Characterization of dengue virus isolates from patients experiencing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome

The four serotypes of dengue virus (DENV) belong to the genus flavivirus, and have a positive sense, single-stranded RNA genome of ~11 kb. The DENVs cause the most common arthropod-borne viral disease in man with ~100 million infections per year. The sole measure of control is limiting the mosquito vectors Aedes aegypti and Ae. albopictus, and there is an urgent need for an effective vaccine and potent anti-viral drugs. DENV infection can be asymptomatic or a self-limited, acute febrile disease ranging in severity. The classical form, dengue fever (DF), is characterized by high fever, headache, stomach ache, rash, myalgia and arthralgia. Severe dengue, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are accompanied by thrombocytopenia, vascular leakage and hypotension. The fatal condition DSS is characterized by systemic shock. Dengue research has been hampered by a lack of appropriate animal models of infection and disease. Furthermore, fundamental knowledge such as host cell tropism and virulence markers are still not established. This thesis focuses on the characterization of clinical DENV isolates from all four serotypes and clinical conditions (DF, DHF, and DSS) aimed at identifying viral features involved in pathogenesis. Attempts to develop a strand-specific qRT-PCR to identify primary target cells for DENV replication, failed due to the self-priming phenomenon of the DENV genome. Selfpriming was not restricted to any particular regions of the viral genome, nor to contaminating cellular nucleic acids, nor the lack of a poly(A)-tail at the 3‟ end. Firststrand synthesis in situ of the DENV genome is believed to arise due to spontaneous loopback structures functioning as transient primers for the reverse transcriptase. In vitro studies in mammalian Vero cells revealed a decreased level of replication for all DENV isolates from DSS patients compared to DENV isolates from DF patients. The replication patterns of the DHF isolates resembled either the DF- or DSS-derived DENV isolates depending on serotype. The DSS isolates were further distinguished from milder case DENV isolates by induction of apoptosis in mosquito C6/36 cells. Three DENV-1 isolates representing a DF, DHF, and a DSS case, were further characterized in vivo in BALB/c mice. Infection with the DF and DHF isolates peaked during the first week with viral RNA found primarily in lungs, liver, and to a certain extent in brain. In contrast, the DSS isolate was primarily neurotropic and persisted longer compared to the DF and DHF isolates. Genomic sequencing revealed a preference for amino acid substitutions in the viral envelope protein and the non-structural (NS) protein NS1 and NS5. Thus, these viral proteins may influence pathogenesis either by immunomodulation, and/or host cells tropism and replication. In conclusion, these results based on clinical DENV isolates indicate that DENVs within the same serotype and genotype may have both different phenotypes and genotypes. These intrinsic viral features could influence virus virulence and disease pathogenesis in humans

Phenotypic characterization of patient dengue virus isolates in BALB/c mice differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

International audienceBACKGROUND: Dengue virus (DENV) infection is the most common arthropod-borne viral disease in man and there are approximately 100 million infections annually. Despite the global burden of DENV infections many important questions regarding DENV pathogenesis remain unaddressed due to the lack of appropriate animal models of infection and disease. A major problem is the fact that no non-human species naturally develop disease similar to human dengue fever (DF) or dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Apart from other risk factors for severe dengue such as host genetics and secondary infection with a heterologous DENV, virus virulence is a risk factor that is not well characterized. RESULTS: Three clinical DENV-1 isolates from Cambodian patients experiencing the various forms of dengue disease (DF, DHF, and DSS) were inoculated in BALB/c mice at three different concentrations. The DENV-1 isolates had different organ and cell tropism and replication kinetics. The DENV-1 isolate from a DSS patient infected the largest number of mice and was primarily neurotropic. In contrast, the DENV-1 isolates from milder clinical dengue cases infected predominantly lungs and liver, and to a lesser extent brain. In addition, infection with the DENV isolate derived from a DSS patient persisted for more than two weeks in a majority of mice compared to the other DENV-1 isolates that peaked during the first week. CONCLUSIONS: These results confirm the in vitro findings of the same DENV-1 isolates, that showed that the isolate derived from a DSS patient can be distinguished based on phenotypic characteristics that differ from the isolates derived from a DF and DHF case 1. We observed in this study that the DSS virus isolate persist longer in vivo with extensive neuroinvasion in contrast to the other DENV-1 isolates originating in milder human cases. Genomic characterization of the three clinical isolates identified six amino acid substitutions unique for the DSS isolates that were located both in structural genes (M and E) and in non-structural genes (NS1, NS3, and NS5). The characterization of these clinically distinct DENV-1 isolates highlight that DENVs within the same genotype may have different in vivo phenotypes. HIGHLIGHTS: * Clinical DENV-1 isolates have different organ tropism in BALB/c mice.* The isolate from a DSS patient is primarily neurotropic compared to the other isolates.* The DENV-1 isolates have different in vivo replication kinetics.* The isolate from a DSS patient persists longer compared to the other isolates.* These phenotypic differences confirm our earlier in vitro findings with the same DENV-1 isolates. Thus, DENVs within the same serotype and genotype may differ enough to affect clinical conditions in vivo

Clinical and Virological Factors Influencing the Performance of a NS1 Antigen-Capture Assay and Potential Use as a Marker of Dengue Disease Severity

Dengue is the most prevalent arthropod-borne disease in tropical regions. The clinical manifestation may vary from asymptomatic to potentially fatal dengue shock syndrome. Early laboratory confirmation of dengue diagnosis is essential since many symptoms are not specific. Dengue non-structural protein 1 (NS1) may be used in simple antigen-capture ELISA for early detection of dengue virus infection. Our result demonstrated that the Platelia NS1 antigen detection kit had a quite low overall sensitivity. However, sensitivity rises significantly when used in combination with MAC-ELISA. When taking into account the various forms of dengue infection, the NS1 antigen detection was found relatively high in patients sampled during the first 3 days of fever onset, in patients with primary infection, DENV-1 infection, with high level of viremia and in mild form of dengue fever. In asymptomatically infected individuals, RT-PCR assay has proved to be more sensitive than NS1 antigen detection. Moreover, the NS1 antigen level correlated significantly with high viremia and low level of NS1 antigen was associated with more severe disease

Dengue viruses – an overview

Dengue viruses (DENVs) cause the most common arthropod-borne viral disease in man with 50–100 million infections per year. Because of the lack of a vaccine and antiviral drugs, the sole measure of control is limiting the Aedes mosquito vectors. DENV infection can be asymptomatic or a self-limited, acute febrile disease ranging in severity. The classical form of dengue fever (DF) is characterized by high fever, headache, stomach ache, rash, myalgia, and arthralgia. Severe dengue, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) are accompanied by thrombocytopenia, vascular leakage, and hypotension. DSS, which can be fatal, is characterized by systemic shock. Despite intensive research, the underlying mechanisms causing severe dengue is still not well understood partly due to the lack of appropriate animal models of infection and disease. However, even though it is clear that both viral and host factors play important roles in the course of infection, a fundamental knowledge gap still remains to be filled regarding host cell tropism, crucial host immune response mechanisms, and viral markers for virulence.dengue virusdengue feverdengue hemorrhagic feverdengue shock syndromeflaviviru

Extensive spinal epidural hematoma as the cause of postpartum headache and neck pain after epidural anesthesia : a case-based report

Spinal epidural hematomas (SEH) are a rare hemorrhagic event occurring after trauma, epidural anesthesia, or operative inventions. However, in 40–50% of cases, they occur spontaneously. Spontaneous spinal epidural hematomas (SSEH) are rare in occurrence with an estimated incidence of 1 case per million annually. Pregnancy is an independent risk factor. Sudden neck or back pain, often in combination with a rapid onset of neurological symptoms, is the most common presentation of SEH (1). A 36-year-old Caucasian female with rheumatoid arthritis (RA) presented to the emergency department approximately 48 h after an uncomplicated vaginal delivery. She sought medical attention due to constant headaches and neck pain that started during active labor. An MRI of the spine revealed an extensive SEH spreading from C1 to L5. The patient was without neurological symptoms or deficits and was successfully treated conservatively without any sequelae. Even though the definitive cause of this case of SEH will remain unknown, several possible synergistic mechanisms have been identified. These include female gender, full-term pregnancy, physical activity with increased intraabdominal pressure (i.e., Valsalva maneuver), systemic administration of platelet aggregation inhibitor (PAI), and iatrogenic manipulation such as spinal epidural anesthesia. Even though autoimmune and inflammatory disorders have been described in the literature to be rare sources of hemorrhage in the spinal canal, it is unclear whether the patient’s RA should be regarded as an individual risk factor

Dengue fever in returned Swedish travelers from Thailand

The dengue viruses (DENV) are endemic in the tropical and sub-tropical countries and cause the most common arthropod-borne viral disease in humans. Travelers visiting endemic areas may both acquire and spread DENV infections, and this is the reason why prevention of mosquito bites is of crucial importance. Dengue fever (DF) has become the most common cause for tropical fever in Swedish tourists. Swedish data from 1995 to 2010 show that the number of DF cases has increased since the beginning of 2000; partly due to improved diagnostics based on IgM detection, and partly due to an increase in the number of tourists traveling to, and between, endemic areas. Young adults aged 20–29 are mostly affected, and epidemiological data indicate increased incidence rates from 2008 onwards. Our data pose a call for attention when traveling to DENV endemic areas as well as an increased awareness among physicians when treating returning travelers

Acute myocarditis caused by Francisella tularensis : a case report

Myocarditis is an inflammatory disease of the myocardium with either focal or diffuse involvement and usually gives rise to chest pain, dyspnea, palpitations, and fatigue. In severe cases, arrythmias, syncope, and cardiogenic shock may occur. Acute myocarditis is most commonly caused by a variety of viruses with cardiotropic properties. Rare causes of myocarditis include bacterial infections. We, herein, describe a case of acute myocarditis caused by the intracellular bacterium Francisella tularensis. A young and previously healthy male in Northern Sweden was referred to the emergency department due to intense upper-chest pain and dyspnea. ECG exhibited minimal ST-segment elevations and laboratory parameters revealed pathological levels of high-sensitivity cardiac troponin and C-reactive protein. Radiological imaging showed increased metabolism in enlarged lymph nodes in the chest and signs that could be compatible with increased metabolism in the left ventricular of the heart. The combination of acute myocarditis and enlarged lymph nodes was believed to be caused by the intracellular bacterium Francisella tularensis, endemic in the Northern Sweden, and was verified with positive serology. The patient showed full recovery after antimicrobial treatment. As this is the fifth published case of myocarditis associated with Francisella tularensis, we suggest considering tularemia in acute myocarditis in tularemia-endemic area