The molecular genetic analysis of the genomes of the virus of porcine reproductive respiratory syndrome (VPRRS) and porcine circovirus type 2 (PCV-2) circulating in the area of the Russian Federation was discussed. The results of this work showed the circulation of the strains of the European genotype VPRRS similar to those found in France and Denmark from 1998 to 2001. The homology of the fragment of one of the genes between the Russian isolates and the vaccine strain Porcilis PRRS (Intervet) was found. It requires further study. The strains representing the North American genotype VPRRS were not found. The PCV-2 genomes fall into three separate goups. One (genotype 2b) is formed by isolates in Malaysia, Brazil, Switzerland, China, Slovakia, UK, USA, isolated during the period from 2004 to the present time. The second group consists of sequences of the viruses isolated in 2000-2012 in Canada, the U.S., China, and South Korea (genotype 2a). The third group is formed by highly pathogenic isolates in 2013 from China (highly pathogenic genotype 2c). The circulation of all three known genotypes of PCV-2: 2a, 2b, and 2c in Russian Federation was demonstrated.
A Chicken anemia virus has been isolated from a chicken flock in Harbin of China. The genome of the ivrus was cloned through polymerase chain reaction(PCR) and sequence of the genome was analyzed. The cycle genome is made of 2298 base pairs including three overlapping open reading frames(vp1, vp2, vp3) and a regulative region. Comparing sequence of the genome through BLAST in GenBank, this sequence exhibits 96.9% identity with other genome of CA Vs and least. Multiple alignment of this genome of this virus, 26p4, strain isolated in Germany, strain isolated in Malaysia and Cux-1 found that this sequence exhibits 98.2% (42/2298), 98.2% (42/2298), 96.9% (72/2298) and 97.5% (60/2319) identify with them, respectively. A new CAV strain was isolated and it has better identify with CAV isolated in Europe countries than is Asia country Malaysia. Multiple alignment of VP1, VP2, VP3 of 26p4, strain isolated in Germany, strain isolated in Malaysia, Cux-1 and strain isolated in Harbin of China found the VP2 the most conservative.
Reassortment of genetic segments between and within influenza B lineages (Victoria and Yamagata) has been shown to generate novel reassortants with unique genetic characteristics. Based on hemagglutinin (HA) and neuraminidase (NA) genes, recent surveillance study has identified reassortment properties in B/Phuket/3073/2013-like virus, which is currently used in the WHO-recommended influenza vaccine. To understand the potential reassortment patterns for all gene segments, four B/Phuket/3073/2013-like viruses and two unique reassortants (one each from Yamagata and Victoria) detected in Malaysia from 2012-2014 were subjected to whole-genome sequencing. Each gene was phylogenetically classified into lineages, clades and sub-clades. Three B/Phuket/3073/2013-like viruses from Yamagata lineage were found to be intra-clade reassortants, possessing PA and NA genes derived from Stockholm/12-like sub-clade, while the remaining genes from Wisconsin/01-like sub-clade (both sub-clades were within Yamagata Clade 3/Yam-3). However, the other B/Phuket/3073/2013-like virus had NS gene that derived from Stockholm/12-like sub-clade instead of Wisconsin/01-like sub-clade. One inter-clade reassortant had Yamagata Clade 2/Yam-2-derived HA and NP, and its remaining genes were Yam-3-derived. Within Victoria Clade 1/Vic-1 in Victoria lineage, one virus had intra-clade reassortment properties: HA and PB2 from Vic-1B sub-clade, MP and NS from a unique sub-clade "Vic-1C", and the remaining genes from Vic-1A sub-clade. Although random reassortment event may generate unique reassortants, detailed phylogenetic classification of gene segments showed possible genetic linkage between PA and NA genes in B/Phuket/3073/2013-like viruses, which requires further investigation. Understanding on reassortment patterns in influenza B evolution may contribute to future vaccine design.
SARS-CoV-2 has spread throughout the world since its discovery in China, and Malaysia is no exception. WGS has been a crucial approach in studying the evolution and genetic diversity of SARS-CoV-2 in the ongoing pandemic. Despite considerable number of SARS-CoV-2 genome sequences have been submitted to GISAID and NCBI databases, there is still scarcity of data from Malaysia. This study aims to report new Malaysian lineages of the virus, responsible for the sustained spikes in COVID-19 cases during the third wave of the pandemic. Patients with nasopharyngeal and/or oropharyngeal swabs confirmed COVID-19 positive by real-time RT-PCR with CT value < 25 were chosen for WGS. The selected SARS-CoV-2 isolates were then sequenced, characterized and analyzed along with 986 sequences of the dominant lineages of D614G variants currently circulating throughout Malaysia. The prevalence of clade GH and G formed strong ground for the presence of two Malaysian lineages of AU.2 and B.1.524 that has caused sustained spikes of cases in the country. Statistical analysis on the association of gender and age group with Malaysian lineages revealed a significant association (p <0.05). Phylogenetic analysis revealed dispersion of 41 lineages, of these, 22 lineages are still active. Mutational analysis showed presence of unique G1223C missense mutation in transmembrane domain of the spike protein. For better understanding of the SARS-CoV-2 evolution in Malaysia especially with reference to the reported lineages, large scale studies based on WGS are warranted.
Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.
The DNA genomes of isolates of rice tungro bacilliform virus from Bangladesh, India, Indonesia, Malaysia and Thailand were cloned and compared with that of the type isolate from the Philippines. Restriction endonuclease maps revealed differences between the isolates and cross-hybridization showed that they fell into two groups, those from the Indian subcontinent and those from south-east Asian countries. The genomes of isolates from the Indian subcontinent contained a deletion of 64 bp when compared with those from south-east Asia. The implications of this variation are discussed.
Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.
Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.
Duck Tembusu virus (DTMUV), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT-PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV, which was the most closely related DTMUV. It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUVs, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUVs; however, it was genetically distinctive to any of the currently circulating DTMUVs. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUVs in Asia.
The human gut contains a complex microbiota dominated by bacteriophages but also containing other viruses and bacteria and fungi. There are a growing number of techniques for the extraction, sequencing, and analysis of the virome but currently no standardized protocols. This study established an effective workflow for virome analysis to investigate the virome of stool samples from two understudied ethnic groups from Malaysia: the Jakun and Jehai Orang Asli. By using the virome extraction and analysis workflow with the Oxford Nanopore Technology, long-read sequencing successfully captured close to full-length viral genomes. The virome composition of the two indigenous Malaysian communities were remarkably different from those found in other parts of the world. Additionally, plant viruses found in the viromes of these individuals were attributed to traditional food-seeking methods. This study establishes a human gut virome workflow and extends insights into the healthy human gut virome, laying the groundwork for comparative studies.
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
Dengue virus belongs to the Flaviviridae family which also includes viruses such as the Zika, West Nile and yellow fever virus. Dengue virus generally causes mild disease, however, more severe forms of the dengue virus infection, dengue haemorrhagic fever (DHF) and dengue haemorrhagic fever with shock syndrome (DSS) can also occur, resulting in multiple organ failure and even death, especially in children. The only dengue vaccine available in the market, CYD-TDV offers limited coverage for vaccinees from 9-45 years of age and is only recommended for individuals with prior dengue exposure. A number of mutations that were shown to attenuate virulence of dengue virus in vitro and/or in vivo have been identified in the literature. The mutations which fall within the conserved regions of all four dengue serotypes are discussed. This review hopes to provide information leading to the construction of a live attenuated dengue vaccine that is suitable for all ages, irrespective of the infecting dengue serotype and prior dengue exposure.
A neurological disease of young Pekin ducks characterized by ataxia, lameness, and paralysis was observed at several duck farms in Malaysia in 2012. Gross pathological lesions were absent or inconsistent in most of the cases, but severe and consistent microscopic lesions were found in the brain and spinal cord, characterized by non-purulent panencephalomyelitis. Several virus isolates were obtained in embryonated duck eggs and in cell cultures (Vero and DF-1) inoculated with the brain homogenates of affected ducks. After exclusion of other viruses, the isolates were identified as a flavivirus by flavivirus-specific reverse transcription-polymerase chain reaction (RT-PCR) assays. Inoculation of 2-week-old Pekin ducks with a flavivirus isolate by the subcutaneous or intramuscular route resulted in typical clinical signs and histological lesions in the brain and spinal cord. The inoculated virus was detected by RT-PCR from organ samples of ducks with clinical signs and histological lesions. With a few days delay, the disease was also observed among co-mingled contact control birds. Phylogenetic analysis of NS5 and E gene sequences proved that the isolates were representatives of a novel phylogenetic group within clade XI (Ntaya virus group) of the Flavivirus genus. This Malaysian Duck Tembusu Virus (DTMUV), named Perak virus, has moderate genomic RNA sequence similarity to a related DTMUV identified in China. In our experiment the Malaysian strain of DTMUV could be transmitted in the absence of mosquito vectors. These findings may have implications for the control and prevention of this emerging group of flaviviruses.
Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major health concern globally. Genomic epidemiology is an important tool to assess the pandemic of coronavirus disease 2019 (COVID-19). Several mutations have been reported by genome analysis of the SARS-CoV-2. In the present study, we investigated the mutational and phylogenetic analysis of 30 whole-genome sequences for the virus's genomic characteristics in the specimens collected in the early phase of the pandemic (March-June, 2020) and the sudden surge of local transmission (August-September, 2020). The four samples in the early phase of infection were B.6 lineage and located within a clade of the samples collected at the same time in Singapore and Malaysia, while five returnees by rescue flights showed the lineage B. 1.36.1 (three from India), B.1.1 (one from India) and B.1.80 (one from China). However, there was no evidence of local spread from these returnees. Further, all 19 whole-genome sequences collected in the sudden surge of local transmission showed lineage B.1.36. The surge of the second wave on SARS-CoV-2 infection was linked to the single-introduction of a variant (B.1.36) that may result from the strict restriction of international travel and containment efforts. These genomic data provides the useful information to disease control and prevention strategy.
Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
Viruses with small circular ssDNA genomes encoding a replication initiator protein can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two such viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCV-SL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing metagenomics datasets revealed closely related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cyclovirus and gemycircularvirus reported here include humans or other cellular sources in or on the human body remains to be determined.