Essi M. Korhonen

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities

The current outbreak of Zika virus (ZIKV) infection has been associated with an apparent increased risk of congenital microcephaly. We describe a case of a pregnant woman and her fetus infected with ZIKV during the 11th gestational week. The fetal head circumference decreased from the 47th percentile to the 24th percentile between 16 and 20 weeks of gestation. ZIKV RNA was identified in maternal serum at 16 and 21 weeks of gestation. At 19 and 20 weeks of gestation, substantial brain abnormalities were detected on ultrasonography and magnetic resonance imaging (MRI) without the presence of microcephaly or intracranial calcifications. On postmortem analysis of the fetal brain, diffuse cerebral cortical thinning, high ZIKV RNA loads, and viral particles were detected, and ZIKV was subsequently isolated.

Zika virus infection in a traveller returning from the Maldives, June 2015

We report a Zika virus (ZIKV) infection in a patient with fever and rash after returning to Finland from Maldives, June 2015. The patient had dengue virus (DENV) IgG and IgM antibodies but pan-flavivirus RT-PCR and subsequent sequencing showed presence of ZIKV RNA in urine. Recent association of ZIKV with microcephaly highlights the need for laboratory differentiation of ZIKV from DENV infection and the circulation of ZIKV in areas outside its currently known distribution range.

Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.

Approach to non-invasive sampling in dengue diagnostics: exploring virus and NS1 antigen detection in saliva and urine of travelers with dengue.

BACKGROUND Dengue diagnostics currently relies on serum and plasma tests. Although the proof of concept for detecting dengue virus (DENV) RNA and nonstructural protein 1 (NS1) antigen from urine and saliva has been demonstrated, few studies have explored their use in diagnostics. OBJECTIVES To investigate the occurrence, excretion kinetics, and diagnostic potential of DENV-RNA and NS1 antigen in the urine and saliva of dengue patients. STUDY DESIGN We examined serial serum, urine (n=50) and saliva (n=48) samples of 14 Finnish travelers with dengue. All samples were analyzed by NS1 ELISA and DENV RT-PCR, and the first and last serum specimens were tested for DENV IgG and IgM. In addition, biochemical parameters were studied from the urine and clinical and laboratory data of the patients were collected. RESULTS DENV-NS1 protein and RNA proved detectable from saliva and urine using tests developed for serum samples. RNA/NS1 detection showed a diagnostic sensitivity of 64%/54% and 60%/56% for urine and saliva, respectively. RNA analyses performed on days 7-13 after onset of symptoms revealed the sensitivity for urine (72%) to be greater than for serum (31%) or saliva (50%). The concentration of urine samples had no impact on RNA detection. CONCLUSIONS Noninvasive sampling enables an alternative approach to dengue diagnostics. The performance of the NS1 antigen assay may be improved by optimizing it for urine and saliva samples. The prolonged excretion of DENV-RNA in urine extends the sampling time window for molecular diagnostics and surveillance.

Dengue in Travelers: Kinetics of Viremia and NS1 Antigenemia and Their Associations with Clinical Parameters

Despite the increasing numbers of travel-acquired dengue, few studies have assessed virologic markers of the disease in non-endemic populations. We examined the kinetics of diagnostic markers and their associations with clinical parameters in 93 patients with travel-acquired dengue fever. Kinetics analyses suggested a longer average duration for viremia (9 days, CI95%: 8–10) and non-structural protein 1 (NS1) antigenemia (15 days, CI95%: 12–20) than reported in endemic populations. While none of the tests sufficed alone, the best diagnostic coverage was achieved by combining antibody detection with RNA or NS1 testing. Studied by regression models, early relative levels of viremia and NS1 antigenemia proved to be significantly associated with several clinical parameters: high viremia predicted greater likelihood and increased length of hospitalization, the degree of NS1 antigenemia correlated positively with hematocrit and liver transaminases, and both viremia and NS1 antigenemia levels negatively with platelet counts in follow-up. Levels of viremia and NS1 antigenemia may serve as predictors of the clinical manifestations in travel-acquired dengue.

Molecular detection of Bartonella spp. in deer ked pupae, adult keds and moose blood in Finland

The deer ked (Lipoptena cervi) is a haematophagous ectoparasite of cervids that harbours haemotrophic Bartonella. A prerequisite for the vector competence of the deer ked is the vertical transmission of the pathogen from the mother to its progeny and transstadial transmission from pupa to winged adult. We screened 1154 pupae and 59 pools of winged adult deer keds from different areas in Finland for Bartonella DNA using PCR. Altogether 13 pupa samples and one winged adult deer ked were positive for the presence of Bartonella DNA. The amplified sequences were closely related to either B. schoenbuchensis or B. bovis. The same lineages were identified in eight blood samples collected from free-ranging moose. This is the first demonstration of Bartonella spp. DNA in a winged adult deer ked and, thus, evidence for potential transstadial transmission of Bartonella spp. in the species.

Isolation and full genomic characterization of Batai virus from mosquitoes, Italy 2009

In 2009, 2589 mosquitoes were collected in northwest Italy and screened for orthobunyavirus RNA by RT-PCR. One pool of Anopheles maculipennis complex mosquitoes was found to be positive and a virus was isolated from that pool. The isolate was identified as Batai virus (BATV) by sequencing. Previously, BATV was detected in Italy, but limited data and no prior isolates existed. Full-length sequences of the S, M and L segments were determined for the newly isolated Italian strain. For comparison, partial sequences were also determined for the BATV strain Calovo (former Czechoslovakia, 1960). Phylogenetic analyses revealed clustering of the newly derived Italian BATV along with a recent isolate from Germany and the historic strain Calovo. To the best of our knowledge, this represents the first isolation of BATV from Italy, which confirms a broader geographical distribution of BATV in Europe than was previously verified by isolation.

Differences in the growth properties of Zika virus foetal brain isolate and related epidemic strains in vitro

Zika virus (ZIKV) has recently emerged into new areas in the Americas and Asia, causing an epidemic characterized by severe congenital infections. While ZIKV infection is usually asymptomatic or causes mild symptoms, it has now caused a high rate of foetal brain and ocular abnormalities. The underlying reasons for the varying severity of disease outcomes is poorly understood. In this study, we compared the infectivity and replication of three disease-associated Zika viruses of Asian lineage, as well as the prototypic ZIKV strain from Africa. The recent foetal brain isolate FB-GWUH-2016 demonstrated enhanced infectivity and replication over the serum-origin isolates from French Polynesia and Martinique, suggesting differences in the pathogenic properties.

Validation of serological and molecular methods for diagnosis of zika virus infections

The laboratory confirmation of Zika virus (ZIKV) infection, and the differential diagnosis from other flavivirus infections such as dengue virus (DENV), often requires the use of several diagnostic test types. Cross-reactions and secondary infections complicate the serological diagnosis and specific viral RNA detection assays are often needed for confirming the diagnosis. The aim of this study was to validate serological and molecular methods for diagnosing ZIKV infection. This included the evaluation of a ZIKV RT-qPCR assay for diagnostics that was previously set up for research use and to compare the ZIKV, DENV and TBEV EIA methods. External and in-house controls and pre-characterized sample panels were tested, and also automated and manual nucleic acid extraction methods were compared. A total of ten Finnish traveler patients were diagnosed with acute ZIKV infection during 2015-2017 including one suspected dual DENV and ZIKV infection. These samples along with panels of DENV and tick-borne encephalitis virus (TBEV) infections were used to test the cross-reactive properties of ZIKV, DENV and TBEV IgM assays. Additionally, the diagnosed acute ZIKV patient samples were tested using commercially available diagnostic DENV NS1 antigen assay and a ZIKV NS1 antigen assay intended for research use. The ZIKV RT-qPCR assay was demonstrated to be both specific and sensitive (one genome per reaction) and suitable for routine diagnostic use utilizing automated nucleic acid extraction. Of the tested IgM tests the NS1 antigen-based ZIKV IgM (Euroimmun) assay performed with least cross-reactivity with a specificity of 97.4%. The DENV IgM assay (Focus Diagnostics) had specificity of only 86.1%. The results are in line with previous studies and additionally highlight that also acute TBEV patients may give a false positive test result in DENV and ZIKV IgM assays.