Improving molecular tools for global surveillance of measles virus.

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® 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® 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.

Molecular characterization of the 2011 Hong Kong scarlet fever outbreak.

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.

Virulence potential of fusogenic orthoreoviruses.

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.

Status of global virologic surveillance for rubella viruses.

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.

Coxsackievirus B3-associated aseptic meningitis: an emerging infection in Hong Kong.

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.

Comparison of laboratory diagnostic methods for measles infection and identification of measles virus genotypes in Hong Kong.

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 >or=5 days after rash onset. Virus isolation gave the highest sensitivity when samples were collected

A HIV-1 heterosexual transmission chain in Guangzhou, China: a molecular epidemiological study.

BACKGROUND: We conducted molecular analyses to confirm four clustering HIV-1 infections (Patient A, B, C & D) in Guangzhou, China. These cases were identified by epidemiological investigation and suspected to acquire the infection through a common heterosexual transmission chain. METHODS: Env C2V3V4 region, gag p17/p24 junction and partial pol gene of HIV-1 genome from serum specimens of these infected cases were amplified by reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequenced. RESULTS: Phylogenetic analyses indicated that their viral nucleotide sequences were significantly clustered together (bootstrap value is 99%, 98% and 100% in env, gag and pol tree respectively). Evolutionary distance analysis indicated that their genetic diversities of env, gag and pol genes were significantly lower than non-clustered controls, as measured by unpaired t-test (env gene comparison: p < 0.005; gag gene comparison: p < 0.005; pol gene comparison: p < 0.005). CONCLUSION: Epidemiological results and molecular analyses consistently illustrated these four cases represented a transmission chain which dispersed in the locality through heterosexual contact involving commercial sex worker.

Oseltamivir- and amantadine-resistant influenza viruses A (H1N1).

Surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in Hong Kong in 2006. In 2008, while both A/Brisbane/59/2007-like and A/Hong Kong/2652/2006-like viruses (H1N1) were cocirculating, we detected amantadine and oseltamivir resistance among A/Hong Kong/2652/2006-like viruses (H1N1), caused by genetic reassortment or spontaneous mutation.

Molecular epidemiology of hepatitis E virus in Hong Kong.

Hepatitis E virus (HEV) is one of the major causes of acute and self-limiting hepatitis in human. In Hong Kong, the number of notifications increased from 26 to 62 from year 2001 to 2007. This study describes the molecular epidemiology of HEV in Hong Kong in order to determine the movement and distribution of HEV. HEV in 171 serum samples from HEV IgM positive cases from year 2001 to 2007 were amplified using RT-PCR and subjected to nucleotide sequencing. Phylogenetic analysis showed 162 of 171 HEV detected cases (94.7%) belonged to genotype IV and 8 (4.7%) to genotype I. Interestingly, a cluster of 10 cases in year 2007 that had the same sequence of HEV was identified. Epidemiological data however did not detect any relationship between these cases. Since zoonotic transmission is a well known route of HEV infection, close monitoring of the circulating HEV strains in human and food source animals may help to provide additional information on the transmission of HEV and possible source of infection in Hong Kong.

Clinical and molecular epidemiology of human parainfluenza virus 4 infections in hong kong: subtype 4B as common as subtype 4A.

In this 1-year study, 35 (1.2%) of 2,912 nasopharyngeal aspirates were positive for human parainfluenza virus 4 (HPIV4) by reverse transcription-PCR. Patients with HPIV4 infection were mainly young children and immunocompromised adults. In contrast to the reported predominance of HPIV4A infection, molecular subtyping revealed that 15 (44%) cases were caused by HPIV4B.

High prevalence of HCV in a cohort of injectors on methadone substitution treatment.

BACKGROUND: In Hong Kong, methadone treatment is widely accessible. Injecting drug users (IDU) have a relatively low risk behavioural profile and low HIV prevalence (0.3%). The corresponding Hepatitis C (HCV) level, however, is unclear. OBJECTIVES: To determine the HCV prevalence in IDU in Hong Kong and to identify any associated factors. STUDY DESIGN: A community-based HCV prevalence study of IDU was conducted in methadone clinics. Demographics and drug use pattern were collected through a questionnaire survey and blood samples were obtained for HCV serological tests. RESULTS: Data of 567 IDU were analyzed. Most were male (84%) and ethnic Chinese (98%). The median age was 49 years and median injection duration 17 years. Two-thirds (62%) admitted ever sharing injecting equipments. Most (76%) reported having injection drug use in the preceding 3 months, and 44% abused midazolam/triazolam in addition to heroin. Prevalence of HCV antibodies was 85% (95% confidence interval 82.5-88.3%). Injection duration, recent injection, ever sharing injecting equipments and concomitant use of other drugs were independent factors associated with HCV infection. CONCLUSIONS: HCV prevalence is high in IDU despite a low HIV prevalence and widely available substitution treatment, which has probably slowed but not prevented the HCV epidemic in IDU in Hong Kong.

Atypical norovirus epidemic in Hong Kong during summer of 2006 caused by a new genogroup II/4 variant.

An atypically high level of norovirus activity was noticed in Hong Kong beginning in early May 2006. A study was carried out to investigate whether this was caused by a new norovirus variant. Epidemiological data including monthly positivity rates and the numbers of outbreaks per month from January to July 2006 were analyzed and compared to those from 2002 to 2005. In a comparison with the epidemiological data from 2001 to 2005, an atypical peak of norovirus-associated gastroenteritis outbreak was observed beginning in May 2006, concurring with a striking increase in norovirus activity. Most of the outbreaks (>60%) were located in homes for the elderly. Phylogenetic analysis for both RdRp and 5' capsid regions showed that this epidemic was caused by a new genogroup II/4 variant. This variant was genetically distinct from the predominant variants of 2002 and 2004 but was closely related to one of the 95/96-subset variants which caused an epidemic in Hong Kong in 2001, suggesting that the 95/96 subset may be starting to recirculate.

One-step rapid reverse transcription-PCR assay for detecting and typing dengue viruses with GC tail and induced fluorescence resonance energy transfer techniques for melting temperature and color multiplexing.

BACKGROUND: Dengue fever is an arthropod-borne infection caused by dengue viruses (DVs; DEN-1 to DEN-4). Early diagnosis is critical to prevent severe disease progression and the spreading of DV because no vaccine or specific treatment is available; therefore, a rapid and specific diagnostic assay capable of detecting and typing all serotypes would be ideal. METHODS: We amplified RNA samples from all 4 DV serotypes and Japanese encephalitis virus with 4 serotype-specific forward primers and a universal species-specific reverse primer. DEN-1 and DEN-3 forward primers were labeled at their 5' ends with BODIPY 630/650 and Cy5.5, respectively. DEN-1 and DEN-3 amplicons were detected by their characteristic emission generated from induced fluorescence resonance energy transfer. The presence of DEN-2 and DEN-4 amplicons was indicated by SYBR Green I (SGI) signals at specific amplicon melting temperatures (T(m)s). RESULTS: Fluorescence signals with specific emission wavelengths were obtained from DEN-1 and DEN-3. SGI melting profiles showed a T(m) difference between DEN-2 and DEN-4 of 4.7 degrees C, which was sufficient for differentiating these 2 serotypes. The primers did not amplify the Japanese encephalitis virus. The detection limits of DEN-1 to DEN-4 were 1.64 x 10(-4), 1.05 x 10(-3), 8.15 x 10(-4), and 5.80 x 10(-3) plaque-forming units per reaction, respectively. The assay had a dynamic range of 10(3)-10(8) plaque-forming units/L and could be performed in 2 h. CONCLUSIONS: A single-tube, 1-step reverse transcription-PCR assay based on T(m) and color multiplexing was developed for detecting and typing all 4 DV serotypes.

Correlation of norovirus variants with epidemics of acute viral gastroenteritis in Hong Kong.

Norovirus (NV) (formerly called Norwalk-like virus) is the most common etiological agent of acute viral gastroenteritis outbreaks worldwide. Recent reports have shown that two new GII.4 variants caused epidemics in Europe. To investigate if it is also the case in Hong Kong, a molecular epidemiological study was undertaken between January 2002 and June 2005. During this period, there was a substantial increase in acute cases of gastroenteritis caused by NV. Phylogenetic analysis showed that GII.2 and GII.4 are the major circulating genotypes. Two new GII.4 variants (variants C and D) were identified in 2002 and 2004, which quickly became the predominant strains. They were almost identical to the variants causing epidemics in Europe recently. Since geographically distinct areas were involved within a short period of time, it is possible that GII.4 has a particular propensity for causing pandemics.

Respiratory infections during SARS outbreak, Hong Kong, 2003.

The effect of community hygienic measures during the outbreak of severe acute respiratory syndrome in Hong Kong was studied by comparing the proportion of positive specimens of various respiratory viruses in 2003 with those from 1998 to 2002. Community hygienic measures significantly reduced the incidence of various respiratory viral infections.

Cytokine profiles in human immunodeficiency virus-infected children treated with highly active antiretroviral therapy.

CONTEXT: There have been few longitudinal studies of cytokine production in neonatally acquired HIV-1 infection and none in Asian or Chinese children. OBJECTIVE: To determine whether monitoring cytokine production could contribute to the better management of pediatric patients with HIV-1 infection. SETTING: Clinical Immunology Laboratory and Pediatrics Department, University Hospital, Hong Kong. PATIENTS: Ten Asian and 2 Eurasian children infected with HIV-1 by mother-to-child transmission were followed for up to 5 years while on treatment with highly active antiretroviral therapy (HAART). MAIN OUTCOME MEASURES: Numbers of unstimulated and mitogen-activated cytokine-secreting cells (IFN-gamma, interleukin [IL]-2, IL-4, IL-6, IL-10, IL-12, and TNF-alpha) were measured by ELISPOT assay at frequent intervals, and correlations were sought with CD4+ and CD8+ cell counts and viral loads. RESULTS: Mitogen-stimulated IL-2-secreting cells were directly associated with recovery of CD4+ cells. Correlations with viral load were found for Con A-induced IFN-gamma, Con A-induced IL-4, and unstimulated IL-10, suggesting that these cytokines were either suppressed by high virus levels or that higher cytokine levels suppressed virus. IFN-gamma, IL-2-, IL-4-, and IL-12-secreting cells induced by PHA, Con A, and/or SAC tended to increase for the first 3-4 years of treatment but declined thereafter. CONCLUSIONS: Alterations in cytokine profiles were not associated with adverse clinical events and there was little evidence to indicate that monitoring cytokine enzyme-linked immunospots (ELISPOTs) could contribute to pediatric patient management.

Survival of severe acute respiratory syndrome coronavirus.

BACKGROUND: The primary modes of transmission of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) appear to be direct mucus membrane contact with infectious droplets and through exposure to formites. Knowledge of the survival characteristics of the virus is essential for formulating appropriate infection-control measures. METHODS: Survival of SARS-CoV strain GVU6109 was studied in stool and respiratory specimens. Survival of the virus on different environmental surfaces, including a laboratory request form, an impervious disposable gown, and a cotton nondisposable gown, was investigated. The virucidal effects of sodium hypochlorite, house detergent, and a peroxygen compound (Virkon S; Antec International) on the virus were also studied. RESULTS: SARS-CoV GVU6109 can survive for 4 days in diarrheal stool samples with an alkaline pH, and it can remain infectious in respiratory specimens for >7 days at room temperature. Even at a relatively high concentration (10(4) tissue culture infective doses/mL), the virus could not be recovered after drying of a paper request form, and its infectivity was shown to last longer on the disposable gown than on the cotton gown. All disinfectants tested were shown to be able to reduce the virus load by >3 log within 5 min. CONCLUSIONS: Fecal and respiratory samples can remain infectious for a long period of time at room temperature. The risk of infection via contact with droplet-contaminated paper is small. Absorbent material, such as cotton, is preferred to nonabsorptive material for personal protective clothing for routine patient care where risk of large spillage is unlikely. The virus is easily inactivated by commonly used disinfectants.

Human parainfluenza virus 4 outbreak and the role of diagnostic tests.

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.