Sudarat Damrongwatanapokin

Probable tiger-to-tiger transmission of avian influenza H5N1.

During the second outbreak of avian influenza H5N1 in Thailand, probable horizontal transmission among tigers was demonstrated in the tiger zoo. Sequencing and phylogenetic analysis of those viruses showed no differences from the first isolate obtained in January 2004. This finding has implications for influenza virus epidemiology and pathogenicity in mammals.

H5N1 influenza A virus and infected human plasma.

To the Editor: Since January 2004, a total of 22 persons have been confirmed infected with avian influenza A virus (H5N1) in Thailand; 14 of these patients died. Three waves of outbreaks occurred during the past 2 years. The last patient of the third wave was a 5-year-old boy whose symptoms developed on November 28, 2005; he was hospitalized on December 5 and died 2 days later. The child resided in the Ongkharak District, Nakhon Nayok Province, ≈70 km northeast of Bangkok. Villagers informed the Department of Livestock after the patients illness was diagnosed. Five dead chickens had been reported in this area from November 28 to December 1, 2005. Samples from these chickens could not be obtained, thus, no H5N1 testing was performed. The boy had fever, headache, and productive cough for 7 days before he was admitted to the Her Royal Highness Princess Maha Chakri Sirindhorn Medical Center. Clinical examination and chest radiograph showed evidence of lobar pneumonia. He was treated with antimicrobial drugs (midecamycin and penicillin G) and supportive care, including oxygen therapy. On December 7, the patients condition worsened, and severe pneumonia with adult respiratory distress syndrome developed. Laboratory tests showed leukopenia (2,300 cells/mm3), acidosis, and low blood oxygen saturation by cutaneous pulse oximetry (81.6%). Oseltamivir was administered after his parents informed hospital staff about the boys contact with the dead chicken. However, the boy died the same day; no autopsy was performed. On December 9, the cause of death was declared by the Ministry of Public Health to be H5N1 influenza virus. A blood sample was collected from the patient on December 7; anticoagulation was accomplished with ethylenediaminetetraacetic acid (EDTA) for repeated biochemistry analysis and complete blood count. The plasma from the EDTA blood sample was separated 2 days later and stored at –20°C for 12 days. The sample was subsequently given to the Center of Excellence in Viral Hepatitis, Faculty of Medicine, Chulalongkorn University, for molecular diagnosis and then stored at –70°C, where specific precautions implemented for handling highly infectious disease specimens such as H5N1 influenza virus were observed. Plasma was examined by multiplex reverse transcription–polymerase chain reaction (RT-PCR) (1) and multiplex real-time RT-PCR (2), both of which showed positive results for H5N1 virus. The virus titer obtained from the plasma was 3.08 × 103 copies/mL. The plasma specimen was processed for virus isolation by embryonated egg injection, according to the standard protocol described by Harmon (3). Briefly, 100 μL 1:2 diluted plasma was injected into the allantoic cavity of a 9-day-old embryonated egg and incubated at 37°C. The infected embryo died within 48 hours, and the allantoic fluid was shown to contain 2,048 hemagglutinin (HA) units; also, subtype H5N1 was confirmed (1,2). Whole genome sequencing was performed and submitted to the GenBank database under the strain A/Thailand/NK165/05 accession no. DQ 372591-8. The phylogenetic trees of the HA and neuraminidase (NA) genes were constructed by using MEGA 3 (4) for comparison with H5N1 viruses isolated from humans, tigers, and chickens from previous outbreaks in 2004 and 2005 (Figure). The sequence analyses of the viruses showed that the HA cleavage site contained SPQREKRRKKR, which differed from the 2004 H5N1 virus by an arginine-to-lysine substitution at position 341. That finding had also been observed in wild bird species during earlier outbreaks in Thailand in 2004 (5). Similar to the 2004–2005 H5N1 isolates from Thailand, a 20–amino acid deletion at the NA stalk region was observed. Moreover, the amino acid residues (E119, H274, R292, and N294) of the NA active site were conserved, which suggests that the virus was sensitive to oseltamivir. In addition, a single amino acid substitution from glutamic acid to lysine at position 627 of PB2 showed increased virus replication efficiency in mammals (6). Figure Phylogenetic analysis of the hemagglutinin and neuraminidase genes of H5N1 from study patient compared with sequences from previous outbreaks (2004–2005). Observing live influenza virus in human serum or plasma is unusual. However, in 1963, low quantities of virus were isolated from blood of a patient on day 4 of illness (7), and in 1970, the virus was cultivated from blood specimens from 2 patients (8). Recently, a fatal case of avian influenza A (H5N1) in a Vietnamese child was reported. The diagnosis was determined by isolating the virus from cerebrospinal fluid, fecal, throat, and serum specimens (9); viral RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness (10). In this case, the H5N1 virus could be isolated from plasma on day 10 after symptoms developed. This case showed the virus in the patients blood, which raises concern about transmission among humans. Because probable H5N1 avian influenza transmission among humans has been reported (11), this case should be a reminder of the necessity to carefully handle and transport serum or plasma samples suspected to be infected with H5N1 avian influenza. Because viable virus has been detected in blood samples, handling, transportation, and testing of blood samples should be performed in a biosafety (category III) containment laboratory to prevent the spread of the virus to healthcare and laboratory workers. We express our thanks to the Thailand Research Fund (Senior Research Scholar), Royal Golden Jubilee PhD Program and Center of Excellence in Viral Hepatitis Research, and Prasert Auewarakul for their generous support of our study.

Genetic diversity of swine influenza viruses isolated from pigs during 2000 to 2005 in Thailand

Background  Recent studies have revealed the existence of genetic diversity in swine influenza viruses (SIVs) in the world. In Thailand, there has been a little information on the molecular characteristics of the SIVs since the first isolation of viruses of H1N1 and H3N2 subtypes in the late 1970s. Our previous study demonstrated that Thai H1N1 SIVs possessed the classical swine H1 and avian‐like swine N1 genes (Takemae et al., Proceedings of the Options for the Control of Influenza VI.2007;350–353).

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.

Development of a TaqMan real-time RT-PCR assay for the detection of rabies virus.

Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. The mouse brain inoculation test is used to confirm FAT but requires weeks until the result is known. TaqMan real-time PCR has been described for rabies viral RNA detection; however, this is burdened by primer and probe binding site mismatches. The purpose of this study was to develop a TaqMan real-time RT-PCR assay as an adjunct to FAT, based on national data of 239 rabies nucleoprotein sequences from rabies-infected brain specimens collected between 1998 and 2003. Two showed as many as 3 mismatches. However, mismatches on primer and/or probe binding sites did not affect amplification or detection. One hundred and forty-three brain samples submitted for rabies diagnosis from all over the country between 2005 and 2007 were also tested. Results were concordant with FAT. Thirteen rabies proven samples from Myanmar, Cambodia, Indonesia and India; 3 of which had up to 7 mismatches at primer/probe binding sites, could be detectable. Challenge Virus Standard, a fixed virus strain with 4 mismatches at probe binding site, could not be detected but remained amplified. This assay could be used as an adjunct to FAT and may serve as a rabies surveillance tool.

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

Abstract A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/μl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.

Qualitative detection of avian influenza A (H5N1) viruses: A comparative evaluation of four real-time nucleic acid amplification methods

The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

Genetic grouping of classical swine fever virus by restriction fragment length polymorphism of the E2 gene.

A method for genetic grouping of classical swine fever viruses (CSFV) was developed based on the restriction fragment length polymorphism (RFLP) revealed by AvaII, BanII and PvuII digestion of RT-PCR amplified segments of the E2 gene. From inspection of the genetic sequences of Thai isolates and reference strains, the RFLP method was designed to be capable of differentiating all known genogroups and subgenogroups suggested by phylogenetic analysis of the CSFV E2 gene. The method was applied to 60 CSFV samples which included three genogroups and seven subgenogroups. Unlike previously described RFLP methods, the agarose gel patterns obtained from these samples were completely in agreement with the predicted RFLP patterns and enabled accurate genetic grouping of CSFV at the subgenogroup level. The simplicity of this method allows rapid CSFV genogrouping at diagnostic laboratories without sequencing facilities and provides a useful method for diagnosis as well as epidemiological investigation and control of classical swine fever outbreaks.

Genetic variations of nucleoprotein gene of influenza A viruses isolated from swine in Thailand

BackgroundInfluenza A virus causes severe disease in both humans and animals and thus, has a considerably impact on economy and public health. In this study, the genetic variations of the nucleoprotein (NP) gene of influenza viruses recovered from swine in Thailand were determined.ResultsTwelve influenza A virus specimens were isolated from Thai swine. All samples were subjected to nucleotide sequencing of the complete NP gene. Phylogenetic analysis was conducted by comparing the NP gene of swine influenza viruses with that of seasonal and pandemic human viruses and highly pathogenic avian viruses from Thailand (n = 77). Phylogenetic analysis showed that the NP gene from different host species clustered in distinct host specific lineages. The NP gene of swine influenza viruses clustered in either Eurasian swine or Classical swine lineages. Genetic analysis of the NP gene suggested that swine influenza viruses circulating in Thailand display 4 amino acids unique to Eurasian and Classical swine lineages. In addition, the result showed 1 and 5 amino acids unique to avian and human lineages, respectively. Furthermore, nucleotide substitution rates showed that the NP gene is highly conserved especially in avian influenza viruses.ConclusionThe NP gene sequence of influenza A in Thailand is highly conserved within host-specific lineages and shows amino acids potentially unique to distinct NP lineages. This information can be used to investigate potential interspecies transmission of influenza A viruses. In addition, the genetic variations of the NP gene will be useful for monitoring the viruses and preparing effective prevention and control strategies for potentially pandemic influenza outbreaks.

Assay to Detect Oseltamivir Resistance

To the Editor: Oseltamivir is a neuraminidase inhibitor approved for treatment and prevention of influenza virus infection. Oseltamivir resistance caused by a single amino acid substitution from histidine (H) to tyrosine (Y) at position 274 of the neuraminidase active site has been reported in persons infected both experimentally and naturally with influenza A virus subtype H5N1 (1,2). Evidence suggests that using lower doses of oseltamivir or shorter-than-recommended treatment periods can contribute to emergence of viral resistance (1,3) Currently, oseltamivir is being used in several countries that may be affected by epidemics of H5N1. Therefore, monitoring for oseltamivir-resistant strains of H5N1 during oseltamivir administration is essential for outbreak management and prevention. Although real-time PCR or pyrosequencing is more rapid than high-throughput assays for mutation detection (4,5), the conventional PCR technique can be applied to detect drug-resistant mutation (6) in areas lacking real-time PCR or pyrosequencing capabilities. Therefore, to discriminate between oseltamivir-sensitive and oseltamivir-resistant strains, we developed a simple method, based on PCR, which takes advantage of the H274Y substitution. The forward primer was designed from the conserved region common to both wild-type and mutant strains; the reverse primers were designed specifically for wild-type and mutant strains, respectively, derived from the 3´ terminal base of each primer. The primers consisted of a forward primer N1f (nt 517-534: 5´-GGGGCTGTGGCTGTATTG-3´) and reverse primer H274r (nt 759-784: 5´-GGATAACAGGAGCAYTCCTCATAGTG -3´) for wild-type strain detection or Y274r (nt 759-784: 5´-GGATAACAGGAGCAYTCCTCATAGTA-3´) for mutant strain detection. Both strains yielded products of ≈267 bp; hence, the assay consisted of 2 separate reactions for detecting wild-type and mutant strains, respectively. For each reaction, 1.0 μL cDNA was combined with a reaction mixture that contained 10 μL 2.5× MasterMix (Eppendorf, Hamburg, Germany), forward and reverse primers at a final concentration of 0.15 μM, and nuclease-free water to a final volume of 20 μL. Thermocycling conditions comprised initial denaturation at 94°C for 3 min and 35 cycles of amplification including denaturation (94°C, 30 s), annealing (65°C, 50 s), extension (72°C, 45 s), and final extension (72°C, 7 min). Subsequently, 10 μL of the amplified products was analyzed by using 2% agarose gel electrophoresis. To optimize the assay, we performed PCR-based H274Y mutagenesis of the N1 fragment of the H5N1 virus (primers on request). The resulting mutagenic and wild-type products were cloned into the pGEM-T Easy Vectors (Promega, Madison, WI, USA), confirmed by direct sequencing, and then used as positive controls. Preliminary results showed that the wild-type primer was specific for the oseltamivir-sensitive strain, whereas the mutant primer can be used to detect the oseltamivir-resistant strain exclusively because no significant cross-amplification had been observed. To establish sensitivity, serial 10-fold dilutions of the standard N1 plasmids (wild-type and mutant) ranging from 109 to 101 copies/μL were used as a template. The threshold concentrations for detection of wild-types and mutants were 103 copies/μL. To provide semiquantitative data to detect subpopulations of the resistant variants, the 2 control plasmids were mixed at wild-type:variant ratios of 108:102, 107:103, 106:104, 105:105, 104:106, 103:107, and 102:108. The result showed that the density of the expected bands depended on the amount of DNA templates (Figure, B). However, the mixing experiments indicated that the predominant mixtures of wild-type:resistant variant were 80:20, which is the lowest ratio of resistant variants that the assay can reliably detect (data not shown). To assess specificity, human DNA and viral cDNA extracted from other subtypes of influenza A virus (N2–N9) were subjected to this assay. No cross-reaction occurred with human DNA or other subtypes of influenza A virus. Figure A) Representative result from conventional PCR that used H274r primer for oseltamivir-sensitive and Y274r primer for oseltamivir-resistant detection in samples isolated from human plasma (P), tiger (T), and Vietnamese patient (V). Plasmids containing ... We further validated the assay by testing 3 specimens from hosts treated with oseltamivir and 17 specimens from untreated hosts; infection with H5N1 was detected by using multiplex real-time PCR (7). The specimens from oseltamivir recipients were isolated from a Vietnamese patient on the third day after he received a prophylactic dose (1) and from 2 tigers (CU-T7 and KU-11) (8). The specimens from untreated hosts were isolated from the plasma of an H5N1-infected human (9) and from virus-containing allantoic fluid isolated from infected chickens, ducks, and cats (n = 16) during a 2005 outbreak in Thailand. The specimen isolated from the Vietnamese patient yielded detectable bands after amplification by wild-type and mutant primer sets, which indicates that this specimen contains mixed populations of wild-type and resistant strains (Figure, A). The result was confirmed by cloning the amplicon of the Vietnamese strain into the pGEM-T EASY vector (Promega). Ten colonies were randomly picked and sequenced; 9 clones were mutant, and 1 was wild type. The other specimens produced a visible positive band only on amplification using the wild-type primer set, which indicates that samples containing these strains were oseltamivir sensitive. The assay described here provides an accurate, cost-effective, and highly efficient approach to detecting oseltamivir-sensitive and oseltamivir-resistant H5N1 strains; it is based on conventional PCR and takes advantage of the H274Y substitution within the neuraminidase gene. This simple technique can be applied in all laboratories that lack more advanced and expensive instruments; thus, it provides a valuable tool for managing and preventing influenza A H5N1 outbreaks. Concerning the spread of mutant viruses, however, the fitness of the viruses needs further investigation.