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.
Porcine circovirus type 2 is the primary etiological agent associated with a group of complex multi-factorial diseases classified as Porcine Circovirus Associated Diseases (PCVAD). Sporadic cases reported in Malaysia in 2007 caused major economic losses to the 2.2 billion Malaysian ringgit (MYR) (approximately 0.7 billion US dollar) swine industry. The objective of the present study was to determine the association between the presence of PCV2 and occurrences of PCVAD.
Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). Study on the type of CAV isolates present and their genetic diversity, transmission to their progeny and level of protection afforded in the breeder farms is lacking in Malaysia. Hence, the present study was aimed to detect CAV from commercial broiler breeder farms and characterize CAV positive samples based on sequence and phylogenetic analysis of partial VP1 gene.
Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.
Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in Vietnam in recent years. In this work, six serotype A foot-and-mouth disease viruses (FMDV), collected from endemic outbreaks during January and February of 2009 in four different provinces in Vietnam, were genetically characterized for their complete genome sequences. Genetic analysis based on the complete viral genome sequence indicated that they were closely related to each other and shared 99.0-99.8% amino acid (aa) identity. Genetic and deduced aa analysis of the capsid coding gene VP1 showed that the six Vietnamese strains were all classified into the genotype IX from a total of 10 major genotypes worldwide, sharing 98.1-100% aa identity each other. They were most closely related to the type A strains recently isolated in Laos (A/LAO/36/2003, A/LAO/1/2006, A/LAO/6/2006, A/LAO/7/2006, and A/LAO/8/2006), Thailand (A/TAI/2/1997 and A/TAI/118/1987), and Malaysia (A/MAY/2/2002), sharing 88.3-95.5% nucleotide (nt) identities. In contrast, Vietnamese type A strains showed low nt identities with the two old type A FMDVs, isolated in 1960 in Thailand (a15thailand iso43) and in 1975 in the Philippines (aphilippines iso50), ranging from 77.3 to 80.9% nt identity. A multiple alignment based on the deduced amino acid sequences of the capsid VP1 coding gene of type A FMDV revealed three amino acid substitutions between Vietnamese strains and the strains of other Southeast Asian countries (Laos, Thailand, Malaysia, and the Philippines). Alanine was replaced by valine at residue 24, asparagine by arginine at residue 85, and serine by threonine at residue 196. Furthermore, type A FMDV strains recently isolated in Vietnam, Laos, Thailand, and Malaysia all have one amino acid deletion at residue 140 of the capsid VP1 protein compared with the two old type A FMDV strains from Thailand and the Philippines as well as most other type A representatives worldwide. This article is the first to report on the comprehensive genetic characterization of type A FMDV circulating in Vietnam.
Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in cats with infection in its progressive form. Although there are numerous reports on the occurrence of FeLV in the feline population worldwide, there is a paucity of data in Asia. In this study, we assessed the circulation of FeLV by ELISA and nested PCR in cats from different countries in Southeast Asia (i.e., Thailand, Malaysia, Singapore, Philippines, Indonesia and Vietnam) and Taiwan during 2017-2018. Forty-seven cats were positive to FeLV by antigen or provirus detection, but 32 samples were considered truly positive on the basis of positive molecular testing. Frequency of occurrence of FeLV proviral DNA ranged from 0% (0/43 positive samples) in Indonesia to 18.5% (22/119 positive samples) in Thailand. A statistically significant association (p < 0.05) was found between country of cats origin, age, lifestyle, abnormal oral mucosa, and FeLV molecular positive results. In-depth studies are needed in other countries in Southeast Asia to elucidate the mosaic of knowledge about FeLV epidemiology.
Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.
It is widely accepted that Newcastle disease is endemic in most African countries, but little attention has been afforded to establishing the sources and frequency of the introductions of exotic strains. Newcastle disease outbreaks have a high cost in Africa, particularly on rural livelihoods. Genotype VIIh emerged in South-East Asia and has since caused serious outbreaks in poultry in Malaysia, Indonesia, southern China, Vietnam and Cambodia. Genotype VIIh reached the African continent in 2011, with the first outbreaks reported in Mozambique. Here, we used a combination of phylogenetic evidence, molecular dating and epidemiological reports to trace the origins and spread of subgenotype VIIh Newcastle disease in southern Africa. We determined that the infection spread northwards through Mozambique, and then into the poultry of the north-eastern provinces of Zimbabwe. From Mozambique, it also reached neighbouring Malawi and Zambia. In Zimbabwe, the disease spread southward towards South Africa and Botswana, causing outbreaks in backyard chickens in early-to-mid 2013. In August 2013, the disease entered South Africa's large commercial industry, and the entire country was infected within a year, likely through fomites and the movements of cull chickens. Illegal poultry trading or infected waste from ships and not wild migratory birds was the likely source of the introduction to Mozambique in 2011.
A retrospective study was carried out to determine the frequency of the pre-core stop codon mutant virus in a group of chronic hepatitis B carriers: 81 cases were considered [33 hepatits B e antigen (HBe) positive and 48 HBe negative]. All of the HBe positive cases had detectable viral DNA by hybridization analysis; in the case of the HBe negative cases, one third had detectable viral DNA by hybridization analysis and two thirds had HBV DNA detectable by polymerase chain reaction (PCR) amplification. Pre-core stop codon mutant detection was carried out on all specimens using allele-specific oligonucleotide hybridization following PCR amplification of the target sequence. The pre-core mutant was detected in 13/33 (39.4%) of HBe positive cases and in 32/48 (66.7%) of HBe negative cases. Sequence analysis was carried out on 8 of the 16 HBe negative specimens that did not carry the pre-core mutant virus to determine the molecular basis for the HBe minus phenotype in these cases: the 1762/1764 TA paired mutation in the second AT rich region of the core promoter was detected in five cases; a start codon mutation was detected in one case. The predominant mutation resulting in the HBe minus phenotype in our isolates was the 1896A pre-core ("pre-core stop codon") mutation; other mutations responsible for the phenotype included the core promoter paired mutation and pre-core start codon mutation. In view of the high frequency of the pre-core mutant virus, sequence analysis was performed to determine the virus genotype on the basis of the nucleotide sequence of codon 15. The sequences of 21 wild type virus (14 HBe positive and 7 HBe negative cases) were examined: 15 were found to be codon 15 CCT variants (71.4%); the frequency in the HBe positive group was 12/14 (85.7%), while that in the HBe negative group was 3/7 (42.9%). The high frequency of the codon 15 CCT variant in association with the frequent occurrence of the pre-core mutant in our isolates concurs with the results of other studies.
Infectious agent(s) causing the fatal Sarawak acute childhood viral infection (SACVI) has not been identified. In the present study, results indicating that inocula prepared from the fatal cases of SACVI induced apoptosis in Vero cell cultures are presented. These findings suggest the possible involvement of apoptotic cellular responses in SACVI.
Between 2005 and 2013 six severe pneumonia cases (all requiring mechanical ventilation, two fatal outcomes) caused by human adenovirus type 21 (HAdV-B21) were observed in Germany. So far, HAdV-B21 was mainly associated with non-severe upper and lower respiratory tract infections. However, a few highly virulent HAdV types, e.g. HAdV-B14p1, were previously associated with severe, fatal pneumonia. Complete genomic sequences of the German HAdV-B21 pneumonia isolates formed a single phylogenetic cluster with very high sequence identity (≥ 99.897%). Compared to the HAdV-B21 prototype (only 99.319% identity), all isolates had a unique 15 amino acid deletion and a 2 amino acid insertion in the RGD loop of the penton base which may affect binding to the secondary receptor on the host cells. Moreover, a recombinant E4 gene region derived of HAdV-B3 was identified by bootscan analysis. Thus, the highly virulent, pneumotropic HAdV-B21 was denominated as subtype 21a. Surprisingly, there was 99.963% identity with agent Y/SIBU97 (only 13.4 kb available in GenBank of the 35.4 kb genome) which was associated with 10 fatalities due to cardiopulmonary failure in Sarawak, Malaysia, in 1997. In conclusion, a HAdV-B21 subtype (21a) associated with severe pneumonia in Germany was phylogenetically linked to an adenovirus isolated in Malaysia.
Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/μL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.
As most published studies on precore mutants have been carried out on isolates from patients with liver diseases, and it is unclear whether HBsAg carriers with viraemia in the absence of HBeAg are also generally infected by such mutants, it was decided to sequence the precore region in some HBV-DNA isolated from HBsAg-positive carriers. Precore sequences of HBV-DNA from 43 HBsAg carriers in Malaysia were studied. Three HBV subtypes were identified according to the nucleotide sequence of the precore region. Most of the carriers were found to be infected by the subtype adr. Mutations were detected in the precore regions. The most common conserved mutation was a silent mutation involving conversion from T to C (CCT to CCC) at position 1858 at codon 15 (proline). It was found that 4/43 (9.3%) had a mutation at the penultimate codon where TGG was changed to TAG. All 4 isolates with the TAG mutation had nt T at position 1858. Of the 4 carriers who were infected by these mutant viruses, 2 were coinfected with the wild type, 1 was infected only by a variant with the mutation at position 1896, while another was infected by a variant with mutations at positions 1896 and 1899. Three of the 4 were anti-HBe positive while 1 was HBeAg positive. Alanine aminotransaminase activities in all 4 carriers were normal. This study therefore demonstrated that variants with stop codons at the penultimate codon could be found in asymptomatic carriers in Malaysia.
Both wild-type virulent and mutant strains of pseudorabies virus (PrV) were used in this study. Mutants used were derived from the plaque purified strain PrVmAIP. A total of six drug resistant mutants, three bromodeoxyuridine (BUdR) resistant and three iododeoxyuridine (IUdR) resistant, respectively, were isolated and passaged in chicken embryo fibroblast (CEF) cells. The DNA of these PrVs were compared with the wild-type isolates by means of the restriction fragment pattern (RFP) findings produced with Bam HI, Kpn I, Hind III and Bgl II restriction enzymes (RE). Compared to the wild-type PrVs (PrV-VBA1-parental strain of PrVmAIP; PrV-VBA2; PrV-VBA3), the RFP of PrVmAIP showed the presence of mutations within the RE sites studied. Both PrV-VBA1 and PrV-VBA2 appeared to be closely related but their RFPs differed from PrV-VBA3. Significant differences either in the number, size or migrations of the DNA fragments could also be detected in the BUdR resistant strains. Even though different features of cytopathic effect (GPE) were observed in the IUdR resistant PrVs, the RFP findings remained identical. The PrVs studied showed considerable differences from the reference PrV (PrV-CD).
Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3' ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71.
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.
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.
Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.
Shewanella is a prevalent bacterial genus in deep-sea environments including marine sediments, exhibiting diverse metabolic capabilities that indicate its significant contributions to the marine biogeochemical cycles. However, only a few Shewanella phages were isolated and deposited in the NCBI database. In this study, we report the isolation and characterization of a novel Shewanella phage, vB_SbaS_Y11, that infects Shewanella KR11 and was isolated from the sewage in Qingdao, China. Transmission electron microscopy revealed that vB_SbaS_Y11 has an icosahedral head and a long tail. The genome of vB_SbaS_Y11 is a linear, double-stranded DNA with a length of 62,799 bp and a G+C content of 46.9%, encoding 71 putative open reading frames. No tRNA genes or integrase-related feature genes were identified. An uncharacterized anti-CRISPR AcrVA2 gene was detected in its genome. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analyses indicate that vB_SbaS_Y11 has a novel genomic architecture and shares low similarity to Pseudomonas virus H66 and Pseudomonas phage F116. vB_SbaS_Y11 represents a potential new family-level virus cluster with eight metagenomic assembled viral genomes named Ranviridae.IMPORTANCEThe Gram-negative Shewanella bacterial genus currently includes about 80 species of mostly aquatic Gammaproteobacteria, which were isolated around the globe in a multitude of environments, such as freshwater, seawater, coastal sediments, and the deepest trenches. Here, we present a Shewanella phage vB_SbaS_Y11 that contains an uncharacterized anti-CRISPR AcrVA2 gene and belongs to a potential virus family, Ranviridae. This study will enhance the knowledge about the genome, diversity, taxonomic classification, and global distribution of Shewanella phage populations.
Hepatitis B is a major public health problem worldwide which may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. An interaction between hepatitis B virus (HBV) envelope protein, particularly the PreS1 region, and a specific cell surface receptor is believed to be the initial step of HBV infection through attachment to hepatocytes. In order to develop a gene delivery system, bacteriophage T7 was modified genetically to display polypeptides of the PreS1 region. A recombinant T7 phage displaying amino acids 60-108 of the PreS1 region (PreS1(60-108)) was demonstrated to be most effective in transfecting HepG2 cells in a dose- and time-dependant manner. The phage genome was recovered from the cell lysate and confirmed by PCR whereas the infectious form of the internalized phage was measured by a plaque-forming assay. The internalized phage exhibited the appearance of green fluorescent dots when examined by immunofluorescence microscopy. Surface modification, particularly by displaying the PreS1(60-108) enhanced phage uptake, resulting in more efficient in vitro gene transfer. The ability of the recombinant phage to transfect HepG2 cells demonstrates the potential of the phage display system as a gene therapy for liver cancer.