One cluster arose following a southward expansion into Southeast Asia. Three additional clusters arose from two independent eastward expansions: one expansion from Russia to the Aleutian Archipelago, and a second to western North America. Westward expansion resulted in the colonization of Europe from which subsequent rapid colonization of Africa, the Americas and Australasia occurred, and multiple evolutionary clusters were detected. An astonishing degree of fine-grained clustering between and within sampling sites underscored the extent to which urban heterogeneity shaped genetic structure of commensal rodents. Surprisingly, few individuals were recent migrants, suggesting that recruitment into established populations is limited. Understanding the global population structure of R. norvegicus offers novel perspectives on the forces driving the spread of zoonotic disease, and aids in development of rat eradication programmes.

First evidence of Seoul hantavirus in the wild rat population in the Netherlands

Abstract We report the first detection of Seoul hantavirus (SEOV)-specific antibodies in the wild brown rat population in the Netherlands. SEOV-reactive antibodies were found in three rats out of 16 in a repeated series of tests including immunofluorescence assay, immunoblot, and enzyme-linked immunosorbent assay. Focus reduction neutralization test confirmed the presence of SEOV-specific antibodies, and reverse-transcription polymerase chain reaction (RT-PCR) confirmed the presence of hantaviral RNA. This discovery follows the recent findings of SEOV infections in wild and pet brown rats and humans in England, Wales, France, Belgium, and Sweden, indicating an even higher importance of this hantavirus for public health in large areas of Europe.