Nina Lagerqvist

Vaccination with virus-like particles protects mice from lethal infection of Rift Valley Fever Virus

Rift Valley Fever virus (RVFV) regularly accounts for severe and often lethal outbreaks among livestock and humans in Africa. Safe and effective veterinarian and human vaccines are highly needed. We present evidence that administration of RVF virus-like particles (VLPs) induces protective immunity in mice. In an accompanying paper, (Habjan, M., Penski, N., Wagner, V., Spiegel, M., Overby, A.K., Kochs, G., Huiskonen, J., Weber, F., 2009. Efficient production of Rift Valley fever virus-like particles: the antiviral protein MxA can inhibit primary transcription of Bunyaviruses. Virology 385, 400-408) we report the production of these VLPs in mammalian cells. After three subsequent immunizations with 1x10(6) VLPs/dose, high titers of virus-neutralizing antibodies were detected; 11 out of 12 mice were protected from challenge and only 1 out of 12 mice survived infection in the control groups. VLP vaccination efficiently suppressed replication of the challenge virus, whereas in the control animals high RNA levels and increasing antibody titers against the nucleocapsid protein indicated extensive viral replication. Our study demonstrates that the RVF VLPs are highly immunogenic and confer protection against RVFV infection in mice. In the test groups, the vaccinated mice did not exhibit any side effects, and the lack of anti-nucleocapsid protein antibodies serologically distinguished vaccinated animals from experimentally infected animals.

Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs

BackgroundAffecting both livestock and humans, Rift Valley Fever is considered as one of the most important viral zoonoses in Africa. However, no licensed vaccines or effective treatments are yet available for human use. Naked DNA vaccines are an interesting approach since the virus is highly infectious and existing attenuated Rift Valley Fever virus vaccine strains display adverse effects in animal trials. In this study, gene-gun immunisations with cDNA encoding structural proteins of the Rift Valley Fever virus were evaluated in mice. The induced immune responses were analysed for the ability to protect mice against virus challenge.ResultsImmunisation with cDNA encoding the nucleocapsid protein induced strong humoral and lymphocyte proliferative immune responses, and virus neutralising antibodies were acquired after vaccination with cDNA encoding the glycoproteins. Even though complete protection was not achieved by genetic immunisation, four out of eight, and five out of eight mice vaccinated with cDNA encoding the nucleocapsid protein or the glycoproteins, respectively, displayed no clinical signs of infection after challenge. In contrast, all fourteen control animals displayed clinical manifestations of Rift Valley Fever after challenge.ConclusionThe appearance of Rift Valley Fever associated clinical signs were significantly decreased among the DNA vaccinated mice and further adjustment of this strategy may result in full protection against Rift Valley Fever.

Kinetics of Rift Valley Fever Virus in experimentally infected mice using quantitative real-time RT-PCR.

Rift Valley Fever (RVF) is an important viral zoonosis in Africa affecting animals and humans. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for surveillance of the disease in order to implement adequate public health actions. To study the kinetics of the RVF Virus (RVFV) infection, a SYBR Green-based quantitative real-time RT-PCR assay was developed. By using primers targeting the S-segment of RVFV, the detection limit of this assay was estimated to 30 RNA templates. Blood and organs of experimentally infected mice were sampled at different time points and RVFV RNA was quantified. High amounts of RVFV RNA were found in blood, brain, and liver samples shortly after infection with a 1-4 days post infection window for viral RNA detection. Mice developed symptoms after the appearance of serum antibodies, indicating that the host response plays an important role in the outcome of the disease. The RVFV quantitative RT-PCR proved to be a valuable diagnostic tool during the first days of infection, before detectable antibody levels and visual symptoms of RVF were observed.

Specificity and Dynamics of Effector and Memory CD8 T Cell Responses in Human Tick-Borne Encephalitis Virus Infection

Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases.

Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

Background Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1–4. Methodology/Principal Findings The primers and probe used in our RT-PCR assay were designed to target the 3′ untranslated region of all complete genome sequences of dengue virus available in GenBank (n = 3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 104–1011 GCE/mL, and the detection limit was between 6.0×102 and 1.1×103 GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n = 163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1–9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms.

Antibodies against Rift Valley fever virus in cattle, Mozambique.

To the Editor: During the past 2 decades, several countries in Africa and the Arabian Peninsula, to which Rift Valley fever virus (RVFV) is endemic, have reported outbreaks of Rift Valley fever in humans and livestock. The first evidence of RVFV in Mozambique was documented as early as the 1960s (1). Endemicity was subsequently confirmed in the 1980s by a prevalence study that identified virus-specific antibodies in 2% of pregnant women (2) and in the 1990s by serosurveillance in Zambezia Province, which showed that cattle had been infected with RVFV (3).

Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests.

Introduction Avian influenza viruses circulate in bird populations, and it is important to maintain and uphold our knowledge of the viral strains that are currently of interest in this context. Here, we describe the use of hemagglutinin-pseudotype retroviruses based on highly pathogenic influenza viruses for the screening of avian sera for influenza A antibodies. Our aim was also to determine whether the pseudovirus neutralization tests that we assessed were sensitive and simple to use compared to the traditional methods, including hemagglutination inhibition assays and microneutralization tests. Material and methods H5 and H7 pseudovirus neutralization tests were evaluated by using serum from infected rabbits. Subsequently, the assays were further investigated using a panel of serum samples from avian species. The panel contained samples that were seropositive for five different hemagglutinin subtypes as well as influenza A seronegative samples. Results and discussion The results suggest that the pseudovirus neutralization test is an alternative to hemagglutination inhibition assays, as we observed comparable titers to those of both standard microneutralizations assays as well as hemagglutinin inhibition assays. When evaluated by a panel of avian sera, the method also showed its capability to recognize antibodies directed toward low-pathogenic H5 and H7. Hence, we conclude that it is possible to use pseudoviruses based on highly pathogenic avian influenza viruses to screen avian sera for antibodies directed against influenza A subtypes H5 and H7.

Seroepidemiologic Screening for Zoonotic Viral Infections, Maputo, Mozambique.

To the Editor: In sub-Saharan Africa, febrile patients are often assumed to have, and are treated for, malaria, but when tested, many are malaria-negative. Because emerging diseases, such as chikungunya virus (CHIKV) and dengue virus (DENV) infections, cause outbreaks around the world (1–3), the importance of these pathogens has become more evident. However, low-income countries have limited epidemiologic data on alternative diagnoses to malaria (4,5) and poor laboratory capacity (1), which restrict further diagnostic investigations. An early study in Mozambique during the 1980s found antibodies to Rift Valley fever virus (RVFV) in 2% of pregnant women (6). More recently, a RVFV seroprevalence of 36.9% among cattle in the Maputo Province was shown in 2010–2011 (7). Furthermore, the movement of humans from rural areas to major cities, particularly to the capital of Maputo, might affect human illnesses and disease pattern of zoonotic viruses (3). We conducted a pilot study on CHIKV, DENV, hantavirus, RVFV, and West Nile virus (WNV) epidemiology in Mozambique. Ethical approval (registration no. IRB00002657) was granted by the National Bioethics Committee in Mozambique and by the Regional Ethical Review Board at Karolinska Institutet, Stockholm, Sweden (permit no. 2012/974–31/3). During 2012–2013, a total of 78 febrile patients were prospectively enrolled when they sought medical attention at the Polana Canico Health Center and Mavalane Health Center (catchment area 4,663 km2, estimated population 46,184 inhabitants) in the suburban area of Maputo city. All included patients answered a questionnaire and were initially screened for malaria by blood smear light microscopy; 15 were positive for malaria (Table). Patients’ median age was 29 years (37 years for seropositive patients) and ranged from 5 to 78 years. Forty-six (59%) were female. Fifty-eight (74%) reported recent exposure to mosquitoes. None of these persons had a history of international travel, and none had received a yellow fever vaccination. Table Results of screening for viral antibodies and malaria parasites in 78 febrile patients, Maputo, Mozambique, 2012–2013* Sixty (77%) patients provided paired acute- and convalescent-phase blood samples, with a minimum of 14 days (median 33 days) between samples. Serum samples were sent to the Public Health Agency of Sweden and blindly screened at a titer of 1:20 for IgG to CHIKV, DENV, hantavirus, RVFV, and WNV by using in-house indirect immunofluorescence assays as described for DENV by Vene et al. (8). Screening for IgG was done on convalescent-phase serum samples or, when those were not available, on acute-phase serum samples. Further immunofluorescence analyses for titer increases were performed for patients for whom paired serum samples were available and screening results were positive for IgG; however, no titer increases were found. Serum from admittance were tested for DENV IgM and WNV IgM by using commercial assays according to manufacturers’ instructions (Panbio Dengue IgM Capture ELISA E-DEN01M/E-DEN01M05, Standard Diagnostics, Inc., Yongin-si, South Korea; Serion ELISA classic ESR14M West Nile Virus IgM, Institut Virion/Serion GmbH, Wurzburg, Germany); 2 samples were positive for DENV IgM but none for WNV IgM. All acute serum samples were screened by using 1-step real-time reverse transcription PCR for CHIKV, RVFV, WNV (in-house validated assays), and DENV (9). Results were negative for viral RNA. Twenty-three (29%) of the 78 patients had a positive serology result from acute- or convalescent-phase serum samples for >1 of the tested viral pathogens (Table). The main finding was CHIKV IgG in 15 (19%) patients. Ten (13%) patients had positive results for DENV, including 2 DENV IgM–positive samples. The seroepidemiologic findings in this pilot study in Maputo strongly suggest possible and neglected alternative causes of febrile illness in Mozambique. Antibodies to CHIKV were found in 19% of the patients, which was a novel finding for Mozambique but corresponded well with other reports on the spread of CHIKV in tropical and subtropical areas of the world (2,3). DENV antibodies were present in 13% of the study population, representing a new finding in southern Mozambique; previous outbreaks have been reported from the northern part of the country (5). The median age of the seropositive patients (37 years) was higher than for the group as a whole (29 years), which might reflect increased exposure to zoonotic viruses over time. One patient was IgG positive for RVFV, a potentially emerging cause of fever in Mozambique, especially in view of recent reports of RVFV in cattle (7). The samples positive for both DENV and WNV IgG could represent previous independent infections with these viruses, co-infection, or cross-reactivity, which are common for flavivirus IgG (10). Overall, results indicate that exposure to vectorborne viruses in persons living in suburban areas of Maputo city is frequent, suggesting that infections with CHIKV, DENV, and RVFV infection should be considered as alternative diagnoses for patients with febrile illness in these settings. On the basis of these results, more extensive research is planned on the epidemiology of zoonotic viral infections in Mozambique.

Multiplex Nucleic Acid Suspension Bead Arrays for Detection and Subtyping of Filoviruses

ABSTRACT Here we describe multiplex suspension bead array systems that allow fast and reliable detection of reverse transcriptase (RT) PCR amplified filovirus genomes and also enable subtyping of Ebola virus species and Marburg virus strains. These systems have an analytical sensitivity equivalent to that of RT-PCR.

Complete Genome Sequence of a Sapporo Virus GV.2 Variant from a 2016 Outbreak of Gastroenteritis in Sweden

ABSTRACT During an outbreak of acute gastroenteritis in Sweden when laboratory routine diagnostics failed to detect a causative agent, Sapporo virus was detected in stool specimens using electron microscopy (M.-P. Hergens, J. Nederby Öhd, E. Alm, H. Hervius Askling, S. Helgesson, M. Insulander, N. Lagerkvist, B. Svennungsson, M. Tihane, T. Tolfvenstam, P. Follin, unpublished data). Whole-genome sequencing revealed a Sapporo virus variant clustering with genogroup V.