Stephanie Jansen

Prolonged detection of Zika virus RNA in urine samples during the ongoing Zika virus epidemic in Brazil.

Zika virus (ZIKV) is an emerging mosquito-borne virus that elongs to the genus Flavivirus. ZIKV may cause Zika virus disease ZVD) in humans that is characterized by fever, headache, myalgia nd rash. The first autochthonous transmission of ZIKV in Brazil was emonstrated in 2015 for patients from Rio Grande do Norte [1] and t was shown that the epidemic ZIKV strain in Brazil belong to the sian lineage [1,2]. Diagnosis of ZVD in humans is mainly based on NA detection in serum or plasma samples. Specific antibody detecion is mostly hampered due to strong serological cross-reactivity ith other circulating flaviviruses such as dengue virus or yellow ever virus [3–6]. Thus, there is an urgent need for a ZIKV diagostic protocol in Brazil that is effective in any ZIKV epidemic cenario, leading to a rapid, reliable and prolonged ZIKV RNA detecion. Paired samples of urine and serum from seven Brazilian ZVD atients were used for this study. For two additional ZVD patients nly urine samples were collected for a longer period after the onset f symptoms (ethical committee 80709). Viral RNA was extracted sing RTP® Pathogen Kit (Stratec, Birkenfeld—Germany), 400 l as used (for both, sera and urine samples), eluted in 50 l Elution uffer and ZIKV RT-PCR was performed according to Waehre et al. 7] using the Thermal Cycler peqLab—model peqstar 96X Univeral Gradient. Amplified fragments ∼200 pb were Sanger sequenced or confirmation. ZIKV RNA was detected in sera collected 2 days fter onset of symptoms, however urine samples from the same day ere tested negative. ZIKV RNA was first detectable in urine 4 days fter onset of symptoms, suggesting the beginning of renal excreion of ZIKV. For one patient ZIKV RNA excretion was observed until 4 days after onset of symptoms. In addition, our study demontrated higher levels of ZIKV RNA in urine when compared to serum. his was also observed by Gourinat et al. [8] during the ZVD epiemic in French Polynesia, reinforcing the evidence that the use f urine can increase the number of laboratory confirmed cases in n epidemic setting. The nucleotide sequences obtained with the mplicons confirmed the presence of the Asian lineage of ZIKV in io de Janeiro. In conclusion, urine samples should be considered s an important alternative to serum or plasma for the detection of IKV RNA because of a longer period of RNA detection, higher RNA evels, and less invasive sample collection.

Experimental transmission of Zika virus by mosquitoes from central Europe

Mosquitoes collected in Germany in 2016, including Culex pipiens pipiens biotype pipiens, Culex torrentium and Aedes albopictus, as well as Culex pipiens pipiens biotype molestus (in colony since 2011) were experimentally infected with Zika virus (ZIKV) at 18 °C or 27 °C. None of the Culex taxa showed vector competence for ZIKV. In contrast, Aedes albopictus were susceptible for ZIKV but only at 27 °C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016

Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.

Inactivation and Environmental Stability of Zika Virus.

To the Editor: Zika virus is an emerging virus that has spread to most countries in Latin America and the Caribbean (1,2). It is transmitted by mosquitoes and through sexual intercourse (3). Most persons infected with Zika virus are asymptomatic or experience mild symptoms (4). However, in a pregnant woman, infection may cause severe pregnancy and birth complications, most notably microcephaly in children infected in utero (5–7). Zika virus infection might also be associated with an increased incidence of Guillain-Barre syndrome (8). Thus, the virus represents a threat to healthcare workers who manage infected patients or diagnostic samples and researchers who work with infectious virus in laboratories.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25 °C as well as 18 °C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25 °C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18 °C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.

Development of a high-throughput colorimetric Zika virus infection assay

Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.

RNA interference restricts Rift Valley fever virus in multiple insect systems

Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies. ABSTRACT The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Genetic Diversity and New Lineages of Dengue Virus Serotypes 3 and 4 in Returning Travelers, Germany, 2006–2015

During 2006–2015, we analyzed 70 dengue virus (DENV) strains isolated from febrile travelers returning to Germany. High genetic diversity, including multiple co-circulating DENV lineages and emerging new lineages of DENV-3 and DENV-4, was demonstrated. Our passive surveillance system based on returning travelers yielded substantial information on DENV diversity.

No serological evidence for Zika virus infection and low specificity for anti-Zika virus ELISA in malaria positive individuals among pregnant women from Madagascar in 2010

It was previously reported that a malaria infection may interfere with the specificity of a commercial ELISA test against Zika virus (ZIKV). We analyzed 1,216 plasma samples from healthy, pregnant women collected in two sites in Madagascar in 2010 for ZIKV antibodies using a commercial ELISA and for Plasmodium infection by PCR. This screen revealed six putative ZIKV-positive samples by ELISA. These results could not be confirmed by indirect immunofluorescence assays or virus neutralization tests. Four of these six samples were also positive for P. falciparum. We noted that the frequency of malaria positivity was higher in ZIKV-ELISA positive samples (50% and 100% in the two study sites) than ZIKV-negative samples (17% and 10%, respectively), suggesting that malaria may have led to false ZIKV-ELISA positives.

Indoor development of Aedes aegypti in Germany, 2016

In spring 2016, a German traveller returning from Martinique cultivated imported plant offsets in her home, and accidentally bred Aedes aegypti. Thirteen adult mosquito specimens submitted for identification and the traveller were tested for Zika, dengue and chikungunya virus infections, with negative results. The detection of Ae. aegypti by the ‘Mueckenatlas’ project demonstrates the value of this passive surveillance scheme for potential public health threats posed by invasive mosquitoes in Germany.