Jarika Makkoch

High prevalence of human rhinovirus C infection in Thai children with acute lower respiratory tract disease

Summary Objective To determine the prevalence of human rhinoviruses (HRV) infections in children with lower respiratory disease in Thailand and monitor the association between species of HRV and clinical presentation in hospitalized paediatric patients. Method Two hundred and eighty-nine nasopharyngeal (NP) suction specimens were collected from hospitalized paediatric patients admitted to King Chulalongkorn Memorial Hospital, Thailand during February 2006–2007. Nucleic acids were extracted from each sample with subsequent amplification of VP4/2 by semi-nested RT-PCR for HRV detection. Other viral respiratory pathogens were also detected by PCR, RT-PCR or real time PCR. Nucleotide sequences of the VP4 region were used for genotyping and phylogenetic tree construction. Result In total, 87 of 289 specimens were positive for HRV indicating an annual prevalence of 30%. Wheezing or asthma exacerbation was the most common clinical presentation observed in infected patients. Sequence analysis and phylogenetic tree showed that 29 (33%) and 8 (9%) specimens belonged to HRV-A and HRV-B, respectively. Most of the HRV positive samples were HRV-C (58%). Moreover, species C was predominantly found in the paediatric population of Thailand in raining season (p <0.05). The frequency of co-infection of HRV-C with other respiratory viral pathogens was approximately 40%. Conclusion HRV-C represents the predominant species and is one of the etiologic agents in acute lower respiratory tract infection, causes of wheezing and asthma exacerbation in infants and young children in Thailand.

Global alert to avian influenza virus infection: From H5N1 to H7N9

Abstract Outbreak of a novel influenza virus is usually triggered by mutational change due to the process known as ‘antigenic shift’ or re-assortment process that allows animal-to-human or avian-to-human transmission. Birds are a natural reservoir for the influenza virus, and subtypes H5, H7, and H9 have all caused outbreaks of avian influenza in human populations. An especially notorious strain is the HPAI influenza virus H5N1, which has a mortality rate of approximately 60% and which has resulted in numerous hospitalizations, deaths, and significant economic loss. In March 2013, in Eastern China, there was an outbreak of the novel H7N9 influenza virus, which although less pathogenic in avian species, resulted in 131 confirmed cases and 36 deaths in humans over a two-month span. The rapid outbreak of this virus caused global concern but resulted in international cooperation to control the outbreak. Furthermore, cooperation led to valuable research-sharing including genome sequencing of the virus, the development of rapid and specific diagnosis, specimen sharing for future studies, and vaccine development. Although a H7N9 pandemic in the human population is possible due to its rapid transmissibility and extensive surveillance, the closure of the live-bird market will help mitigate the possibility of another H7N9 outbreak. In addition, further research into the source of the outbreak, pathogenicity of the virus, and the development of specific and sensitive detection assays will be essential for controlling and preparing for future H7N9 outbreaks.

Perinatal Pandemic (H1N1) 2009 Infection, Thailand

To the Editor: Infection with influenza A pandemic (H1N1) 2009 has been reported worldwide following initial identification of the virus in April 2009 (1). The groups at highest risk for infection or influenza-related complications include pregnant women and children (2). We report a case of pandemic (H1N1) 2009 infection in a newborn whose mother became ill with pandemic (H1N1) 2009 during the perinatal period. A newborn girl showed signs of respiratory distress. The relevant perinatal history was maternal illness with pandemic (H1N1) 2009 7 days before delivery. The infant, who had a birth weight of 1,560 grams, was delivered by emergency cesarean section after the mother experienced cardiopulmonary failure at the gestational age of 31 weeks. Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. Physical examination at birth showed a premature infant girl with mild subcostal retraction. Oxygen saturation at room air was 91%–99%. Other results of the physical examination were unremarkable. Initial management included routine care for premature infants. On the basis of the perinatal history, a throat swab specimen was collected for pandemic (H1N1) 2009 testing by PCR and oseltamivir, 6 mg, was administered every 12 hours (4 mg/kg/day). The specimen obtained from the throat swab was positive for pandemic (H1N1) 2009 by real-time PCR. The infant required oxygen supplementation. At day 2 of life, acute renal failure with an elevated plasma creatinine level of 1.1 mg/dL developed in the infant. Chest radiograph showed minimal pulmonary infiltrations. She was started on cefotaxime for suspected sepsis. Oseltamivir dosage was adjusted based on the glomerular filtration rate estimated by the formula of Schwartz et al. (3) of 10.5 mL/min/1.73m2 to 3 mg every 12 hours to complete 10 doses (2 mg/kg/day). Infection of the patient was confirmed by real-time reverse transcription–PCR of the throat swab specimen and by a 4× increase in antibodies against the virus by hemagglutination inhibition test (HI). Antibody titers against pandemic influenza (H1N1) 2009 by HI with turkey erythrocytes (4) on days 10, 24, and 42 of life were 10, 160, and 320, respectively (Figure). At day 4 of life, repeated PCR performed on a throat swab specimen was negative for pandemic (H1N1) 2009. Oxygen supplementation was gradually decreased and finally discontinued. Her room air oxygen saturation was 95%–98%. Her clinical symptoms gradually improved. Hemoculture was negative after 72 hours. The antimicrobial drugs were given over an 8-day course. Plasma creatinine decreased to 0.9 mg/dL and 0.6 mg/dL at days 6 and 7 of life, respectively. Her average urine output was 2–3 mL/kg/h. She was discharged at the age of 28 days with a body weight of 2,070 grams. Figure Antibody titer against influenza A pandemic (H1N1) 2009 by hemagglutination inhibition (HI) test on days 10, 24, and 42 of life of the patient. Pregnant women are one of the highest risk groups for influenza A infection and influenza-associated complications, including increased maternal and perinatal illness and death rates (5). Thus, pregnant women are given first priority to receive influenza vaccination. When influenza develops in mothers during the perinatal period, newborns can be infected transplacentally during maternal viremia or by respiratory droplets after birth. Transplacental infection of influenza A is rare (5), however, and there have been only a few case reports (6,7). Viremia is more frequent and more extensive in pregnant women due to depressed cell-mediated immune response during the pregnancy (8). Our patient was likely infected in utero because she was delivered by cesarean section and was never exposed to her mother, who required intensive cardiopulmonary support at the time of delivery. (The mother died from respiratory failure 7 days after the cesarean section.) Clinical manifestations in this patient, including respiratory distress and acute renal failure, were nonspecific. The high plasma creatinine level in the newborn sometimes reflects the mother’s plasma creatinine level (9). However, kidney function of the mother of the newborn was within normal limits at the time of cesarean section; plasma creatinine level of 0.7 mg/dL. An elevated plasma creatinine level is observed frequently in premature infants due to immaturity of the kidney tissue and will usually decrease within a few weeks. Oseltamivir was administered with dose adjustment based on the infant’s estimated glomerular filtration rate. The recommended dose of oseltamivir for glomerular filtration rate <30 mL/min/1.73 m2 is 2–3 mg/kg/day, based on preliminary data obtained by a National Institutes of Health–funded Collaborative Antiviral Study Group (10). The success of our management strategy for this case suggests early treatment with oseltamivir can prevent severe illness in newborns with perinatal influenza A pandemic (H1N1) 2009 infection.

Molecular Evolution of Human H1N1 and H3N2 Influenza A Virus in Thailand, 2006–2009

Background Annual seasonal influenza outbreaks are associated with high morbidity and mortality. Objective To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006–2009, using complete genome sequences. Methods Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006–2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches. Results Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains. Conclusion In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006–2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3′-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3′-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.

Epidemiology of seasonal influenza in Bangkok between 2009 and 2012.

INTRODUCTION This study investigated influenza activity in Bangkok, Thailand between June 2009 and July 2012. METHODOLOGY Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect influenza viruses among patients with influenza-like illnesses. RESULTS Of the 6417 patients tested, influenza virus infection was detected in 42% (n = 2697) of the specimens. Influenza A pH1N1 viruses comprised the predominant strain between 2009 and 2010, and seasonal influenza (H3) had a high prevalence in 2011. Laboratory data showed a prevalence and seasonal pattern of influenza viruses. In 2009, influenza activity peaked in July, the rainy season. In 2010, influenza activity happened in two phases, with the initial one at the beginning of the year and another peak between June and August 2010, which again corresponded to the rainy period. Influenza activity was low for several consecutive weeks at the beginning of 2011, and high H3N2 activity was recorded during the rainy season between July and September 2011. However, from the beginning of 2012 through July 2012, pH1N1, influenza H3N2, and influenza B viruses continuously circulated at a very low level. CONCLUSION The seasonal pattern of influenza activity in Thailand tended to peak during rainy season between July and September.

Whole genome characterization, phylogenetic and genome signature analysis of human pandemic H1N1 virus in Thailand, 2009-2012.

Background Three waves of human pandemic influenza occurred in Thailand in 2009–2012. The genome signature features and evolution of pH1N1 need to be characterized to elucidate the aspects responsible for the multiple waves of pandemic. Methodology/Findings Forty whole genome sequences and 584 partial sequences of pH1N1 circulating in Thailand, divided into 1st, 2nd and 3rd wave and post-pandemic were characterized and 77 genome signatures were analyzed. Phylogenetic trees of concatenated whole genome and HA gene sequences were constructed calculating substitution rate and dN/dS of each gene. Phylogenetic analysis showed a distinct pattern of pH1N1 circulation in Thailand, with the first two isolates from May, 2009 belonging to clade 5 while clades 5, 6 and 7 co-circulated during the first wave of pH1N1 pandemic in Thailand. Clade 8 predominated during the second wave and different proportions of the pH1N1 viruses circulating during the third wave and post pandemic period belonged to clades 8, 11.1 and 11.2. The mutation analysis of pH1N1 revealed many adaptive mutations which have become the signature of each clade and may be responsible for the multiple pandemic waves in Thailand, especially with regard to clades 11.1 and 11.2 as evidenced with V731I, G154D of PB1 gene, PA I330V, HA A214T S160G and S202T. The substitution rate of pH1N1 in Thailand ranged from 2.53×10−3±0.02 (M2 genes) to 5.27×10−3±0.03 per site per year (NA gene). Conclusions All results suggested that this virus is still adaptive, maybe to evade the hosts immune response and tends to remain in the human host although the dN/dS were under purifying selection in all 8 genes. Due to the gradual evolution of pH1N1 in Thailand, continuous monitoring is essential for evaluation and surveillance to be prepared for and able to control future influenza activities.

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus–host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.

Detection of oseltamivir sensitive/resistant strains of pandemic influenza A virus (H1N1) from patients admitted to hospitals in Thailand

Oseltamivir has been used widely for prophylaxis or treatment during outbreaks of the pandemic influenza virus (H1N1) in several countries. The aim of this study was to develop a real-time RT-PCR (reverse transcription-polymerase chain reaction) to be applied for detection and monitoring of the oseltamivir resistant strains of this virus during three outbreaks (May 2009 to October 2010) in Thailand. The real-time RT-PCR assay for detecting H275Y proved highly specific for the pandemic influenza virus (H1N1) as no cross-amplification was detected with other respiratory viruses or human total RNA. The assay was also highly sensitive with a detection limit as low as 100 copies/μL for both wild-type and resistant strains. The performance of the assay was evaluated in terms of amplification efficiency (100%). The results obtained by real-time RT-PCR were in complete agreement with direct nucleotide sequencing. However, real-time RT-PCR provided more detail on the relative quantities of ratios between resistant and sensitive strains in each individual. The results revealed that four of 1288 (0.31%) patients were infected with the oseltamivir resistant strain. The number of patients infected by resistant strains was higher during the third (0.61%) and second (0.24%) waves than during the first (0%) outbreak. In conclusion, the real-time RT-PCR assay for H275Y detection is advantageous because it is specific, sensitive, and provides quantitative data. And it would be useful for large-scale testing and monitoring of oseltamivir resistant strains of the pandemic influenza A virus (H1N1).

Original Research: Analysis of hepatic microRNA alterations in response to hepatitis B virus infection and pegylated interferon alpha-2a treatment.

Interferons play important roles in defense mechanisms against viral infection, and thus interferon therapy has been a standard treatment in chronic hepatitis B patients. Interferons signaling pathways promote interferon-inducible genes including microRNAs. In this research, we aimed to determine microRNAs expression profiles in vitro and in vivo. For in vitro model, Huh7 cells were transfected with or without hepatitis B virus plasmid for 6 h, and then treated with 100 ng of pegylated-interferon alpha-2a for 24 h. In vivo, we defined microRNAs expression profiles in pair-liver tissues of chronic hepatitis B patients in comparison between before and after treatment of pegylated-interferon alpha-2a for 48 weeks. Cellular small RNAs were extracted followed by library preparation. To determine microRNAs expression profiles, the next-generation sequencing was carried out on MiSeq platform (Illumina®). In vitro analysis demonstrated that microRNAs can be classified into up-regulated and down-regulated microRNAs in response to hepatitis B virus, interferon, and combination of hepatitis B virus and interferon. Moreover, in vivo analysis revealed microRNAs profiles in non-responders, responders without hepatitis B surface antigen clearance, and responders with hepatitis B surface antigen clearance. The target genes of the candidate microRNAs were determined in terms of roles in cellular pathways and immune response, which might be related to treatment in chronic hepatitis B patients. Results revealed that two down-regulated microRNAs including miR-185-5p and miR-186-5p were correlated in both in vitro and in vivo studies. These two microRNAs might be represented as specific hepatic microRNAs responding to hepatitis B virus and pegylated-interferon alpha-2a treatment, which may remarkable and attractive for further study involving in the association of their target genes and prediction of pegylated-interferon alpha-2a response. Interestingly, microRNAs expression patterns might be useful for understanding the response mechanism and serve as biomarkers for prediction of pegylated-interferon alpha-2a treatment response in patients with chronic hepatitis B.