Naoko Fuji

Enterovirus 68 among Children with Severe Acute Respiratory Infection, the Philippines

TOC summary: Enterovirus 68 was found in 21 children with severe pneumonia.

Acute respiratory infections due to enterovirus 68 in Yamagata, Japan between 2005 and 2010

To clarify the epidemiology of enterovirus 68 (EV68), which is one of the most rarely identified enteroviruses, virus isolation and molecular screening using RT‐PCR was performed on 6307 respiratory specimens collected at pediatric clinics in Yamagata, Japan between 2005 and 2010. In the years 2005–2009, 10, 1, 2, 0, and 2 (40) EV68‐positive cases, respectively, were identified by RT‐PCR. In 2010, 40 cases were identified altogether: 2 by isolation only, 26 by RT‐PCR only, and 12 by both isolation and RT‐PCR. Phylogenetic analysis indicated that plural genetically distinct clusters co‐circulated. These results suggest that that difficulty in EV68 isolation leads to an underestimation of the prevalence of EV68 infections.

Detection of human rhinovirus C viral genome in blood among children with severe respiratory infections in the Philippines.

Human rhinovirus (HRV) C was recently identified as the third species of HRV using a molecular technique. Infections caused by previously identified HRVs (A and B) are thought to be limited to the respiratory tract; however, pathogenesis of HRVC is still largely unknown. A total of 816 nasopharyngeal swabs from hospitalized children with severe respiratory infections in the Philippines (May 2008–May 2009) were tested for HRV by reverse transcription polymerase chain reaction (RT-PCR), and 243 samples (29.8%) were positive for HRV. Among these patients, serum samples were also tested to determine whether specific HRV species were associated with viremia. Only 30 serum samples (12.3%) were positive for HRV. However, the HRV positive rates were different among HRV species, 3% (4/135) for HRVA, 0% (0/25) for HRVB, and 31% (26/83) for HRVC, and were the highest on 2 days after the onset of symptoms. These results suggest that HRVC may have a different pathogenicity and can more commonly cause viremia than HRVA and HRVB. Serum positive rates for HRV are affected by age, i.e., higher positive rates for those aged 1 year or more. HRVC that were detected from serum exhibited the same level of sequence diversity as those positive only for nasopharyngeal samples in phylogenetic analysis. However, all HRVA which were detected from serum were clustered in a monophyletic clade based on their 5′ non-coding region (NCR) sequences, which is closely related with a certain HRVC genotype (A2) in 5′-NCR. This finding suggests that the 5′NCR region may be associated with viremia.

Respiratory viruses from hospitalized children with severe pneumonia in the Philippines

BackgroundPneumonia remains a leading cause of child death in developing countries. The viruses in severe pneumonia remain poorly defined.MethodsThe study was conducted at the Eastern Visayas Regional Medical Center in Tacloban City, Philippines from May 2008 to May 2009. Patients aged 8 days to 13 years old who were admitted to the Department of Pediatrics with severe pneumonia were enrolled for the study. Upon admission, polymerase chain reaction was performed using nasopharyngeal swabs and blood cultures to detect respiratory viruses and bacteria, respectively.ResultAmong the 819 patients enrolled, at least one virus was detected in 501 cases (61.2%). In addition, 423 cases were positive for a single virus while bacteria were detected in the blood culture sample of 31 cases. The most commonly detected viruses were human rhinoviruses (n = 189), including types A (n = 103), B (n = 17), and C (n = 69), and respiratory syncytial virus (RSV) (n = 165). Novel viruses such as human metapneumovirus, human coronavirus NL63, human bocavirus, and human polyomaviruses WU and KI were also detected. There were 70 deaths, and one or more viruses were detected in 35 (50%) of these cases. Positivity only for influenza A virus (OR = 4.3, 95% CI = 1.3-14.6) was significantly associated with fatal outcome. From the blood culture, Burkholderia cepacia group (n = 9), Streptococcus pneumoniae (n = 4), Staphylococcus aureus (n = 4), Haemophilus influenzae (n = 1), and Salmonella C1 (n = 1) were also isolated.ConclusionViruses were commonly detected in children with severe pneumonia in the Philippines. Hence, viral etiologies should be considered while developing better effective strategies to reduce child pneumonia-related deaths in developing countries.

Molecular evolution of enterovirus 68 detected in the Philippines.

Background Detection of Enterovirus 68 (EV68) has recently been increased. However, underlying evolutionary mechanism of this increasing trend is not fully understood. Methods Nasopharyngeal swabs were collected from 5,240 patients with acute respiratory infections in the Philippines from June 2009 to December 2011. EV68 was detected by polymerase chain reaction (PCR) targeting for 5′ untranslated region (5′UTR), viral protein 1 (VP1), and VP4/VP2. Phylogenetic trees were generated using the obtained sequences. Results Of the 5,240 tested samples, 12 EV68 positive cases were detected between August and December in 2011 (detection rate, 0.23%). The detection rate was higher among inpatients than outpatients (p<0.0001). Among VP1 sequences detected from 7 patients in 2011, 5 in lineage 2 were diverged from those detected in the Philippines in 2008, however, 2 in lineage 3 were not diverged from strains detected in the Philippines in 2008 but closely associated with strains detected in the United States. Combined with our previous report, EV68 occurrences were observed twice in the Philippines within the last four years. Conclusions EV68 detections might be occurring in cyclic patterns, and viruses might have been maintained in the community while some strains might have been newly introduced.

Genetic characterization of human respiratory syncytial virus detected in hospitalized children in the Philippines from 2008 to 2012

BACKGROUND Human respiratory syncytial virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and young children. However, molecular characteristic of HRSV is still unknown in the Philippines. OBJECTIVE To describe the molecular epidemiology of circulating HRSV detected in the Philippines. STUDY DESIGN From May 2008 to April 2012, nasopharyngeal swabs were collected from infants and children aged between 7 days and 14 years who were hospitalized with severe pneumonia. HRSV was detected by nested PCR targeting M2 gene, and C-terminus of the G gene was sequenced for phylogenetic analysis. RESULT Out of total 2150 samples, 19.3% (n = 415) were positive for HRSV, and 65.0% of them (n = 270) were identified as HRSV-A and 35.0% (n = 145) as HRSV-B. There were two major HRSV outbreaks: between June 2008 and February 2009, and between June and March 2012. Majority of HRSV strains detected during the former outbreak were HRSV-A (97.5%, 203/208) whereas during the later outbreak, both HRSV-A (54/158, 34.2%) and HRSV-B (104/158, 65.8%) were detected. All HRSV-A strains were classified as genotype NA1 and all HRSV-B as genotype BA, which had 60-nucleotide duplication in secondary hypervariable region of the G gene. Among HRSV-B positive samples, there were 2 distinct clusters with unique amino acid changes and low homology in compared to other strains in BA, suggesting emergence of new variant of HRSV-B. CONCLUSION The study provides an overview of the genetic variation in circulating HRSV viruses in the Philippines along with identification of possibly a novel variant of HRSV-B.

Detection of Non-Polio Enteroviruses From 17 Years of Virological Surveillance of Acute Flaccid Paralysis in the Philippines

Acute flaccid paralysis (AFP) surveillance has been conducted as part of the World Health Organization (WHO) strategy on poliomyelitis eradication. Aside from poliovirus, which is the target pathogen, isolation, and identification of non‐polio enteroviruses (NPEVs) is also done by neutralization test using pools of antisera which can only identify limited number of NPEVs. In the Philippines, despite the significant number of isolated NPEVs, no information is available with regard to its occurrence, diversity, and pattern of circulation. In this study, a total of 790 NPEVs isolated from stool samples submitted to the National Reference Laboratory from 1992 to 2008 were analyzed; neutralization test was able to type 55% (442) of the isolates. Of the remaining 356 isolates, which were untyped by using neutralization test, 348 isolates were analyzed further by RT‐PCR targeting the VP1 gene. A total of 47 serotypes of NPEV strains were identified using neutralization test and molecular typing, including 28 serotypes of human enterovirus B (HEV‐B), 12 serotypes of HEV‐A, and 7 of HEV‐C. The HEV‐B group (625/790; 79%) constituted the largest proportion of isolates, followed by HEV‐C (108/790; 13.7%), HEV‐A (57/790; 7.2%), and no HEV‐D. Coxsackievirus (CV) B, echovirus (E)6, E11, and E13 were the most frequent isolates. E6, E11, E13, E14, E25, E30, E33, CVA20, and CVA24 were considered as endemic strains, some NPEVs recurred and few serotypes existed only for 1–3 years during the study period. Despite some limitations in this study, plural NPEVs with multiple patterns of circulation in the Philippines for 17 years were identified. J. Med. Virol. 84:624–631, 2012.

Occurrence of Mixed Populations of Influenza A Viruses That Can Be Maintained through Transmission in a Single Host and Potential for Reassortment

ABSTRACT Reassortment, which is the rearrangement of viral gene segments in a host cell infected with two different viruses, is an important mechanism for the evolution of influenza viruses. Mixed infections with multiple virus types could lead to reassortment. To better understand the occurrence of quasispecies in a single host, we investigated mixed infections in individual isolates of seasonal influenza A viruses using amantadine sensitivity as a marker. We cultured viruses with amantadine and performed sequencing, restriction fragment length polymorphism analysis, cloning, and quantitative PCR to detect mixed populations. Culturing with amantadine showed evidence of a high number of mixed populations, while the other assays could hardly detect mixed populations. The existence of quasispecies in each isolate was common. However, the proportion of these, which can be less than 1%, is too low to be detected by conventional methods. Such mixed populations in which reassortment occurs may have a significant role in the evolution of viruses.

Reassortment between Amantadine-Resistant and -Sensitive H1N1 Influenza A Viruses Generated an Amantadine-Sensitive Virus during the 2007–2008 Season

The frequency of the amantadine-resistant H1N1 influenza A virus has been increasing since the 2005-2006 season. It is unclear whether reassortment was involved in this trend. Here, we show that cocirculation of amantadine-resistant and -sensitive strains led to the genesis of amantadine-sensitive reassortant virus during the 2007-2008 season. Thereafter, the reassortant virus predominated. This contrasts with the trend for the H3N2 virus, in which the amantadine-resistant reassortant virus became predominant. The results suggest that it is necessary to monitor genome dynamics to understand the evolution and mechanism of the emergence and spread of antiviral resistance among influenza A viruses.

Interruption of the circulation of an indigenous measles genotype and the introduction of other genotypes after a mass vaccination campaign in the Philippines

Molecular analysis of measles viruses in the Philippines was conducted from 2000 to 2008. No confirmed measles cases were detected in the surveillance in 2005 after the mass vaccination campaign in 2004. However, a re‐emergence of measles cases occurred in 2007, which was caused by other genotypes and the previous circulating genotype had disappeared. J. Med. Virol. 83:1424–1427, 2011.