, 276 (2003); 302 Science et al. Y. Guan Coronavirus from Animals in Southern China Isolation and Characterization of Viruses Related to the SARS This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): October 6, 2014 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/302/5643/276.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2003/10/09/1087139.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/302/5643/276.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/302/5643/276.full.html#ref-list-1 , 2 of which can be accessed free: cites 6 articles This article 521 article(s) on the ISI Web of Science cited by This article has been http://www.sciencemag.org/content/302/5643/276.full.html#related-urls 100 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/virology Virology subject collections: This article appears in the following

Background: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.Objectives: The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels.Study design: A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA (R) cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity.Results: A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA (R) cards completely inactivated measles virus and stabilized RNA for at least six months.Conclusions: These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. Published by Elsevier B.V.

Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent.

Surveillance of respiratory viruses has been conducted for many years at the public health laboratory in Hong Kong. With the occurrence of pandemic influenza A (H1N1) 2009, we observed a change in the seasonality of influenza activity with a seemingly corresponding change in the activity of respiratory syncytial virus, parainfluenza virus, and adenovirus during 2009-2011. This phenomenon could most likely be explained by virus interference.

Influenza A H1N1 2009 virus caused the first pandemic in an era when neuraminidase inhibitor antiviral drugs were available in many countries. The experiences of detecting and responding to resistance during the pandemic provided important lessons for public health, laboratory testing, and clinical management. We propose recommendations for antiviral susceptibility testing, reporting results, and management of patients infected with 2009 pandemic influenza A H1N1. Sustained global monitoring for antiviral resistance among circulating influenza viruses is crucial to inform public health and clinical recommendations for antiviral use, especially since community spread of oseltamivir-resistant A H1N1 2009 virus remains a concern. Further studies are needed to better understand influenza management in specific patient groups, such as severely immunocompromised hosts, including optimisation of antiviral treatment, rapid sample testing, and timely reporting of susceptibility results.

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak.

During the early phase of the influenza pandemic in 2009, all cases of laboratory-confirmed pandemic (H1N1) 2009 (pH1N1) infection required compulsory isolation in hospital. These cases were offered oseltamivir treatment and only allowed to be discharged from the hospital when three consecutive respiratory specimens were negative for the virus by reverse transcription-polymerase chain reaction (RT-PCR). We reviewed the case records of these patients to assess the viral shedding kinetics of the pH1N1 virus. We defined viral shedding duration as the interval from illness onset date to the date of collection of the last positive specimen from the patients. Fifty-six patients were included in the study, of whom 96% received oseltamivir. The median viral shedding duration of pH1N1 virus by viral culture and RT-PCR were 3 days and 4 days, respectively. Patients who started oseltamivir treatment >48 h after onset had a significantly longer median viral shedding duration by viral culture than those who started treatment within 48 h of onset (4 days vs. 2 days, P=0·014).

Enterovirus (EV) infection is a common disease of childhood and associated not uncommonly with aseptic meningitis. In the summer of 2008, laboratory surveillance has detected increased number of coxsackievirus B3 (CVB3) associated aseptic meningitis in Hong Kong, constituting 11.6% of those infected. This study analyzed the epidemiology, circulating pattern, and clinical presentations of CVB3 in Hong Kong over the last 10 years with reference to the circulation of EV in the locality. Enteroviruses (EV) were isolated from respiratory, cerebrospinal fluid (CSF), stool, and vesicular samples using human rhabdomyosarcoma, human laryngeal carcinoma (HEp2‐C), human lung fibroblast (MRC‐5), and African green monkey kidney (Vero) cell lines. Virus isolates were identified and characterized by indirect immunofluorescence (IF) using monoclonal antibodies (mAB), neutralization test as well as partial VP1 sequencing. Different from previous years, IF test result showed that majority of the isolates from 2008 were untypeable by the mAB suggesting antigenic change. Sequence analysis revealed that these isolates were clustered with recent isolate from Fuyang, China. Review of data from 1999 to 2008 showed increased activity of CVB3 in the years 2005 and 2008, and isolates in these 2 years displayed an amino acid change from threonine to alanine at codon 277 of the VP1 gene, which may be associated with central nervous system (CNS) disease. J. Med. Virol. 83:483–489, 2011. © 2011 Wiley‐Liss, Inc.

A critical component of laboratory surveillance for measles is the genetic characterization of circulating wild-type viruses. The World Health Organization (WHO) Measles and Rubella Laboratory Network (LabNet), provides for standardized testing in 183 countries and supports genetic characterization of currently circulating strains of measles viruses. The goal of this report is to describe the lessons learned from nearly 20 years of virologic surveillance for measles, to describe the global databases for measles sequences, and to provide regional updates about measles genotypes detected by recent surveillance activities. Virologic surveillance for measles is now well established in all of the WHO regions, and most countries have conducted at least some baseline surveillance. The WHO Global Genotype Database contains >7000 genotype reports, and the Measles Nucleotide Surveillance (MeaNS) contains >4000 entries. This sequence information has proven to be extremely useful for tracking global transmission patterns and for documenting the interruption of transmission in some countries. The future challenges will be to develop quality control programs for molecular methods and to continue to expand virologic surveillance activities in all regions.

Phylogenetic analysis of the haemagglutinin (HA) gene shows that the influenza A(H1N1)2009 viruses collected in Hong Kong clustered in two main branches characterised by the E391E and E391K amino acids. The main branch E391K evolved in two sub-branches with N142D and S202T mutations that first appeared in March and July 2010, respectively, with the latter becoming the predominant strain. These genetic variants that emerged display similar antigenic characteristics. Concurrent with genetic surveillance, laboratories should continue monitoring the circulating viruses antigenically.

The suspected measles case definition captures rubella cases. Therefore, measles surveillance will be improved in the course of the control and eventual elimination of rubella transmission. One aspect of rubella control, virologic surveillance, is reviewed here. A systematic nomenclature for rubella viruses (RVs) based on 13 genotypes has been established and is updated when warranted by increases in information about RVs. From 2005 through 2010, the genotypes of RVs most frequently reported were 1E, 1G, and 2B, and genotypes 1a, 1B, 1C, 1h, 1j, and 2C were less frequently reported. Virologic surveillance can support rubella control and elimination. Synopses of rubella virologic surveillance in various countries, regions, and globally are given, including characterization of viruses from imported cases in a country that has eliminated rubella and studies of endemic viruses circulating in countries without rubella control objectives. Current challenges are discussed.

The sensitivities of IgM detection, virus isolation, and RT‐PCR for the diagnosis of measles infection were assessed using samples collected from confirmed measles cases from 2006 to 2009. The optimal timing of specimen collection and the preferred specimen type(s) for these tests were also determined. IgM detection showed highest sensitivity when serum samples were collected ≥5 days after rash onset. Virus isolation gave the highest sensitivity when samples were collected ≤3 days after rash onset, with nasopharyngeal aspirate being the best specimen type, followed by urine and throat/combined throat and nasal swab. The highest RT‐PCR positive rate (81.0%) was obtained with serum samples collected ≤3 days after rash onset. RT‐PCR positive rate of 100% was observed with throat/combined throat and nasal swab, urine and nasopharyngeal aspirate collected ≤16, 4–16, and 4–7 days after rash onset, respectively. The genotype of each measles case was confirmed by sequencing. It was shown that the predominant measles viruses detected in Hong Kong during 2006–2009 belonged to genotype H1 (subtype a) and these strains were related closely to those detected in China. J. Med. Virol. 82:1773–1781, 2010. © 2010 Wiley‐Liss, Inc.

Background: To diagnose influenza A(H1N1)v virus infection, accurate and rapid detection are important. However, there is scanty data on the performance of various laboratory diagnostics.Objective: To compare the performance of rapid antigen test (RAT), viral culture and RT-PCR for the detection of influenza A(H1N1) v virus and to correlate their performance with the time after symptom onset and viral load.Study Design: From May 1, 2009 to June 25, 2009, respiratory samples were collected from 5740 individuals suspected of having influenza A(H1N1) v infection. The performance of viral culture and RT-PCR were investigated and correlated with the time after symptom onset. The sensitivity of RAT ESPLINE influenza A & B-N (Fujirebio Inc, Tokyo) was evaluated using a subset of 60 samples from patients diagnosed as having influenza A(H1N1) v infection.Results: Using respiratory samples from 587 patients diagnosed with influenza A(H1N1) v infection, comparison of laboratory diagnostics showed viral culture and RT-PCR gave comparable results with overall sensitivity of 93.9% and 98.1%, respectively. For RAT, when testing a subset of 60 samples collected <= 3 days following symptom onset, the sensitivity was 62%.Conclusions: Although viral shedding is prolonged and of higher titre in influenza A(H1N1) v infection, RAT showed a low sensitivity of 62% among patients presenting <= 3 days after symptom onset. Viral culture showed comparable performance with RT-PCR and with sensitivity better than that documented for seasonal influenza. (C) 2009 Elsevier B. V. All rights reserved.

To the editor: The preferential binding of influenza virus to sialic acid-α2,3-galactose (α2,3 receptor) or sialic acid-α2,6-galactose (α2,6 receptors) may determine its tropism as α2,3 and α2,6 receptors are dominant on lower and upper respiratory cells respectively (1). The recent glycan microarray analysis suggested that the haemagglutinin (HA) D222G substitution could cause a shift from α2,6 receptors to the mixed α2,3/α2,6 recep- tors specificity which might increase binding to α2,3 receptors (2) and contribute to severity of disease. This substitution in the HA gene has been reported in sam- ples of viruses obtained from cases with mild to severe illness from around 20 countries, areas and territories (3). A recent study from Norway has evaluated the clini- cal relevance of this substitution with severe and mild cases (4). In an attempt to understand the relevance of HA D222G substitution among pandemic influenza A (H1N1) caus- ing infections in Hong Kong, HA gene sequences from respiratory specimens and virus isolates of severe and non-severe cases were examined. Cases were indi- viduals who had laboratory confirmed pandemic H1N1 influenza virus by either viral culture or reverse tran- scription PCR (RT-PCR) of respiratory specimens (5). The severe cases were individuals classified by the attending physician as being in a serious or critical condition.

To study the serologic response to the new pandemic influenza A (H1N1) 2009 virus in Hong Kong, the level of immunity was measured before and after the occurrence of the outbreak, and the titer of antibody to the pandemic influenza A (H1N1) 2009 virus in serum samples of laboratory confirmed cases. The presence of pre-outbreak pandemic influenza A (H1N1) 2009 virus antibodies in 37% of individuals older than >65 years suggested previous exposures to heterologous virus strains may have elicited cross-reacting antibody. Following large outbreaks of pandemic influenza A 2009 virus that peaked in September 2009, there is a change in immunity level in various age groups consistent with the attack rates among population in Hong Kong. Among individuals with mild clinical presentation, the antibody response to pandemic influenza A (H1N1) 2009 virus was stronger in those individuals aged ≤ 24 years but took more time to reach a titer of 40 when compared with those aged >24 years; however, the antibody level declined slower among individuals aged ≤ 24 years. Regardless of age, the antibody response rose rapidly and reached much higher titer among individuals with severe clinical presentation. Further study is required to collect additional data on antibody persistence and determine how much protection is conferred by previous exposure to seasonal influenza A (H1N1) viruses.

Objectives: To determine virologically confirmed hospitalization rates associated with respiratory syncytial virus (RSV), adenovirus, and parainfluenza viruses in Hong Kong children. Methods: All patients <18 years of age living on Hong Kong Island (within Hong Kong SAR) admitted for a febrile acute respiratory infection to 1 of the 2 public hospitals on 1 fixed day of the week between October 2003 and September 2006 were prospectively recruited. Hong Kong Island has a known population denominator and these 2 hospitals managed 72.5% of all general pediatric admissions for this population. Nasopharyngeal aspirates were tested for RSV, adenovirus, and parainfluenzae types 1, 2, and 3 by direct antigen detection and culture. Results: The annual hospitalization rate for RSV in infants <6 months of age was 233.4 to 311.2 per 10,000. Parainfluenza type 3 had a hospitalization rate of 27.3 to 122.8 per 10,000 in the 1 to <2 years group. Adenovirus was associated with significant hospitalization in those 6 months to 1 year (25.9–77.8 per 10,000), and in those 2 to <5 years (38.1–59.2 per 10,000). The mean duration of hospitalization for RSV was 4.04 ± 2.61 days, significantly longer than the 3.12 ± 1.41 days for adenovirus and the 2.93 ± 2.54 days for parainfluenza infections (P = 0.013 and P = 0.038, respectively). Conclusion: We documented that the overall pediatric hospitalization burden of RSV was high and comparable to that of influenza. The burden for all the studied viruses was mainly in previously healthy children <5 years of age.

Background. Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII. 4. We studied the global patterns of GII. 4 epidemiology in relation to its genetic diversity.Methods. Data from NoV outbreaks with dates of onset from January 2001 through March 2007 were collected from 15 institutions on 5 continents. Partial genome sequences (n = 775) were collected, allowing phylogenetic comparison of data from different countries.Results. The 15 institutions reported 3098 GII. 4 outbreaks, 62% of all reported NoV outbreaks. Eight GII. 4 variants were identified. Four had a global distribution-the 1996, 2002, 2004, and 2006b variants. The 2003Asia and 2006a variants caused epidemics, but they were geographically limited. Finally, the 2001Japan and 2001Henry variants were found across the world but at low frequencies.Conclusions. NoV epidemics resulted from the global spread of GII. 4 strains that evolved under the influence of population immunity. Lineages show notable (and currently unexplained) differences in geographic prevalence. Establishing a global NoV network by which data on strains with the potential to cause pandemics can be rapidly exchanged may lead to improved prevention and intervention strategies.