Faizatul Lela Jafar

Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis

BackgroundBrucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.ResultsSingle sequencing runs of each genome resulted in draft genome sequences of MY1483/09 and Phil1136/12, which covered 99.85% and 99.92% of the complete genome sequences, respectively. The B. melitensis genome sequences, and two B. abortus strains used as the outgroup strains, yielded a total of 13,728 SNP sites. Phylogenetic analysis using whole-genome SNPs and geographical distribution of the isolates revealed spatial clustering of the B. melitensis isolates into five genotypes, I, II, III, IV and V. The Mediterranean strains, identified as genotype I, occupied the basal node of the phylogenetic tree, suggesting that B. melitensis may have originated from the Mediterranean regions. All of the Asian B. melitensis strains clustered into genotype II with the SEA strains, including the two isolates sequenced in this study, forming a distinct clade denoted here as genotype IId. Genotypes III, IV and V of B. melitensis demonstrated a restricted geographical distribution, with genotype III representing the African lineage, genotype IV representing the European lineage and genotype V representing the American lineage.ConclusionWe showed that SNPs retrieved from the B. melitensis draft full genomes were sufficient to resolve the interspecies relationships between B. melitensis strains and to discriminate between the vaccine and endemic strains. Phylogeographic reconstruction of the history of B. melitensis global spread at a finer scale by using whole-genome SNP analyses supported the origin of all B. melitensis strains from the Mediterranean region. The possible global distribution of B. melitensis following the ancient trade routes was also consistent with whole-genome SNP phylogeny. The whole genome SNP phylogenetics analysis, hence is a powerful tool for intraspecies discrimination of closely related species.

Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5‐year study period, with special emphasis on isolates that gave unsatisfactory identification.

Evolution of Influenza B Virus in Kuala Lumpur, Malaysia, between 1995 and 2008.

ABSTRACT Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.

Diverse human rhinoviruses A and C from children with respiratory infections in Kuala Lumpur, Malaysia

We read with interest the recent paper by Xiang and colleagues describing the molecular epidemiology of human rhinovirus in China. Human rhinoviruses (HRVs) were first discovered more than 50 years ago. HRVs are a common cause of upper respiratory tract infections, but are also associated with asthma exacerbations and more severe respiratory infections. HRVs can be divided into three groups, A, B and C, with the latter, HRV-C, only reported in 2007. There are more than 100 types of HRV-A and B, while discovery of new HRV-C still continues. The high genetic and antigenic heterogeneity amongst the HRV results in lack of protective immunity and repeated infections. With the wider availability of molecular diagnostic methods, there is great interest in determining the global range of HRV variants, for subsequent development of antivirals and vaccines. As data from tropical Asian countries is relatively lacking, we report here the clinical manifestations of HRV infection and the molecular typing of HRV detected at the University Malaya Medical Centre, a major 900-bed referral and teaching hospital in Kuala Lumpur, Malaysia. As part of ongoing studies on respiratory virus infections in children seen at our hospital, nasopharyngeal aspirates or throat swabs collected from children with respiratory signs and symptoms between June 2007 and May 2011 were tested for HRV. Over the four years, 1096 samples were tested and 87 (7.9%) were positive for HRV. Viral RNA was extracted from 30 randomly selected specimens using QIAamp viral RNA kit (Qiagen, Germany) and stored at 80 C. RNA was first transcribed to cDNA using Superscript III (Invitrogen, USA). PCR was performed using previously published VP4/VP2 primers. The same primers were used for sequencing on an ABI 3730XL Genetic Analyzer (Applied Biosystems, USA). The partial VP4/VP2 sequences have been shown to correlate with serotype classification. Sequences of 400 nucleotides obtained were aligned with all reference HRV genomes available in GenBank, using Clustal X version 2.12. Phlyogenetic trees were constructed using the neighbour-joining method with 1000 bootstrap replicates and depicted with MEGA version 5. This study was approved by the hospital’s Medical Ethics Committee (no. 788.3). Based on the sequences of partial VP4/VP2 obtained from 30 children, 40% (n Z 12) were HRV-A and 60% (n Z 18) were HRV-C, and no HRV-B was identified. These sequences have been deposited into GenBank (accession numbers JQ356547eJQ356576). The absence of HRV-B is consistent with the findings of others where HRV-B are often the least commonly detected species (0e11%). However, it could also be due to the small number of HRV sequenced in the present study. In most other studies, however, the predominant species reported were HRV-A, followed by HRV-C. Among the HRV positive patients, 60% (n Z 18/30) were males and the median age was 1.1 years (range 2 monthse10 years). Medical records were available for 23 of the patients. Ten (43.5%) had pre-existing medical conditions, including 6 with asthma. The commonest (82.6%) presentation was wheezing and this was as previously described, either as an exacerbation of asthma or bronchiolitis. Other frequently reported symptoms were coryza (69.6%), fever (43.5%), and diarrhoea or vomiting (34.8%). One patient died during admission, but this was most likely due to underlying leukaemia. Excluding two nosocomial cases, the average duration of admission was 3.5 days. Pairwise sequence alignment against the reference HRVA and HRV-B types in the VP4/VP2 region suggests 75e88% and 75e89% sequence similarities amongst all the isolates, respectively. The nucleotide sequence divergence of the 12 HRV-A from this study ranged from 4 to 11%, and belonged to 11 different subtypes, including HRV-49 (n Z 2), HRV-2, HRV-10, HRV-12, HRV-15, HRV-21, HRV-46, HRV-55, HRV-56 and HRV-60 (Fig. 1). One isolate had 90% nucleotide similarity to both HRV-29 and HRV-44, and requires further confirmation using VP1 or 50 untranslated region sequences. Comparison with 20 previously published HRV-A sequences from Malaysia from 2009 showed little overlap, except for clusters of 5 and 2 strains of HRV-12 and HRV-56, respectively. Currently, the molecular classification of HRV-C is still not agreed upon. Using the proposed classification with a cut-off of >10% nucleotide divergence in partial

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5′ and 3′ non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63–81% among themselves and 63–96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.

Seasonal influenza activity based on laboratory surveillance in Malaysia, 2011-2016: SAM et al.

Influenza seasonality in equatorial countries is little understood. Seasonal and alert influenza thresholds were determined for Malaysia, using laboratory‐based data obtained from the Malaysia Influenza Surveillance System and a major teaching hospital, from 2011 to 2016. Influenza was present year‐round, with no clear annual seasons. Variable periods of higher transmission occurred inconsistently, in November to December, January to March, July to September, or a combination of these. These coincide with seasons in the nearby southeast Asian countries or winter seasons of the northern and southern hemispheres. Changes in the predominant circulating influenza type were only sometimes associated with increased transmission. The data can provide public health interventions such as vaccines.