Taiwan Centres for Disease Control.

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

BackgroundInfluenza A viruses are major human and animal pathogens with huge economic and societal impact from illness, hospitalizations, and deaths. Virus-like particles (VLPs) of influenza virus have been suggested as a vaccine candidate offering improved safety and efficacy. To develop this concept further, we established a flexible platform to efficiently generate different subtypes of mammalian-expressed influenza VLPs. Here we demonstrate that these mammalian VLPs strongly resemble the authentic viruses in structure, particle size and composition of host factors, and even glycosylation of viral antigens.Methodology/Principal FindingsIn this study, a mammalian VLP system was established by stable co-expression of four influenza structural proteins (HA, NA, M1, and M2) in a Vero cell line. By replacing the surface glycoproteins of HA and NA, we converted the H3N2-VLP subtype to H5N1-VLP. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were identified by LC/MS/MS. Protease protection assays demonstrated that specific cellular proteins that co-purified with influenza virions were integrated into mammalian VLPs. The glycosylation profiles of mammalian VLPs as revealed by deglycosylation assays were similar to that of progeny viruses produced from Vero cells. Vaccination of mice with 2.5 µg and above of H5N1-VLP elicited H5-specific IgG1 antibodies and resulted in full protection against lethal infection with homologous virus. These results provide compelling evidence that mammalian VLPs closely emulate the exterior of authentic virus particles not only in antigen presentation but also in biological properties and should provide promising vaccine candidates.Conclusions/SignificanceThis flexible mammalian influenza VLP system offers a superior alternative to the conventional reverse genetic vaccine platform without concerns over inadequate presentation of immune antigens or limitations imposed by the manipulation of real viruses.

Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.

A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007–2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005–2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007–2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008–2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes.

BackgroundHuman enterovirus 71 (EV-71) is known of having caused numerous outbreaks of hand-foot-mouth disease, and other clinical manifestations globally. In 2008, 989 EV-71 strains were isolated in Taiwan.ResultsIn this study, the genetic and antigenic properties of these strains were analyzed and the genetic diversity of EV-71 subgenogroups surfacing in Taiwan was depicted, which includes 3 previously reported subgenogroups of C5, B5, and C4, and one C2-like subgenogroup. Based on the phylogenetic analyses using their complete genome nucleotide sequences and neutralization tests, the C2-like subgenogroup forms a genetically distinct cluster from other subgenogroups, and the antisera show a maximum of 128-fold decrease of neutralization titer against this subgenogroup. In addition, the subgenogroup C4 isolates of 2008 were found quite similar genetically to the Chinese strains that caused outbreaks in recent years and thus they should be carefully watched.ConclusionsOther than to be the first report describing the existence of C2-like subgenogroup of EV-71 in Taiwan, this article also foresees a potential of subgenogroup C4 outbreaks in Taiwan in the near future.

We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.Rapid and accurate detection of dengue virus (DENV) infection from acute-phase viremic blood samples from patients with a fever contributes greatly to patient management in hospitals and control measures in public health. In addition, rapid detection of imported dengue cases at airports can help to reduce the annual local outbreaks in a country where dengue is not endemic, such as Taiwan. We have previously reported on screening for fever at airports in Taiwan as part of active surveillance for a panel of notifiable infectious diseases, such as dengue, gastroenteritis caused by enteric bacteria, malaria, and chikungunya (10, 12). A total of 298 imported dengue cases were identified at airports in Taiwan from July 2003 to August 2008 using real-time one-step reverse transcription-PCR (RT-PCR) and envelope/membrane-specific capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) (8, 9, 11).For molecular diagnosis, a real-time one-step RT-PCR was performed using two sets of consensus primers, one primer set targeting a region of the nonstructural protein 5 (NS5) genes to detect all flaviviruses and the other primer set targeting a region of the capsid gene to detect all DENV serotypes. Positive samples were then confirmed by DENV serotyping using four sets of serotype-specific primers targeting the capsid gene to differentiate the DENV serotypes (8, 11). For serological diagnosis, envelope/membrane-specific capture IgM and IgG ELISAs were used to detect and differentiate primary and secondary DENV infections in acute-phase and convalescentphase serum samples (9, 11). Differentiation of primary and secondary DENV infections was defined by the ratio of the IgM-to-IgG readings, Ն1.2 or Ͻ1.2, respectively. In addition, a simple and sensitive nonstructural protein 1 (NS1) serotypespecific IgG ELISA was used to differentiate the immunological status of individuals into naïve, primary, or secondary DENV infections using acute-phase, convalescent-phase, or postinfection serum samples (3, 9, 11). A unique feature of this assay is that differentiation of primary and secondary DENV infections can be made when DENV-specific IgG antibody has not been produced in the early acute-phase serum samples.Most of these imported dengue cases were viremic when arriving at airports. It would be desirable to reduce the time gap between clinical and laboratory diagnoses to prevent the local transmission of the imported DENVs. Recent advances on the development of DENV NS1 antigen (Ag) assay offer promising new perspectives on the rapid diagnosis of dengue, although limited sensitivity was reported in patients with acute febrile illness in an area where dengue is...

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