Lisa F. P. Ng

Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection

Summary Background The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. Methods We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). Findings We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. Interpretation Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations. Published online May 9, 2003 http://image.thelancet.com/extras/03art4454web.pdf

IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity

Background Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity. Methods and Findings Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity. Conclusions This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.

Persistent Arthralgia Induced by Chikungunya Virus Infection is Associated with Interleukin-6 and Granulocyte Macrophage Colony-Stimulating Factor

Background. Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients. Methods. A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated. Results. When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)–6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor. Conclusions. The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.

Catching bird flu in a droplet

It is assumed that a timely mass administration of antiviral drugs, backed by quarantines and social distancing, could contain a nascent influenza epidemic at its source, provided that the first clusters of cases were localized within a short time. However, effective routine surveillance may be impossible in countries lacking basic public health resources. For a global containment strategy to be successful, low-cost, easy-to-use handheld units that permit decentralized testing would be vital. Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. In a sequential process, the viral RNA is isolated, purified, preconcentrated by 50,000% and subjected to ultrafast real-time RT-PCR. Compared to commercially available tests, the bioassay is equally sensitive and is 440% faster and 2,000–5,000% cheaper.

Active Infection of Human Blood Monocytes by Chikungunya Virus Triggers an Innate Immune Response

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-α were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

Early Appearance of Neutralizing Immunoglobulin G3 Antibodies Is Associated With Chikungunya Virus Clearance and Long-term Clinical Protection

Background. Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia. Methods. We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity. Results. We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage. Conclusions. Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease.

A Pathogenic Role for CD4+ T Cells during Chikungunya Virus Infection in Mice

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Injury to the joint is believed to occur because of viral and host immune-mediated effects. However, the exact involvement of the different immune mediators in CHIKV-induced pathogenesis is unknown. In this study, we assessed the roles of T cells in primary CHIKV infection, virus replication and dissemination, and virus persistence, as well as in the mediation of disease severity in adult RAG2−/−, CD4−/−, CD8−/−, and wild-type CHIKV C57BL/6J mice and in wild-type mice depleted of CD4+ or CD8+ T cells after Ab treatment. CHIKV-specific T cells in the spleen and footpad were investigated using IFN-γ ELISPOT. Interestingly, our results indicated that CHIKV-specific CD4+, but not CD8+, T cells are essential for the development of joint swelling without any effect on virus replication and dissemination. Infection in IFN-γ−/− mice demonstrated that pathogenic CD4+ T cells do not mediate inflammation via an IFN-γ–mediated pathway. Taken together, these observations strongly indicate that mechanisms of joint pathology induced by CHIKV in mice resemble those in humans and differ from infections caused by other arthritogenic viruses, such as Ross River virus.

Viperin restricts chikungunya virus replication and pathology

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally‐acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope ‘E2EP3’. E2EP3 is located at the N‐terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3‐specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV‐specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non‐human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre‐clinical basis for the design of effective vaccine against arthralgia‐inducing CHIKV and other alphaviruses.

An Essential Role of Antibodies in the Control of Chikungunya Virus Infection

In recent years, Chikungunya virus (CHIKV) was responsible for epidemic outbreaks in intertropical regions. Although acquired immunity has been shown to be crucial during CHIKV infection in both humans and mice, their exact role in the control of CHIKV infection remains unclear. In this study, wild-type (WT), CD4−/−, and B cell (μMT) knockout mice were infected with CHIKV. Sera were taken at different days postinfection and measured for anti-CHIKV Ab levels. Isotype and neutralizing capacity of these Abs were assessed in vitro, and specific linear epitopes were mapped. Viremia in CHIKV-infected μMT mice persisted for more than a year, indicating a direct role for B cells in mediating CHIKV clearance. These animals exhibited a more severe disease than WT mice during the acute phase. Characterization of CHIKV-specific Abs revealed that anti-CHIKV Abs were elicited early and targeted epitopes mainly at the C terminus of the virus E2 glycoprotein. Furthermore, CD4−/− mice could still control CHIKV infection despite having lower anti-CHIKV Ab levels with reduced neutralizing capacity. Lastly, pre-existing natural Abs in the sera of normal WT mice recognized CHIKV and were able to partially inhibit CHIKV. Taken together, natural and CHIKV infection–induced specific Abs are essential for controlling CHIKV infections.