Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Ixodiphagus hookeri (Hymenoptera: Encyrtidae) and Tick-Borne Pathogens in Ticks with Sympatric Occurrence (and Different Activities) in the Slovak Karst National Park (Slovakia), Central Europe.

Ticks are involved in the transmission a plethora of pathogens. To effectively control ticks and mitigate the risks associated with tick-borne diseases, it is important to implement tick control measures. These may include the use of acaricides as well as the development and implementation of an alternative, environmentally friendly tick management program that include practices such as habitat modification or establishing biological control. Ixodiphagus hookeri Howard is a tick-specific parasitoid wasp that predates on several species of ixodid ticks and could contribute to the control of the tick population. This work aimed to detect the presence of parasitoid wasps in ticks (Ixodidae) using genetic approaches. Several tick species of the genera Ixodes, Haemaphysalis, and Dermacentor, with a sympatric occurrence in the Slovak Karst National Park in southeastern Slovakia, were screened for the presence of wasps of the genus Ixodiphagus. The DNA of the parasitoids was detected in four tick species from three genera. This work presents the first molecular detection of parasitoids in two Dermacentor tick species, as well as the first molecular identification of Ixodiphagus wasps in Ixodes ricinus and Haemaphysalis concinna ticks from the Karst area. In the given area, it was observed that I. ricinus and H. concinna ticks are hyper-parasitized by wasps. Moreover, it was observed that wasps here can parasitize several tick species, some of which are of less significance for human and animal health (as they transmit fewer pathogens).

Towards reliable whole genome sequencing for outbreak preparedness and response.

BACKGROUND: To understand the dynamics of infectious diseases, genomic epidemiology is increasingly advocated, with a need for rapid generation of genetic sequences during outbreaks for public health decision making. Here, we explore the use of metagenomic sequencing compared to specific amplicon- and capture-based sequencing, both on the Nanopore and the Illumina platform for generation of whole genomes of Usutu virus, Zika virus, West Nile virus, and Yellow Fever virus. RESULTS: We show that amplicon-based Nanopore sequencing can be used to rapidly obtain whole genome sequences in samples with a viral load up to Ct 33 and capture-based Illumina is the most sensitive method for initial virus determination. CONCLUSIONS: The choice of sequencing approach and platform is important for laboratories wishing to start whole genome sequencing. Depending on the purpose of genome sequencing the best choice can differ. The insights presented in this work and the shown differences in data characteristics can guide labs to make a well informed choice.

An efficient molecular approach to distinguish chains of measles virus transmission in the elimination phase.

Measles viruses continue to spread globally, despite the availability of a safe and effective vaccine. Molecular surveillance of measles virus has become an essential tool to demonstrate whether cascades of infections in a certain region or country are the result of endemic spread or the repeatedly introduction of the virus in contained outbreaks. Currently, molecular surveillance of measles viruses worldwide is mainly based on 450 nucleotides of the C-terminal region of the nucleoprotein (N450). However, as a result of the disappearance of particular measles virus clades over the past decades, this gene segment does not provide sufficient resolution anymore to answer these questions. To increase the molecular resolution, sequence data were collected from three regions of the measles virus genome, the partial non-coding region between the M and F gene (M-F NCR4465-4754), partial H gene (H8022-8621) and the partial L gene (L10724-11438) for measles viruses detected in 2018 and 2019 in the Netherlands. Analysis of obtained sequence data indicated that sequencing of these three regions resulted in an increase in molecular resolution for measles virus genotype B3 and D8 viruses, two of the four global genotypes currently predominant in the European region. Furthermore, this improved resolution was sufficient to support an epidemiology characterized by repeat introduction of measles virus rather than endemic virus spread. In conclusion, sequencing of the M-F NCR4465-4754, H8022-8621 and L10724-11438 regions of the measles virus is an efficient and useful approach for molecular surveillance of measles viruses.

Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol.

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

First molecular analysis of rabies virus in Qatar and clinical cases imported into Qatar, a case report.

Identifying the origin of the rabies virus (RABV) infection may have significant implications for control measures. Here, we identified the source of a RABV infection of two Nepalese migrants in Qatar by comparing their RABV genomes with RABV genomes isolated from the brains of a RABV infected camel and fox from Qatar.

MERS-CoV in Camels but Not Camel Handlers, Sudan, 2015 and 2017.

We tested samples collected from camels, camel workers, and other animals in Sudan and Qatar in 2015 and 2017 for evidence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV antibodies were abundant in Sudan camels, but we found no evidence of MERS-CoV infection in camel workers, other livestock, or bats.

Assessment of serological evidence for mumps virus infection in vaccinated children.

It is estimated that at least one-third of mumps virus infections in non-vaccinated individuals are asymptomatic. Little information is available whether this proportion is the same among those vaccinated. We validated a commercial oral fluid mumps IgG-specific Enzyme Immunoassays (EIA) with vaccinated control groups to identify symptomatic and asymptomatic mumps virus infections in vaccinated individuals during a mumps outbreak in The Netherlands. A vaccinated control group was required to define a new cutoff value for the assay, because of the presence of low but significant levels of IgG antibodies in oral fluid as a result of mumps vaccination in the past. With a new cutoff, calculated using receiver operator characteristic analysis, we identified an attack rate of 7-10% compared to 2.7% based on clinical symptoms among vaccinated children. This finding has important implications when studying transmission patterns, strain virulence, as well as mumps vaccine effectiveness to protect from infection rather than disease.

Mumps outbreak in a highly vaccinated student population, The Netherlands, 2004.

In September 2004 a mumps outbreak occurred at an international hotel school in The Netherlands. We investigated this outbreak to identify risk factors for mumps. There were 105 mumps cases (overall mumps attack rate (AR) 12% (95% CI: 10-15%)). The AR for Dutch vaccinated and unvaccinated participants was 12% (95% CI: 10-15%) and 15% (95% CI: 3-42%), respectively. Independent risk factor was mumps contact. Explanations for the relatively high AR among vaccinated participants include primary vaccine failure, waning immunity and incomplete vaccine-induced immunity in the context of high mumps virus exposure in a school party and a crowded boarding school.

Rubella outbreak in the Netherlands, 2004-2005: high burden of congenital infection and spread to Canada.

BACKGROUND: In The Netherlands and Canada the measles, mumps, rubella vaccine coverage is high. In 2004 a rubella outbreak started in the Netherlands in a population subgroup with low coverage, with subsequent spread to Canada. METHODS: We examined data on rubella cases in the Netherlands and Canada reported between September 2004 and July 2005. In The Netherlands we established enhanced surveillance for congenital rubella while in Canada we carried out a cohort study to estimate vaccine effectiveness. RESULTS: In The Netherlands and Canada, 387 and 309 rubella cases were reported, respectively. Of these, 97% were in unvaccinated individuals of orthodox protestant denomination. Reported consequences of rubella in pregnancy were 2 fetal deaths and 14 infants with congenital infection. Of the latter, 11 had clinical defects including deafness in all but eye defects in none. The estimated vaccine effectiveness was 99.3% (95% CI: 95.3%-99.9%). Closely related strains of rubella virus genotype 1G were found in Dutch and Canadian cases. CONCLUSIONS: A large rubella outbreak occurred in The Netherlands with spread to Canada in a population subgroup with religious objections to vaccination. Its major public health importance was due to the high burden of congenital disease, international spread and implications for measles and rubella surveillance and elimination. Congenital deafness occurred more frequently and eye defects less frequently than expected. The estimated rubella vaccine effectiveness was very high. Our results demonstrate the risks associated with heterogeneity in rubella vaccine coverage. High rubella vaccine coverage in all population subgroups and sensitive surveillance are crucial for elimination of rubella and CRS.

Radiation-induced skin injury in the animal model of scleroderma: implications for post-radiotherapy fibrosis.

BACKGROUND: Radiation therapy is generally contraindicated for cancer patients with collagen vascular diseases (CVD) such as scleroderma due to an increased risk of fibrosis. The tight skin (TSK) mouse has skin which, in some respects, mimics that of patients with scleroderma. The skin radiation response of TSK mice has not been previously reported. If TSK mice are shown to have radiation sensitive skin, they may prove to be a useful model to examine the mechanisms underlying skin radiation injury, protection, mitigation and treatment. METHODS: The hind limbs of TSK and parental control C57BL/6 mice received a radiation exposure sufficient to cause approximately the same level of acute injury. Endpoints included skin damage scored using a non-linear, semi-quantitative scale and tissue fibrosis assessed by measuring passive leg extension. In addition, TGF-beta1 cytokine levels were measured monthly in skin tissue. RESULTS: Contrary to our expectations, TSK mice were more resistant (i.e. 20%) to radiation than parental control mice. Although acute skin reactions were similar in both mouse strains, radiation injury in TSK mice continued to decrease with time such that several months after radiation there was significantly less skin damage and leg contraction compared to C57BL/6 mice (p < 0.05). Consistent with the expected association of transforming growth factor beta-1 (TGF-beta1) with late tissue injury, levels of the cytokine were significantly higher in the skin of the C57BL/6 mouse compared to TSK mouse at all time points (p < 0.05). CONCLUSION: TSK mice are not recommended as a model of scleroderma involving radiation injury. The genetic and molecular basis for reduced radiation injury observed in TSK mice warrants further investigation particularly to identify mechanisms capable of reducing tissue fibrosis after radiation injury.

Air travel as a risk factor for introduction of measles in a highly vaccinated population.

Epidemiological and molecular investigation of two small measles clusters in The Netherlands in July/August 2007 revealed an association with travel by air of the index cases and nosocomial spread in the first cluster. Although these importations did not result in an outbreak among unvaccinated subjects, the observations illustrate the challenges for measles control in a country with high measles vaccination coverage (> 95%) but with pockets of low coverage.

Gender differences in pretreatment quality of life in a prospective lung cancer trial.

The purpose of this analysis was to assess the impact of pretreatment factors on quality of life (QOL) in patients with locally advanced nonsmall cell lung cancer (NSCLC). In particular, this study focused on the possible interaction between gender-specific baseline health-related QOL and Karnofsky performance score (KPS) in a prospective randomized lung cancer trial. QOL information, using validated instruments (Functional Assessment of Cancer Therapy-Lung [FACT-L], version 2, and Functional Living Index-Cancer [FLIC]), was prospectively collected in patients with locally advanced NSCLC treated on Radiation Therapy Oncology Group (RTOG) trial 89-01. Between April 1990 and April 1994, 70 eligible patients participated in a phase III trial comparing a regimen containing sequential chemotherapy and radiation therapy versus sequential chemotherapy plus surgery. Of these 70 patients, 46 underwent pretreatment FLIC and 49 underwent pretreatment FACT-L. There was a significant interaction between gender and KPS using FLIC (P = 0.009), which also showed a trend toward significance with FACT (P = 0.09). Significant KPS-by-gender interactions were noted for FACT-L in the physical well-being and additional concerns-lung subscales (P = 0.012 and P = 0.0003, respectively). The results of both the FLIC and FACT-L demonstrated significantly lower scores corresponding to lower KPS values (P = 0.009 and P = 0.016, respectively). Results of this randomized study incorporating prospective QOL measurements suggested that in patients with locally advanced NSCLC, analyzing QOL data by either gender or performance status alone may not accurately reflect how these factors depend upon each another. Understanding the interaction between gender and performance status could lead to better prognosticators and potentially could tailor interventions for specific groups of patients with lung cancer.

Differential radiation effect in tumor and normal tissue after treatment with ramipril, an angiotensin-converting enzyme inhibitor.

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy x 2 or 6 Gy x 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.

Evaluation of serological and virological tests in the diagnosis of clinical and subclinical measles virus infections during an outbreak of measles in The Netherlands.

We evaluated different approaches for diagnosing measles virus (MV) infection in unvaccinated children and in healthy contact persons (n=194) during a measles epidemic in The Netherlands. MV RNA was detected by reverse-transcriptase polymerase chain reaction in throat-swab specimens from 93% of the patients with clinical symptoms. MV RNA was detected from 5 days before until 12 days after the onset of symptoms. Most patients (88%) also secreted MV RNA in their urine until 5 weeks after the onset of symptoms. Oral fluid proved to be the most practical specimen for the simultaneous detection of MV-specific IgM antibody and viral RNA, which, together, confirmed 93% of measles cases. Viral RNA was also detected in oropharyngeal specimens from 3 healthy contact persons with serological proof of MV infection. The results of this study emphasize the feasibility of combined detection of viral RNA and MV-specific IgM antibodies in oropharyngeal specimens for the diagnosis of clinical and subclinical MV infection.