Malaysian Strain Research Articles

Genomic analysis revealing the resistance mechanisms of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from pig and humans in Malaysia.

Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae has been associated with a wide range of infections in humans and animals. The objective of this study was to determine the genomic characteristics of two multiple drug resistant, ESBLs-producing K. pneumoniae strains isolated from a swine in 2013 (KP2013Z28) and a hospitalized patient in 2014 (KP2014C46) in Malaysia. Genomic analyses of the two K. pneumoniae strains indicated the presence of various antimicrobial resistance genes associated with resistance to β-lactams, aminoglycosides, colistin, fluoroquinolones, phenicols, tetracycline, sulfonamides, and trimethoprim, corresponding to the antimicrobial susceptibility profiles of the strains. KP2013Z28 (ST25) and KP2014C46 (ST929) harbored 5 and 2 genomic plasmids, respectively. The phylogenomics of these two Malaysian K. pneumoniae, with other 19 strains around the world was determined based on SNPs analysis. Overall, the strains were resolved into five clusters that comprised of strains with different resistance determinants. This study provided a better understanding of the resistance mechanisms and phylogenetic relatedness of the Malaysian strains with 19 strains isolated worldwide. This study also highlighted the needs to monitor the usage of antibiotics in hospital settings, animal husbandry, and agricultural practices due to the increase of β-lactam, aminoglycosides, tetracycline, and colistin resistance among pathogenic bacteria for better infection control.

Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia

BackgroundA new domestic cat hepadnavirus (DCH, family Hepadnaviridae) was first reported from whole blood samples of domestic cats in Australia in 2018, and from cat serum samples in Italy in 2019. The pathogenesis of DCH is unknown, but it was reported in cats with viraemia (6.5–10.8%), chronic hepatitis (43%) and hepatocellular carcinoma (28%). Recent reports suggest that DCH resembles the human hepatitis B virus (HBV) and its related hepatopathies. This study aims to detect and characterize DCH among domestic cats in Malaysia. A cross-sectional study was performed on 253 cats, of which 87 had paired blood and liver samples, entailing whole-genome sequencing and phylogenetic analysis of DCH from a liver tissue sample.ResultsAmong the 253 cats included in this study, 12.3% of the whole blood samples tested positive for DCH. The detection rate was significantly higher in pet cats (16.6%, n = 24/145) compared to shelter cats (6.5%, n = 7/108). Liver tissues showed higher a DCH detection rate (14.9%, n = 13/87) compared to blood; 5 out of these 13 cats tested positive for DCH in their paired liver and blood samples. Serum alanine transaminase (ALT) was elevated (> 95 units/L) in 12 out of the 23 DCH-positive cats (52.2%, p = 0.012). Whole-genome sequence analysis revealed that the Malaysian DCH strain, with a genome size of 3184 bp, had 98.3% and 97.5% nucleotide identities to the Australian and Italian strains, respectively. The phylogenetic analysis demonstrated that the Malaysian DCH genome was clustered closely to the Australian strain, suggesting that they belong to the same geographically-determined genetic pool (Australasia).ConclusionsThis study provided insights into a Malaysian DCH strain that was detected from a liver tissue. Interestingly, pet cats or cats with elevated ALT were significantly more likely to be DCH positive. Cats with positive DCH detection from liver tissues may not necessarily have viraemia. The impact of this virus on inducing liver diseases in felines warrants further investigation.

Co-Infections of Tilapia Lake Virus, Aeromonas hydrophila and Streptococcus agalactiae in Farmed Red Hybrid Tilapia.

Simple SummaryTilapia is a freshwater fish that is commercially cultured around the world. However, intensification of tilapia culture often results in diseases, occasionally with co-infections of multiple pathogens. This paper reports the first case of red hybrid tilapia that naturally co-infected with Tilapia Lake Virus (TiLV), Aeromonas hydrophila and Streptococcus agalactiae in Malaysia. In January 2020, a tilapia farm in Selangor, Malaysia, reported a mass mortality of adult red hybrid tilapias, with 70% mortality. Bacterial isolation, PCR and sequencing analysis confirmed the presence of TiLV, A. hydrophila and S. agalactiae in the affected fish. As tilapia is widely cultured throughout the world, detection of multiple infections might signal a potential threat to the industry.A high death rate among red hybrid tilapias was observed in a farm in Selangor, Malaysia, in January 2020. The affected fish appeared lethargic, isolated from schooling group, showed loss of appetite, red and haemorrhagic skin, exophthalmia and enlarged gall bladders. Histopathological assessment revealed deformation of kidney tubules, and severe congestion with infiltrations of inflammatory cells in the brains and kidneys. Syncytial cells and intracytoplasmic inclusion bodies were occasionally observed in the liver and brain sections. Tilapia Lake Virus (TiLV), Aeromonas hydrophila and Streptococcus agalactiae were identified in the affected fish, either through isolation or through PCR and sequencing analysis. The phylogenetic tree analysis revealed that the TiLV strain in this study was closely related to the previously reported Malaysian strain that was isolated in 2019. On the other hand, A. hydrophila and S. agalactiae were closer to Algerian and Brazilian strains, respectively. The multiple antibiotic resistance index for A. hydrophila and S. agalactiae was 0.50 and 0.25, respectively. Co-infections of virus and bacteria in cultured tilapia is a new threat for the tilapia industry.

Detection of Nigrospora sphaerica in the Philippines and the susceptibility of three Hylocereus species to reddish-brown spot disease

Diseases are among the major problems that negatively affect dragon fruit profitability worldwide. Diseases of dragon fruit in the Philippines are yet to be identified and reported. This study elucidates the causal agent of a disease infecting stems of dragon fruit grown in Los Baños, Laguna, Philippines. The fungus was isolated and identified as Nigrospora sp. based on morphological and cultural characteristics in potato dextrose agar medium. Using the DNA sequence of the internal transcribed spacer (ITS) gene region, isolate MBDF0016b was identified as Nigrospora sphaerica. The Philippines strain was closely related to the Malaysian strain, which also causes reddish-brown spot in dragon fruit (H. polyrhizus), and to other N. sphaerica isolates from other host-plant species. Nigrospora sphaerica MBDF0016b was pathogenic to H. megalanthus, H. undatus, and H. polyrhizus in detached stem and glasshouse assays. The same fungus was re-isolated from the inoculated stems and thus, establishing Koch’s postulate. This paper is the first confirmed scientific record of a dragon fruit disease in the Philippines and the first report of N. sphaerica as a dragon fruit pathogen causing reddishbrown spot disease in H. megalanthus.

First molecular detection and complete sequence analysis of porcine circovirus type 3 (PCV3) in Peninsular Malaysia.

Porcine circovirus type 3 (PCV3) is a newly emerging virus in the swine industry, first reported recently in 2016. PCV3 assembles into a 2000 bp circular genome; slightly larger than PCV1 (1758–1760 bp), PCV2 (1766–1769 bp) and PCV4 (1770 bp). Apart from being associated with porcine dermatitis and nephropathy syndrome (PDNS), PCV3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (PRDC) and neurologic disease. Given that PCV3 is increasingly reported in countries including Thailand and U.S. with whom Malaysia shares trade and geographical relationship; and that PCV3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of PCV3 in Malaysian swine farms. Twenty-four commercial swine farms, three abattoirs and retail shops in Peninsular Malaysia were sampled using convenience sampling method. A total of 281 samples from 141 pigs, including 49 lung archive samples were tested for PCV3 by conventional PCR. Twenty-eight lung samples from wild boar population in Peninsular Malaysia were also included. Nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. Results revealed that PCV3 is present in Peninsular Malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. Despite wide reports of PCV3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. PCV3 strain A1 and A2 were present in Malaysia, and Malaysian PCV3 strains were found to be phylogenetically related to Spanish, U.S. and Mexico strains.

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 β-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

Development of an Effective and Stable Genotype-Matched Live Attenuated Newcastle Disease Virus Vaccine Based on a Novel Naturally Recombinant Malaysian Isolate Using Reverse Genetics

Genotype VII Newcastle disease viruses are associated with huge economic losses in the global poultry industry. Despite the intensive applications of vaccines, disease outbreaks caused by those viruses continue to occur frequently even among the vaccinated poultry farms. An important factor in the suboptimal protective efficacy of the current vaccines is the genetic mismatch between the prevalent strains and the vaccine strains. Therefore, in the present study, an effective and stable genotype-matched live attenuated Newcastle disease virus (NDV) vaccine was developed using reverse genetics, based on a recently isolated virulent naturally recombinant NDV IBS025/13 Malaysian strain. First of all, the sequence encoding the fusion protein (F) cleavage site of the virus was modified in silico from virulent polybasic (RRQKRF) to avirulent monobasic (GRQGRL) motif. The entire modified sequence was then chemically synthesized and inserted into pOLTV5 transcription vector for virus rescue. A recombinant virus termed mIBS025 was successfully recovered and shown to be highly attenuated based on OIE recommended pathogenicity assessment indices. Furthermore, the virus was shown to remain stably attenuated and retain the avirulent monobasic F cleavage site after 15 consecutive passages in specific-pathogen-free embryonated eggs and 12 passages in one-day-old chicks. More so, the recombinant virus induced a significantly higher hemagglutination inhibition antibody titre than LaSota although both vaccines fully protected chicken against genotype VII NDV induced mortality and morbidity. Finally, mIBS025 was shown to significantly reduce both the duration and quantity of cloacal and oropharyngeal shedding of the challenged genotype VII virus compared to the LaSota vaccine. These findings collectively indicate that mIBS025 provides a better protective efficacy than LaSota and therefore can be used as a promising vaccine candidate against genotype VII NDV strains.

Complete Genome Sequences of SARS-CoV-2 Strains Detected in Malaysia.

We sequenced four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Malaysia during the second wave of infection and found unique mutations which suggest local evolution. Circulating Malaysian strains represent introductions from different countries, particularly during the first wave of infection. Genome sequencing is important for understanding local epidemiology.

Evaluation of the antigen relatedness and efficacy of a single vaccination with different infectious bronchitis virus strains against a challenge with Malaysian variant and QX-like IBV strains.

BackgroundThe predominant infectious bronchitis virus (IBV) strains detected in chickens in Malaysia are the Malaysian variant (MV) and QX-like, which are associated with respiratory distress, nephropathy, and high mortality. On the other hand, the antigenic relatedness and efficacy of IBV vaccines against these 2 field IBV strains are not well characterized.ObjectivesThis study aimed to determine the antigen relatedness and efficacy of different IB vaccine strains against a challenge with MV and QX-like strains.MethodsThe antigen relatedness and the ability of different IB vaccine strains in conferring protection against MV and QX-like were assessed based on the clinical signs, macroscopic lesions, and ciliary activity.ResultsThe MV strain IBS037A/2014 showed minor antigenic subtype differences with the vaccine virus Mass H120 and 4/91 strains but showed major antigenic subtype differences with the K2 strain. The Malaysian QX-like strain IBS130/2015 showed major antigenic subtype differences with the MV strain IBS037A/2014 and the vaccine strains except for K2. Chickens vaccinated once with Mass (H120) or with non-Mass (4/91 and K2) developed antibody responses with the highest antibody titer detected in the groups vaccinated with H120 and 4/91. The mean ciliary activities of the vaccinated chickens were between 56 to 59% and 48 to 52% in chickens challenged with IBS037A/2014 and IBS130/2015, respectively. The vaccinated and challenged birds showed mild to severe lesions in the lungs and kidneys.ConclusionsDespite the minor antigenic subtype differences, a single inoculation with Mass or non-Mass vaccines could not protect against the MV IBS037A/2014 and QX-like IBS130/2015.

Toxicity comparison among four strains of Margalefidinium polykrikoides from China, Malaysia, and USA (belonging to two ribotypes) and possible implications

The unarmored dinoflagellate Margalefidinium polykrikoides is a well-known cosmopolitan harmful alga that forms intensive blooms causing massive fish kills and mortality of other marine animals. Globally distributed populations of M. polykrikoides have been categorized into four ribotypes: East Asian, American/Malaysian, Philippines, and Mediterranean, according to their characteristic sequences in LSU rRNA gene and primary geographic distributions, while no discernible morphological feature has been observed to distinguish. Here we examined the toxicity of four strains of M. polykrikoides including MPJZB-C3 and MPJZB-D6 (East Asian ribotype, from China), CP1 (American/Malaysian ribotype, from USA), and MPCoKK23 (American/Malaysian ribotype, from Malaysia), to three aquatic animals including the marine medaka (Oryzias melastigma), rotifer (Brachionus plicatilis, L-type), and brine shrimp (Artemia salina). With equivalent initial cell densities ranged from 1000 to 7000 cells mL−1, CP1 and MPCoKK23 displayed acute toxicity in a dose-response manner to the three target animals (100% mortality at higher cell densities) within 24 h, MPJZB-C3 also displayed acute toxicity to the fish and rotifer in 24 h (up to 50% mortality) when the cell densities were above 3000 cells mL−1. The strain MPJZB-D6 did not exhibit significant toxicity to all three animals in 24 h, but, in 120 h, caused 50–100% mortalities in fish when the cell density was >3000 cells mL−1 and 17% mortality in brine shrimp when the cell density was >5000 cells mL−1. Statistical analysis of the data indicated that the strains CP1 and MPCoKK23 exhibited significantly higher toxicity (50–100% mortalities) to all three animals than the strains MPJZB-C3 and MPJZB-D6 (0–100% mortality) did within 24 h exposure, while, however, the difference in toxicity between the American and Malaysian strains and that between the two Chinese strains were both not significant in terms of the mortality of test animals. While the cell-free culture media (by filtration) and sonicated cultures were less toxic to test animals than the live cultures for all M. polykrikoides strains, sonicated cultures were even less toxic than the cell-free culture media. Among the three target animals, the marine medaka exhibited most sensitive to the toxicity, followed by rotifer and then brine shrimp. Collectively, while our results demonstrated that all strains, or both ribotypes, of M. polykrikoides are toxic, the results may also suggest the differences in toxicity to three test animals among the four cultures of M. polykrikoides reflect the inherent difference in toxicity between East Asian and American/Malaysian ribotypes, which stimulated us to propose a further investigation on whether or not the four different ribotypes also differ inherently in their toxicity in the future, via comparing more strains of all four ribotypes and relevant genetic characterizations.

Immune responses in mice and pigs after oral vaccination with rabies virus vectored Nipah disease vaccines

Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG333E, which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG333E/NiVG and rERAG333E/NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG333E vector platform. Both rERAG333E/NiVG and rERAG333E/NiVF displayed growth properties similar to those of rERAG333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG333E. Oral vaccination with rERAG333E/NiVG either singularly or combined with rERAG333E/NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG333E/NiVG and rERAG333E/NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies.

In-depth spectral characterization of antioxidative (1,3)-β-D-glucan from the mycelium of an identified tiger milk mushroom Lignosus rhinocerus strain ABI in a stirred-tank bioreactor

Abstract A rare Malaysian tiger milk mushroom Lignosus rhinocerus strain ABI (LRSA) was morphologically identified based on its pileus, stipe, and sclerotium. LRSA (515 bp) was sequenced and found to have 99% similar to L. rhinocerus strains CH31 and CH2. Phylogenetically, evolutionary distance (Knuc) and plasmid-matching software (ApE) for sequences of matching fungal species were used to verify that the isolate belonged to the L. rhinocerus species. The strain was cultured in a stirred-tank bioreactor and a mycelial β-glucan (G) was extracted for compound characterization. The structure of exopolysaccharide extract from mycelium of LRSA was studied using Fourier-transform infrared spectroscopy (FT-IR) and one-dimensional (1D) and two-dimensional (2D) Nuclear Magnetic Resonance (NMR). FT-IR spectroscopy showed that G exhibited a similar β-glycosidic structure to the standard (laminarin), and the presence of characteristic bands at 3277, 2919, 1638, 1545, 1400, 1078, and 896 cm−1 confirmed the similarities. 1H and 13C NMR, as well as 2D NMR: homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) spectra, were used for structural elucidation of the β-glucan and confirmed the extracted material as (1,3)-β-D-glucan. In addition, the G compound exhibited antioxidant activities through total phenolic content (4.47 mg gallic acid equivalents/g), 2, 2-diphenyl-1-picrylhydrazyl (11.48 mg/mL), and ferric reducing antioxidant power (0.56 mg/mL) assays. These findings may facilitate the development of rare G production in a high-scale bioreactor using LRSA.

Regression of solid breast tumours in mice by Newcastle disease virus is associated with production of apoptosis related-cytokines

BackgroundDifferent strains of Newcastle disease virus (NDV) worldwide proved to have tumouricidal activity in several types of cancer cells. However, the possible anti-cancer activity of Malaysian NDV AF2240 strain and its mechanism of action remains unknown. The ability of cytokine-related apoptosis-inducing NDV AF2240 to treat breast cancer was investigated in the current study.MethodsA total of 90 mice were used and divided into 15 groups, each group comprising of 6 mice. Tumour, body weight and mortality of the mice were determined throughout the experiment, to observe the effect of NDV and NDV + tamoxifen treatments on the mice. In addition, the toxic effect of the treatments was determined through liver function test. In order to elucidate the involvement of cytokine production induced by NDV, a total of six cytokines, i.e. IL-6, IFN-γ, MCP-1, IL-10, IL12p70 and TNF-α were measured using cytometric bead array assay (plasma) and enzyme-linked immunosorbent spot (isolated splenocytes).ResultsThe results demonstrated that 4 T1 breast cancer cells in allotransplanted mice treated with AF2240 showed a noticeable inhibition of tumour growth and induce apoptotic-related cytokines.ConclusionsNDV AF2240 suppression of breast tumour growth is associated with induction of apoptotic-related cytokines. It would be important to further investigate the molecular mechanism underlaying cytokines production by Newcastle disease virus.

Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China

Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.

Phosphine resistance status in lesser grain borer Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae) strains originating from the tropical countries: Presentation

Stored product beetles that are resistant to the fumigant phosphine (hydrogen phosphide) have been reported for more than 50 years in many places worldwide. The high levels of phosphine resistance in lesser grain borer Rhyzopertha dominica (F.) have been noted from several countries including Bangladesh. This study was designed to evaluate the status of resistance to phosphine in Bangladeshi R. dominica and to verify the possible comparison among other phosphine resistant strains from tropical countries viz. Burkina Faso and Malaysia. The data reported and summarized here showed varied levels of resistance compared to the laboratory phosphine susceptible strain (RDLAB). Rhyzopertha dominica strains originating from Bangladesh (RDBGD) and Burkina Faso (RDBKF) exhibited higher levels of resistance to phosphine compared to the Malaysian strains (RDMAL). Analysis of dose–response data indicated that the RDBGD and RDBKF strains were the most resistant to phosphine under different exposure periods. At LC50, these two strains were more than 80-fold more resistant at all exposures compared to the susceptible strain. Results also revealed that RDBGD and RDBKF strains required a relatively high concentration of 334.94 and 240.081 mg L-1 for 99% mortality. The mean survival time (MST) for the phosphine resistant and susceptible also varied significantly. The maximum MST was recorded for RDBGD and RDBKF strains. The present findings further confirmed that the Bangladeshi originated R. dominica strain contained higher resistance to phosphine compared to strains from other countries. This study could be useful in developing management strategies to prevent stored grain from being infested by resistant strains of R. dominica in tropical countries.

Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher’s exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Comparative Pathogenicity of Malaysian QX-like and Variant Infectious Bronchitis Virus Strains in Chickens at Different Age of Exposure to the Viruses

Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.

In-Vitro Activity of Doxycycline and β-Lactam Combinations Against Different Strains of Burkholderia pseudomallei.

Although doxycycline is active against Burkholderia pseudomallei and has been used in the eradication stage of melioidosis therapy, it is not regularly used during the initial intensive phase. In order to assess its potential use in intensive phase therapy, we investigated in vitro pharmacodynamic activity of doxycycline and β-lactams alone and in combination against four Malaysian strains of B. pseudomallei. Using a checkerboard assay, the combinations of doxycycline and imipenem, doxycycline and ceftazidime, and doxycycline and amoxicillin-clavulanate tested against four strains showed indifferent effects with summation fractional inhibitory concentration values ranging from 0.62 to 2.12. Time-kill experiments also indicated that the combinations of doxycycline/β-lactam antibiotics against four tested strains did not fulfil synergy criteria, in which all combinations showed indifferent effects with -1.36 to 1.26-log CFU/mL compared to the most active monotherapy regimen in each combination. No re-growth of bacteria was detected after the early killing in doxycycline/β-lactam combination regimens compared to β-lactam monotherapy regimens, in which 9 out of 10 were associated with re-growth of bacteria. As no synergistic activity was observed, this in vitro study showed that doxycycline offers no additional benefit to be used in combination with β-lactams in the intensive phase of therapy.

Evaluation of a Malaysian soy sauce koji strain Aspergillus oryzae NSK for γ-aminobutyric acid (GABA) production using different native sugars.

In this study, a selected γ-aminobutyric acid (GABA)-rich Malaysian strain Aspergillus oryzae NSK was collected from soy sauce koji. The strain was used to explore the effect of using renewable native sugar syrup, sugarcane, nipa, and molasses as fermentable substrates for developing a novel functional GABA soy sauce. We evaluated the strain using the chosen native sugars for 7days using shake flask fermentation at 30°C. The results showed optimum GABA concentration was achieved using cane molasses as the fermentable substrate (354.08mg/L), followed by sugarcane syrup (320.7mg/L) and nipa syrup (232.07mg/L). Cane molasses was subsequently utilized as a substrate to determine the most suitable concentration for A. oryzae NSK to enhance GABA production and was determined as 50% g/L of glucose standard cane molasses. Our findings indicate that cane molasses can be used as a GABA-rich ingredient to develop a new starter culture for A. oryzae NSK soy sauce production.

First molecular detection of porcine bocavirus in Malaysia.

Several strains of porcine bocaviruses have been reported worldwide since their first detection in Sweden in 2009. Subsequently, the virus has been reported to be associated with gastrointestinal and respiratory signs in weaner and grower pigs. Although Malaysia is host to a self-sufficient swine livestock industry, there is no study that describes porcine bocavirus in the country. This report is the first to describe porcine bocavirus (PBoV) in Malaysian swine herds. PBoV was identified in various tissues from sick and runt pigs using the conventional PCR method with primers targeting conserved regions encoding for the nonstructural protein (NS1) gene. Out of 103 samples tested from 17 pigs, 32 samples from 15 pigs were positive for porcine bocavirus. In addition, a higher detection rate was identified from mesenteric lymph nodes (52.9%), followed by tonsil (37.0%), and lungs (33.3%). Pairwise comparison and phylogenetic analyses based on a 658-bp fragment of NS1 gene revealed that the Malaysian PBoV strains are highly similar to PBoV3 isolated in Minnesota, USA. The presence of porcine bocavirus in Malaysia and their phylogenetic bond was marked for the first time by this study. Further studies will establish the molecular epidemiology of PBoV in Malaysia and clarify pathogenicity of the local isolates.