Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands.

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

Development of a Comparative European Orthohantavirus Microneutralization Assay With Multi- Species Validation and Evaluation in a Human Diagnostic Cohort.

Orthohantaviruses (family Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the 'Gold Standard' for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.

Hepatitis E virus seroprevalence among the general population in a livestock-dense area in the Netherlands: a cross-sectional population-based serological survey.

BACKGROUND: Recent serological studies indicate that hepatitis E virus (HEV) is endemic in industrialised countries. The increasing trend in the number of autochthonous cases of HEV genotype 3 in Western European countries, stresses the importance to get insight in the exact routes of exposure. Pigs are the main animal reservoir, and zoonotic food-borne transmission of HEV is proven. However, infected pigs can excrete large amounts of virus via their faeces enabling environmental transmission of HEV to humans. This might pose a risk for of neighbouring residents of livestock farming. METHODS: Within a large study on the health of people living in the vicinity of livestock farming we performed a cross-sectional population-based serological survey among 2,494 non-farming adults from the general population in a livestock-dense area in the south of the Netherlands. Participants completed risk factor questionnaires and blood samples of 2,422 subjects (median age 58 years, range 20-72) were tested for anti-HEV IgG using an enzyme immune assay (Wantai). The aim of this study was to determine the HEV seroprevalence and to assess whether seropositivity in adults was associated with living in the vicinity of pig farms. RESULTS: The average seroprevalence of HEV was 28.7% (95% CI: 26.9-30.5). Determinants associated with an increased risk for HEV seropositivity were male gender and low level of education. There was a clear trend of increasing prevalence with increasing age (Chi-square test for linear trend, X2 = 83.1; p < 0.001). A high number of pigs within 1,000 m of the residential address was not a risk factor for seropositivity. CONCLUSIONS: This study confirmed the high HEV seroprevalence (29%) in the general population of the Netherlands, but presence of antibodies was not associated with residential proximity to pig farms. The prevalence increased with age from 10% in adolescents to 33% among those aged 50 and above, supporting the assumption of a cumulative lifetime exposure to HEV in the Netherlands as well as a higher infection pressure in the past. Our findings cannot refute the assumption that transmission is primarily food-borne.

First human case of tick-borne encephalitis virus infection acquired in the Netherlands, July 2016.

In July 2016, the first autochthonous case of tick-borne encephalitis was diagnosed in the Netherlands, five days after a report that tick-borne encephalitis virus (TBEV) had been found in Dutch ticks. A person in their 60s without recent travel history suffered from neurological symptoms after a tick bite. TBEV serology was positive and the tick was positive in TBEV qRT-PCR. TBEV infection should be considered in patients with compatible symptoms in the Netherlands.

Neuralgic amyotrophy and hepatitis E virus infection.

OBJECTIVE: To determine whether there is an association between an acute preceding hepatitis E virus (HEV) infection and neuralgic amyotrophy (NA), and if so, whether patients with HEV-related NA differ from patients without an associated HEV infection. METHODS: HEV testing was conducted in a retrospective cohort of 28 Cornish patients with NA (2011-2013) and a prospective cohort of 38 consecutive Dutch patients with NA (2004-2007). Acute-phase serum samples were analyzed for the presence of anti-HEV immunoglobulin (Ig) M and IgG and HEV RNA (quantitative real-time PCR). RESULTS: Five cases (10.6%) of acute hepatitis E infection were identified in a total group of 47 patients with NA of whom serum samples were available. In 4 patients, HEV RNA was detected in serum samples taken at presentation. All patients with HEV-associated NA had clinical and electrophysiologic evidence of bilateral brachial plexus involvement. Anti-HEV IgM positivity was not related to age, sex, disease severity, disease course, or outcome. CONCLUSIONS: Acute hepatitis E is found in 10% of patients with NA from the United Kingdom and the Netherlands. Further research is required to investigate the role of HEV in NA in other geographical locations and to determine pathophysiologic mechanisms.

Underdiagnosis of chikungunya virus infections in symptomatic dutch travelers returning from the Indian ocean area.

A putative underdiagnosis of clinical chikungunya virus infection in Dutch travelers to the Indian Ocean area was addressed by retrospective screening of all sera for which requested dengue virus serology was negative in the period 2007 to 2010. Evidence for a recent infection was observed in 6.5% of 107 patients, indicating a substantial underdiagnosis and the need for increased awareness among physicians.

Age-dependent patterns of infection and severity explaining the low impact of 2009 influenza A (H1N1): evidence from serial serologic surveys in the Netherlands.

Despite considerable research efforts in specific subpopulations, reliable estimates of the infection attack rates and severity of 2009 influenza A (H1N1) in the general population remain scarce. Such estimates are essential to the tailoring of future control strategies. Therefore, 2 serial population-based serologic surveys were conducted, before and after the 2009 influenza A (H1N1) epidemic, in the Netherlands. Random age-stratified samples were obtained using a 2-stage cluster design. Participants donated blood and completed a questionnaire. Data on sentinel general practitioner-attended influenza-like illness and nationwide hospitalization and mortality were used to assess the severity of infection. The estimated infection attack rates were low in the general population (7.6%, 95% confidence interval: 3.6, 11) but high in children aged 5-19 years (35%, 95% confidence interval: 25, 45). The estimated hospitalization and mortality rates per infection increased significantly with age (5-19 years: 0.042% and 0.00094%, respectively; 20-39 years: 0.12% and 0.0025%; 40-59 years: 0.68% and 0.032%; 60-75 years: >0.81% and >0.068%). The high infection attack rate in children and the very low attack rate in older adults, together with the low severity of illness per infection in children but substantial severity in older adults, produced an epidemic with a low overall impact.

Prevalence of hepatitis E virus infection in liver transplant recipients.

Hepatitis E virus (HEV) infection is known to run a self-limited course. Recently, chronic hepatitis E has been described in several immunosuppressed patients after solid organ transplantation. The prevalence of HEV infection after transplantation, however, is unknown. We studied HEV parameters [HEV RNA, HEV immunoglobulin M (IgM), and HEV immunoglobulin G (IgG) by enzyme-linked immunosorbent assay and confirmatory immunoblotting] in a cohort of 285 adult liver transplant recipients. The most recent freeze-stored sera were investigated, and if they were positive, a retrospective analysis was performed. Samples from 274 patients (96.1%) tested negative for all HEV parameters. This included a patient described earlier as having experienced an episode of chronic HEV hepatitis in the past. One patient was found positive for HEV RNA without HEV antibodies. She presently suffers from chronic HEV hepatitis and has also been described before. Sera from 9 patients tested positive for HEV IgG without HEV IgM or HEV RNA. Six of these 9 patients (2.1% of the total) were found to have HEV IgG antibodies in retrospect related to an HEV infection at some time pre-transplant as they also tested positive in a pretransplant serum sample. One of these 9 patients suffered in retrospect from a chronic HEV infection with mild hepatitis between 2 and 5 years after liver transplantation on the basis of the course of HEV RNA, IgM, and IgG, aminotransferases, and liver histology. Overall, the prevalence of acquired HEV hepatitis after liver transplantation was 1% in this cohort. We conclude that liver transplant recipients have a risk for chronic HEV infection, but the prevalence is low.

Chronic hepatitis E virus infection in liver transplant recipients.

Hepatitis E virus (HEV) infection is known to run a self-limiting course. Sporadic cases of acute hepatitis due to infection with HEV genotype 3, present in pig populations, are increasingly recognized. Zoonotic transmission seems infrequent. The entity of unexplained chronic hepatitis after liver transplantation has been recognized. Detection of HEV in 2 liver transplant recipients triggered a review of these cases. Freeze-stored sera were available for retrospective analysis. HEV antibodies were determined. For virus detection and identification, a fragment of the gene encoding the major capsid protein (open reading frame 2) was amplified by reverse-transcription polymerase chain reaction and sequenced to identify the genotype. Two months after liver transplantation, case A developed unexplained chronic hepatitis, which developed into cirrhosis. Retransplantation followed 7 years later, after which chronic hepatitis recurred. In retrospect, HEV RNA was present in serum 3 weeks after the first transplantation and remained present afterwards. HEV RNA was also present in retransplant liver tissue. HEV antibodies appeared late after retransplantation. Case B developed unexplained chronic hepatitis 7 years after transplantation. Retransplantation was needed 5 years later, after which no signs of hepatitis recurred. In retrospect, the period of chronic hepatitis up to the retransplantation coincided with HEV RNA in serum. In case B, antibodies developed, the viral load was much lower than in case A, and the virus seemed to be cleared after retransplantation. Genotyping in both cases revealed 2 unique strains of genotype 3. In conclusion, chronic HEV infection may develop in immunosuppressed patients, who may then serve as long-term carriers of the virus. We hypothesize that HEV may be the cause of chronic hepatitis in liver transplant recipients.

Systemic immune response after rotavirus inoculation of neonatal mice depends on source and level of purification of the virus: implications for the use of heterologous vaccine candidates.

Rotavirus is an important cause of morbidity and mortality worldwide and vaccines are currently under development, with clinical trails conducted in humans worldwide. The immune responses in infant BALB/c mice were examined following oral inoculation with murine rotavirus EDIM (2 x 10(4) focus-forming units) and with three CsCl gradient-purified fractions of heterologous simian rotavirus SA11 (standardized at 2 x 10(6) CCID(50)) that differed in antigen composition: fraction 1 was enriched for double-layered rotavirus particles, fraction 2 for triple-layered particles and fraction 3 consisted mainly of cell components. Diarrhoea and high IgG responses, but marginal IgA responses, were observed after inoculation with all three SA11 fractions. Virus shedding was observed in all EDIM-inoculated mice, but in none of the SA11-inoculated mice. Rotavirus-specific IgG1 : 2a ratios were similar in mice inoculated with EDIM and SA11 fraction 1, but higher for SA11 fraction 3- and lower for SA11 fraction 2-inoculated mice. A higher IgG1 : 2a ratio indicates a more Th2-like immune response. This undesirable response is apparently mostly induced by inoculation with heterologous rotavirus in the presence of abundant cell-associated and soluble rotavirus proteins, compared with infection with a more purified preparation or with homologous virus. These data show that, following inoculation with a standardized amount of infectious virus, the composition of the fraction influences the outcome of the immune responses significantly.

Homeostasis and function of goblet cells during rotavirus infection in mice.

Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.

Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice.

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.