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Background: Non-dialysis-dependent chronic kidney disease (CKD-ND) patients are recommended to receive a one-dose influenza vaccination annually. However, studies investigating vaccine efficacy in the CKD-ND population are still lacking. In this study, we aimed to evaluate vaccine efficacy between the one-dose and two-dose regimen and among patients with different stages of CKD throughout a 20-week follow-up period. Methods: We conducted a single-center, non-randomized, open-label, controlled trial among patients with all stages of CKD-ND. Subjects were classified as unvaccinated, one-dose, and two-dose groups (4 weeks apart) after enrollment. Serial changes in immunological parameters (0, 4, 8, and 20 weeks after enrollment), including seroprotection, geometric mean titer (GMT), GMT fold-increase, seroconversion, and seroresponse, were applied to evaluate vaccine efficacy. Results: There were 43, 84, and 71 patients in the unvaccinated, one-dose, and two-dose vaccination groups, respectively. At 4–8 weeks after vaccination, seroprotection rates in the one- and two-dose group for H1N1, H3N2, and B ranged from 82.6–95.8%, 97.4–100%, and 73.9–100%, respectively. The concomitant seroconversion and GMT fold-increases nearly met the suggested criteria for vaccine efficacy for the elderly population. Although the seroprotection rates for all of the groups were adequate, the seroconversion and GMT fold-increase at 20 weeks after vaccination did not meet the criteria for vaccine efficacy. The two-dose regimen had a higher probability of achieving seroprotection for B strains (Odds ratio: 3.5, 95% confidence interval (1.30–9.40)). No significant differences in vaccine efficacy were found between early (stage 1–3) and late (stage 4–5) stage CKD. Conclusions: The standard one-dose vaccination can elicit sufficient protective antibodies. The two-dose regimen induced a better immune response when the baseline serum antibody titer was low. Monitoring change in antibody titers for a longer duration is warranted to further determine the current vaccine strategy in CKD-ND population.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

The influenza A (InfA) virus, which poses a significant global public health threat, is routinely classified into "subtypes" based on viral hemagglutinin (HA) and neuraminidase (NA) antigens. Because there are nearly 200 viral subtypes, current diagnostic approaches require multiplexing or array systems to cover various subtypes of HA and NA. A microfluidic chip featuring a HA × NA array was consequently developed herein for diagnosis and subtyping of InfA viruses via the use of glycan-coated magnetic beads followed by reverse transcription (RT) polymerase chain reaction (PCR). Up to 12 InfA subtypes were simultaneously detected in an automated fashion in less than 100 minutes on this microfluidic platform, representing a significant improvement in analysis speed compared to benchtop RT-PCR and chip-based microarray systems. The limits of detection of the RT-PCR assays ranged from 40 to 3000 copy numbers for the different subtypes of InfA viruses, around two orders of magnitude higher than in previous studies using microfluidic technologies. In summary, the array-type microfluidic chip system provides a rapid, sensitive, and fully automated approach for detection and multiple subtyping of InfA.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).

The COVID-19 pandemic negatively affected the economy and health security on a global scale, causing a drastic change on lifestyle, calling a need to mitigate further transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Surface-enhanced Raman spectroscopy (SERS) has shown great potential in the sensitive and rapid detection of various molecules including viruses, through the identification of characteristic peaks of their outer membrane proteins. Accurate detection can be developed through the synergistic integration effect among SERS-active substrate, the appropriate laser wavelength, and the target analyte. In this study, gold nanocavities (Au NC) and Au nanoparticles upon ZrO2 nano-bowls (Au NPs/pZrO2) were tested and used as SERS-active substrates in detecting SARS-CoV-2 pseudovirus containing S protein as a surface capsid glycoprotein (SARS-CoV-2 S pseudovirus) and vesicular stomatitis virus G (VSV-G) pseudo-type lentivirus (VSV-G pseudovirus) to demonstrate their virus detection capability. The optimized Au NCs and Au NPs/pZrO2 substrates were then verified by examining the repetition of measurement, reproducibility, and detection limit. Due to the difference in geometry and composition of the substrates, the characteristic peak-positions of live SARS-CoV-2 S and VSV-G pseudoviruses in the obtained Raman spectra vary, which were also compared with those of inactivated ones. Based on the experimental results, SERS mechanism of each substrate to detect virus is proposed. The formation of hot spots brought by the synergistic integration effect among substrate, analyte, and laser induction may result differences in the obtained SERS spectra., Graphical abstract Image 1

Additional file 2. Amino acid substitutions in NP protein of H3N2 viruses. NP substitutions of 79 randomly chosen Taiwan H3N2 isolates from 1999 to 2017, WHO recommended vaccine strains and reference strains were compared.

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID(50)) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development. IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

The COVID-19 pandemic has caused a significant burden since December 2019 that has negatively impacted the global economy owing to the fact that the SARS-CoV-2 virus is fast-transmitting and highly contagious. Efforts have been taken to minimize the impact through strict screening measures in country borders in order to isolate potential virus carriers. Effective fast-screening methods are thus needed to identify infected individuals. The standard diagnostic methods for screening SARS-CoV-2 virus have always been to perform nucleic acid-based and serological tests. However, with each having drawbacks on producing false results at very early or later stage after symptoms onset, supplementary techniques are needed to back up these tests. Surface-enhanced Raman spectroscopy (SERS) as a detection technique has continuously advanced throughout the years in terms of sensitivity and capability to detect ultralow concentration of analytes ranging from single molecule to pathogens, to present as a highly potential alternative to known sensing methods. SERS technology as a candidate for an alternative and supplementary diagnostic method for the viral envelope of SARS-CoV-2 virus is presented, comparing its pros and cons to the standard methods and what other aspects it could offer that the other methods are not capable of. Factors that contribute to the detection effectivity of SERS is also discussed to show the advantages and limitations of this technique. Despite its promising capabilities, challenges like sources of SARS-CoV-2 virus and its variations, reliable SERS spectra, mass production of SERS-active substrates, and compliance to regulations for wide-scale testing scenario are highlighted.

Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

The first deep sequencing method was announced in 2005. Due to an increasing number of sequencing data and a reduction in the costs of each sequencing dataset, this innovative technique was soon applied to genetic investigations of viral genome diversity in various viruses, particularly RNA viruses. These deep sequencing findings documented viral epidemiology and evolution and provided high-resolution data on the genetic changes in viral populations. Here, we review deep sequencing platforms that have been applied in viral quasispecies studies. Further, we discuss recent deep sequencing studies on viral inter- and intrahost evolution, drug resistance, and humoral immune selection, especially in emerging and re-emerging viruses. Deep sequencing methods are becoming the standard for providing comprehensive results of viral population diversity, and their applications are discussed.

Vascular leakage is one of the salient characteristics of severe dengue. Nonstructural protein 1 (NS1) of dengue virus (DENV) can stimulate endothelial cells to secrete endothelial hyperpermeability factor, macrophage migration inhibitory factor (MIF), and the glycocalyx degradation factor heparanase 1 (HPA-1). However, it is unclear whether MIF is directly involved in NS1-induced glycocalyx degradation. In this study, we observed that among NS1, MIF and glycocalyx degradation-related molecules, the HPA-1, metalloproteinase 9 (MMP-9) and syndecan 1 (CD138) serum levels were all increased in dengue patients, and only NS1 and MIF showed a positive correlation with the CD138 level in severe patients. To further characterize and clarify the relationship between MIF and CD138, we used recombinant NS1 to stimulate human cells in vitro and challenge mice in vivo. Our tabulated results suggested that NS1 stimulation could induce human endothelial cells to secrete HPA-1 and immune cells to secrete MMP-9, resulting in endothelial glycocalyx degradation and hyperpermeability. Moreover, HPA-1, MMP-9, and CD138 secretion after NS1 stimulation was blocked by MIF inhibitors or antibodies both in vitro and in mice. Taken together, these results suggest that MIF directly engages in dengue NS1-induced glycocalyx degradation and that targeting MIF may represent a possible therapeutic approach for preventing dengue-induced vascular leakage., Author summary DENV NS1 induces endothelial glycocalyx degradation and hyperpermeability via HPA-1 and MMP-9 activation in an MIF-dependent manner.

Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCE Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.

Herpes simplex virus (HSV) ocular infection causes significant visual burden worldwide. Despite the fact that dendritic or geographic corneal ulcers are typical findings in HSV epithelial keratitis, conjunctival ulcer as a sign of HSV infection has rarely been reported. Although easily overlooked, this important sign could be enhanced by fluorescein staining. We report two cases of conjunctival geographic ulcers proven to be HSV infection by viral isolation and polymerase chain reaction (PCR). One patient had bilateral disease and blepharitis, and the other had unilateral involvement without skin lesions. With timely diagnosis and proper management, excellent visual outcome can be expected.

Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the Picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and being tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effects of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low-neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability and that surveillance studies should look out for these mutants, which could compromise vaccine efficacy. IMPORTANCE Emerging and reemerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.

RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen. IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro . Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S

It is generally agreed that human influenza virus preferentially binds to α-2,6-linked sialic acid-containing receptors, and mutations that change the binding preference may alter virus infectivity and host tropism. Limited information is available on the glycan-binding specificity of epidemic influenza viruses. In this study, we systemically investigated the glycan-binding preferences of human influenza A(H3N2) viruses isolated from 1999 to 2007 in Taiwan using a high-throughput carbohydrate array. The binding patterns of 37 H3N2 viruses were classified into three groups with significant binding-pattern variations. The results showed that the carbohydrate-binding patterns of H3N2 varied over time. A phylogenetic analysis of the hemagglutinin gene also revealed progressive drift year to year. Of note, the viruses that caused large outbreaks in 1999 and 2003 showed glycan-binding preferences to both α-2,3 and α-2,6 sialylated glycans. Twenty amino acid substitutions were identified primarily at antigenic sites that might contribute to H3N2 virus evolution and the change in the glycan-binding patterns. This study provides not only a systematic analysis of the receptor-binding specificity of influenza clinical isolates but also information that could help to monitor the outbreak potential and virus evolution of influenza viruses.

Viral aerosol infection through cough generates large amounts of viral aerosol and can result in many adverse health effects such as influenza flu and severe acute respiratory syndrome (SARS). To characterize the coughed viral aerosol, the sampler needs to sample at higher flow rate and possess high physical collection efficiency as well as high viral preservation. However, most current inertia-based high flow bioaerosol samplers are not suited for viral aerosol sampling since the viability will be lost doing the sampling process. Current condensation growth methods only have good physical collection efficiency and viral preservation at low flow rate (

Enterovirus 71-induced brainstem encephalitis with pulmonary edema and/or neurogenic shock (stage 3B) is associated with rapid mortality in children. In a small pilot study, we found that milrinone reduced early mortality compared with historical controls. This prospective, randomized control trial was designed to provide more definitive evidence of the ability of milrinone to reduce the 1-week mortality of stage 3B enterovirus 71 infections.Prospective, unicenter, open-label, randomized, controlled study.Inpatient ward of a large tertiary teaching hospital in Ho Chi Minh City, Vietnam.Children (≤ 18 yr old) admitted with proven enterovirus 71-induced pulmonary edema and/or neurogenic shock.Patients were randomly assigned to receive intravenous milrinone (0.5 μg/kg/min) (n = 22) or conventional management (n = 19). Both groups received dopamine or dobutamine and intravenous immunoglobulin.The primary endpoint was 1-week mortality. The secondary endpoints included length of ventilator dependence and hospital stay and adverse events. The median age was 2 years with a predominance of boys in both groups. The 1-week mortality was significantly lower, 18.2% (4/22) in the milrinone compared with 57.9% (11/19) in the conventional management group (relative risk = 0.314 [95% CI, 0.12-0.83], p = 0.01). The median duration of ventilator-free days was longer in the milrinone treatment group (p = 0.01). There was no apparent neurologic sequela in the survivors in either group, and no drug-related adverse events were documented.Milrinone significantly reduced the 1-week mortality of enterovirus 71-induced pulmonary edema and/or neurogenic shock without adverse effects. Further studies are needed to determine whether milrinone might be useful to prevent progression of earlier stages of brainstem encephalitis.

Background Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015. Methods Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay. Results A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 102 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106–109 copies/mL, which was markedly higher than the rate observed in the other age groups. Conclusions The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates., Author Summary The LightMix dengue virus EC qRT-PCR assay is effective for early diagnosis of DENV infection. High viral loads during primary infection in elderly patients may comprise a positive predictor for severe illness, and may contribute to high mortality rates.

International audience; BACKGROUND: The global impact of the 2009 influenza A(H1N1) pandemic (H1N1pdm) is not well understood. OBJECTIVES: We estimate overall and age-specific prevalence of cross-reactive antibodies to H1N1pdm virus and rates of H1N1pdm infection during the first year of the pandemic using data from published and unpublished H1N1pdm seroepidemiological studies.METHODS: Primary aggregate H1N1pdm serologic data from each study were stratified in standardized age groups and evaluated based on when sera were collected in relation to national or subnational peak H1N1pdm activity. Seropositivity was assessed using well-described and standardized hemagglutination inhibition (HI titers ≥ 32 or ≥ 40) and microneutralization (MN ≥ 40) laboratory assays. The prevalence of cross-reactive antibodies to the H1N1pdm virus was estimated for studies using sera collected prior to the start of the pandemic (between 2004 and April 2009); H1N1pdm cumulative incidence was estimated for studies in which collected both pre- and post-pandemic sera; and H1N1pdm seropositivity was calculated from studies with post-pandemic sera only (collected between December 2009-June 2010).RESULTS: Data from 27 published/unpublished studies from 19 countries/administrative regions - Australia, Canada, China, Finland, France, Germany, Hong Kong SAR, India, Iran, Italy, Japan, Netherlands, New Zealand, Norway, Reunion Island, Singapore, United Kingdom, United States, and Vietnam - were eligible for inclusion. The overall age-standardized pre-pandemic prevalence of cross-reactive antibodies was 5% (95%CI 3-7%) and varied significantly by age with the highest rates among persons ≥ 65 years old (14% 95%CI 8-24%). Overall age-standardized H1N1pdm cumulative incidence was 24% (95%CI 20-27%) and varied significantly by age with the highest in children 5-19 (47% 95%CI 39-55%) and 0-4 years old (36% 95%CI 30-43%).CONCLUSIONS: Our results offer unique insight into the global impact of the H1N1 pandemic and highlight the need for standardization of seroepidemiological studies and for their inclusion in pre-pandemic preparedness plans. Our results taken together with recent global pandemic respiratory-associated mortality estimates suggest that the case fatality ratio of the pandemic virus was approximately 0.02%.

Human adenoviruses (HAdVs) are important causes of respiratory infections in children. They usually cause mild upper respiratory symptoms, but they can also produce severe pneumonia and other complications. The aims of this retrospective study were to better define the molecular epidemiology of respiratory adenoviruses circulating in Taiwanese children during 2002 and 2013, detect reinfections and co-infections, and characterize the clinical features and laboratory findings according to the causative genotypes. We collected a representative sample of 182 isolates of adenoviruses from 175 children during the 12-year study period. The most prevalent species was HAdV-B genotype 3 (HAdV-3) (92/182, 50.5%) followed by HAdV-C (HAdV-2) (38/182, 20.9%). A single outbreak of HAdV-E (6/182, 3.3%) was noted in 2007. The mean age of children with adenovirus infections was 3.7 ± 2.0 years, with a slight predominance of males (53.1%). Children with HAdV-B tended to be older, had more lower respiratory tract infections, gastrointestinal symptoms, and a higher rate of hospitalization than those with HAdV-C (P

PURPOSE We verified a multiplex dot hybridization (MDH) assay for the rapid detection and differentiation of Acanthamoeba keratitis (AK) and herpes simplex keratitis (HSK). METHODS Molecular detection of Acanthamoeba and herpes simplex virus in corneal scrapes was performed with the MDH assay and standard diagnostic methods. The experimental group included corneal scrapes (n = 33) from patients with culture- or pathology-confirmed AK (n = 15) and real-time PCR-confirmed HSK (n = 16). The control group included 50 samples from cases of bacterial keratitis (n = 15), fungal keratitis (n = 15), and initially presumed AK (n = 5) or HSK (n = 17) which finally were excluded by culture for Acanthamoeba or by real-time PCR for herpes simplex virus, respectively. Discrepant results between methods were resolved by DNA sequencing of the PCR amplicons. RESULTS After discrepant analysis, the sensitivity for the diagnosis of AK and HSK was both 93.3% by the MDH assay, while the specificity was 100% for the two types of keratitis. The turnaround time of MDH assay was within a working day using an already prepared array. Two false-negatives (one AK case and one HSK case) were obtained by the MDH assay. CONCLUSIONS The MDH assay could effectively prevent missed or delayed diagnosis of AK and HSK and has a potential to be adopted in routine clinical practice if the test is commercialized.

Background Hemodialysis patients have higher risk of mortality and morbidity when infected with 2009 pandemic H1N1 (pH1N1/09) virus. Depending on different methodologies and criteria, previous studies reported variable response rates to adjuvanted vaccines against pH1N1/09 virus in hemodialysis patients, however, the efficacy of non-adjuvanted vaccines, which are currently used in many countries such as the USA and Asian areas, has not been comprehensively evaluated in hemodialysis population before. Methods We evaluated the efficacy of a standard single15 μg-dose of non-adjuvanted monovalent pH1N1/09 vaccine (AdimFlu-S) in vaccine-naive 110 hemodialysis and 173 healthy participants. When enrolling, all participants had not any clinical symptom or sign suggesting pH1N1/09 infection since the index case was identified in Taiwan. Sera from all participants were tested by hemagglutination inhibition (HI) and micro-neutralization-ELISA (microNT-ELISA) tests before and 21 days after vaccination. The outcome parameters were seroconversion rate (≥4-fold in HI titer with titer ≥1:40), seroprotection rate (HI titers ≥1:40), seroresponse rate (≥4-fold increase in HI or microNT-ELISA titer), fold of increase in geometric mean (GM) titers, and adverse effects. Results In method A analyses, we included all participants’ data in final analyses, and the seroconversion rates and the fold increase of GM titer after vaccination were 25.4% and 1.8 in adult (18–60-year olds) hemodialysis subgroup, and 23.4% and 1.8 in elder (>60-year olds) hemodialysis subgroup based on HI titers, which were all significantly lower than those of the corresponding healthy control subgroups. Similar trends were observed based on microNT-ELISA titers, further validating the results. Multivariable analysis revealed hemoglobin and cholesterol levels were significant predictors for seroresponse in hemodialysis patients, suggesting the possible impacts of nutrition status and anemia. In method B analyses, we excluded participants with pre-vaccination seroprotection (based on HI or microNT-ELISA criteria) in final analyses. The response rates in various subgroups from method B analyses were also similar as those from method A analyses. No severe adverse effect was noted. Conclusions According to the European and U.S. criteria, a single 15 μg-dose of non-adjuvanted pH1N1/09 vaccination is safe but ineffective in both adult and elder hemodialysis patients. Further studies using multiple doses or higher antigen amount are warrant to define the most appropriate regimen.

Aims: The outbreak of highly pathogenic avian influenza H5N1 in poultry and the influenza A pandemic have wreaked havoc on public health. Viruses alter their carbohydrate binding preferences, thereby causing pandemics all over the world. In this study, we tried to investigate the carbohydrate binding specificity of influenza clinical isolates. Materials & methods: Biotin-conjugated polyacrylamide-based glycan epitopes were immobilized on UltraBind™ membranes and used to survey the glycan-binding preference of influenza clinical isolates, including seasonal influenza A, A(H1N1)pdm09 and influenza B viruses. In addition, the DNA sequences of influenza B virus hemagglutinin were analyzed. Results: Human influenza A, especially the A(H1N1)pdm09 viruses, accepted α2,6 and α2,3 sialylated glycans, sulfated glycans and α2,8 sialosides. Although all influenza B clinical isolates bound strongly to NeuAc, 6´-sialyl lactose and sialyl biantennary N-glycan, some viruses also recognized sulfated and α2,3 sialylated glycans. According to the nucleotide sequences of viral hemagglutinin, influenza B viruses that exhibited weak interaction with sulfated and α2,3 sialylated glycans showed fewer charged amino acids. Conclusion: The substrate specificities of influenza clinical isolates were surveyed. Influenza A exhibited more complicated glycan-binding patterns than influenza B viruses. Our findings provided a systematic investigation of receptor-binding specificities for influenza clinical isolates, as well as useful information for exploring viral tropism.

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant ( K d ) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.

Enterovirus 71 (EV71), a member of the Enterovirus genus in the Picornaviridae family, has become an emergent infectious disease worldwide, most notably in Asia. As a neurotropic virus, EV71 infection occasionally causes neurological diseases with pulmonary edema, which is fatal for children. In this review, we examine the epidemiology of EV71, with three waves of increased EV71 activity since the 1970s and discuss the genotypic changes in phylogeny between the outbreaks or epidemics. Genetic changes including mutations and recombinations as well as the diversity of antigenic properties among EV71 strains in various outbreaks are described. Furthermore, the impact of genetic changes on viral pathogenesis and vaccine candidate selection are addressed. In conclusion, these genetic and antigenic investigations of EV71 evolution have provided us with new insight into the trend of EV71 epidemiology, which may contribute to a better understanding of the viral pathogenesis and vaccine development.

A novel pandemic influenza H1N1 (pH1N1) virus spread rapidly across the world in 2009. Due to the important role of antibody-mediated immunity in protection against influenza infection, we used an enzyme-linked immunosorbent assay-based microneutralization test to investigate cross-reactive neutralizing antibodies against the 2009 pH1N1 virus in 229 stored sera from donors born between 1917 and 2008 in Taiwan. The peak of cumulative geometric mean titers occurred in donors more than 90 years old and declined sharply with decreasing age. Sixteen of 27 subjects (59%) more than 80 years old had cross-reactive antibody titers of 160 or more against the 2009 pH1N1 virus, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) from 6 months to 9 years old had an antibody titer of 40. We further tested the antibody responses in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed that only the 2 sera from children with antibody responses to the 2009 pH1N1 virus had high titers of neutralizing antibody against recent seasonal influenza virus strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years old or older, and the elderly subjects who may already have been exposed to the 1918 virus had high titers of neutralizing antibody to the 2009 pH1N1 virus. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce a cross-reactive antibody response to the 2009 pH1N1 virus.

Yeh Y-M, Chang K-C, Chen Y-P, Kao L-Y, Tsai H-P, Ho C-L, Wang J-R, Jones D & Chen T-Y (2010) Histopathology57, 785–795 Large B cell lymphoma presenting initially in bone marrow, liver and spleen: an aggressive entity associated frequently with haemophagocytic syndrome Aims: To describe diffuse large B cell lymphoma (DLBCL) presenting initially in bone marrow, liver and spleen (BLS-type) without lymphadenopathy. Methods and results: The clinicopathological and cytogenetic features of 11 such cases (eight men, three women; mean age: 62.7 years are described). Usually presenting with fever and haemophagocytic syndrome suggesting infection and complicating timely diagnosis, bone marrow examination showed patchy and interstitial infiltration of large tumour cells without sinusoidal involvement. All cases had a high Ki-67 index (≥90%), commonly a non-germinal centre/activated B cell immunophenotype and were negative for Epstein–Barr virus and human herpesvirus 6 and 8. The more frequent cytogenetic changes involved chromosomal loci 14q32 and 9p24, as well as del(3)(q21), add(7)(p22), t(3;6), del(8)(p22), +18 and add(19)(p13). Clinical behaviour was very aggressive, with a 2-year survival rate of 18% (45% of patients died within 3 weeks). High-dose chemotherapy with haematopoietic stem cell transplantation prolonged survival in one patient. Conclusions: Although it shares with intravascular LBCL a subtle presentation and an aggressive clinical course, this primary BLS large cell lymphoma variant is distinguished by lacking an intravascular component and having different cytogenetic findings.

In recent years, enterovirus 71 (EV71) has been a cause of numerous outbreaks of hand-foot-and-mouth disease, with severe neurological complications in the Asia-Pacific region. The reemergence in Taiwan of EV71 genotype B5 in 2008 resulted in the largest outbreak of EV71 in Taiwan in the past 11 years. Phylogenetic analyses indicated that dominant genotype changes from B to C or C to B occurred at least three times between 1986 and 2008. Furthermore, antigenic cartography of EV71 by using neutralization tests revealed that the reemerging EV71 genotype B5 strains formed a separate cluster which was antigenically distinct from the B4 and C genotypes. Moreover, analyses of full-length genomic sequences of EV71 circulating in Taiwan during this period showed the occurrence of intra- and interserotypic recombination. Therefore, continuous surveillance of EV71 including the monitoring of genetic evolution and antigenic changes is recommended and may contribute to the development of a vaccine for EV71.

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.

Annual influenza vaccination is recommended, but its efficacy in dialysis population is still controversial. Here we aimed to compare the dynamic changes of immune response between various influenza vaccination protocols in hemodialysis patients. A 18-week open label, non-randomized, controlled trial was conducted during 2011–2012. The efficacy between unvaccinated, one- and two-dose regimens were evaluated in 175 hemodialysis patients. Immunogenic profiles were assessed by hemagglutination-inhibition assays. At 3–9 weeks post-vaccination, antibody responses were similar between the one- and two-dose regimens, while the seroprotection rates (antibody titer ≥1:40) for influenza A were 55.6–82.5% in the adult (18–60 years) and 33.3–66.7% in the elderly (>60 years). Meanwhile, the seroprotection rates for influenza B were low (4.0–25.0%). By 18 weeks post-vaccination, the seroprotection rates for influenza A and B declined (0.0–33.3%) in both the adult and elderly receiving one- or two-dose regimens. Of dialysis patients, at most 2.4% developed moderate to severe adverse effects(myalgia and headache) after vaccination. In conclusion, the two-dose regimen could not improve immune responses than the one-dose regimen in hemodialysis patients; meanwhile the induced protective antibodies of both regimens could not be maintained for more than 4 months. Modification of current influenza vaccination strategy in dialysis population should be re-considered.

Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P

Enterovirus (EV) infections are common. There are more than 60 known serotypes, and each has different epidemiologic or medical importance. Over 700 physicians from 75% of basic administrative units of Taiwan participated in the "Sentinel Physician Surveillance of Infectious Disease" and reported weekly to the Center for Disease Control-Taiwan with data on various infections. Data of laboratory-confirmed EV infections from this surveillance between 2000 and 2005 was analyzed. EV serotypes were determined by immunofluorescence staining and/or viral VP1 sequence analysis. A total of 12,236 EV cases, or approximately 1,300-2,500 per year, were identified, and 52% of the cases occurred between April and July. The median age was 3 years, and 57.6% of patients were male. Coxsackievirus A (CA) 16 and EV71, which primarily manifest as hand-foot-and-mouth disease, were the most prevalent serotypes every year except 2004. Other prevalent serotypes and associated symptoms varied from year to year. Echovirus (E) 30 and E6, which are associated with aseptic meningitis, were prevalent in 2001 and 2002, CA4 and CA10, which cause herpangina, were predominant in 2004, and coxsackievirus B (CB) 4 and CB3, which are associated with neonatal febrile disease, were most common in 2004 and 2005, respectively. Some of these epidemics overlapped with outbreaks of the same serotypes in other Asian Pacific countries. Of all serotypes, EV71 was associated with the highest number of severe complications in patients. Surveying the epidemic pattern, disease spectra, and severity associated with each EV serotype provided important information for public health and medical personnel.

Viral etiologies of respiratory tract infections (RTIs) have been less studied in adult than in pediatric populations. Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinoviruses in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for rhinoviruses and enteroviruses. Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples. Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation, PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and rhinoviruses (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (

PHS154 EStimating tHE HEaltH BEnEfitS of mEEting tHE HEaltHy PEoPlE 2020 oBjEctivES for rEducing invaSivE colorEctal cancEr incidEncE in tHE unitEd StatES Hung M.1, Ekwueme D.2, Wang J.3 1Division of Cancer Prevention and Control, US Centers for Disease Control and Prevention, Chamblee, GA, USA, 2Centers for Disease Control and Prevention, Chamblee, GA, USA, 3Department of Public Health, National Cheng Kung University College of Medicine, Tainan, Taiwan Objectives: This study aims to quantify the expected years of life lost (EYLL) that could be saved if the original Healthy People (HP) 2020 target goal for reducing invasive colorectal cancer (CRC) was met. MethOds: A total of 232,208 patients diagnosed with invasive CRC in 2000–2011 were identified from the Surveillance, Epidemiology, and End Results (SEER) registries. The lifetime survival functions for the cancer cohort and age-/sex-matched reference population were generated using a semiparametric extrapolation method with annual US life tables from 2000-2011. The EYLL for CRC was calculated by subtracting the estimated life expectancy of CRC patients from that of the reference population. The total EYLL that could be prevented was calculated by subtracting the age-adjusted incidence of invasive CRC rate of the base year from that of the target year multiplied by the average EYLL per person and the 2020 population projections. Results: An individual diagnosed with invasive CRC was estimated to have an average life-expectancy of 10.1 years and 6.8 EYLL. If the HP 2020 target goal for invasive CRC was met, the nation could potentially save an estimated 154,848 EYLL. For men and women with an invasive CRC, estimated EYLL were 74,528 and 79,334, respectively. By race, reducing invasive CRC would result in a significantly greater benefit for both black men and women compared to white men and women with p< 0.05. The estimated EYLL was 7.4 and 8.0 years for black men and women and 6.4 and 6.1 years for white men and women per person, respectively. cOnclusiOns: The potential life years saved by successfully meeting the HP 2020 target for invasive CRC rate would be substantial for the nation. The benefit in terms of life years saved per person would be greater among blacks relative to whites.

Genetic and antigenic analyses of influenza B virus field strains isolated in Taiwan from 1998 to 2005 were performed. To investigate the molecular evolution of influenza B viruses, sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase genes was performed. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculating with the Yamagata lineage, was identified in Taiwan in March 2001. Concurrently, there was an increasing prevalence of this lineage in many parts of the world, including North America and Europe, during the 2001-2002 season. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain.

The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-alpha expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.

A laboratory-based surveillance network of 11 clinical virological laboratories for influenza viruses was established in Taiwan under the coordination of the Center for Disease Control and Prevention (CDC), Taiwan. From October 2000 to March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred frequently in winter in the northern hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B also occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from the year 2002 clustered into different groups, and the data indicate that both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were cocirculating. Sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least 2 years before the vaccine strains were introduced. No clear seasonality of influenza reports in Taiwan occurred in contrast to other more continental regions.