Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.
Tomato mosaic virus (ToMV), a member of the genus Tobamovirus, infects several ornamental and horticultural crops worldwide. In this study, the nucleotide sequences of the coat protein gene of worldwide ToMV isolates were analyzed to estimate the genetic structure and diversity of this virus and the involved evolutionary forces. The phylogenetic analysis showed three clades with high bootstrap support: Clade I contained three ToMV isolates from Brazil collected from pepper, Clade II comprised one Brazilian ToMV isolate from pepper, and Clade III was composed of ToMV isolates collected from different plant hosts (pepper, tomato, eggplant, lilac, camellia, dogwood, red spruce, etc.) and water (from melting ice, lakes and streams) from different countries: USA, Brazil, Korea, Germany, Spain, Denmark (Greenland), China, Taiwan, Malaysia, Iran, and Kazakhstan. With the exception of Brazil, nucleotide diversity within and between different geographic regions was very low, although statistical analyses suggested some gene flow between most of these regions. Our analyses also suggested a strong negative selection which could have contributed to the genetic stability of ToMV.
Two Malaysian infectious bronchitis virus isolates, MH5365/95 and V9/04 were characterized based on sequence and phylogenetic analyses of S1, S2, M, and N genes. Nucleotide sequence alignments revealed many point mutations, short deletions, and insertions in S1 region of both IBV isolates. Phylogenetic analysis of S1 gene and sequences analysis of M gene indicated that MH5365/95 and V9/04 belong to non-Massachusetts strain. However, both isolates share only 77% identity. Analysis based on S1 gene showed that MH5365/95 shared more than 87% identity to several Chinese strains. Meanwhile, V9/04 showed only 67-77% identity to all the previously studied IBV strains included in this study suggesting it is a variant of IBV isolate that is unique to Malaysia. Phylogenetic analysis suggests, although both isolates were isolated 10 years apart from different states in Malaysia, they shared a common origin. Analysis based on S2 and N genes indicated that both strains are highly related to each other, and there are fewer mutations which occurred in the respective genes.
Six papaya samples showing downward leaf curling were collected in Guangdong and Guangxi provinces, China. The result of TAS-ELISA showed they were all infected by geminiviruses. Comparison of partial DNA-A sequences reveals that these virus isolates can be classified into two groups. Group I includes isolates G2, G4, G5, G28 and G29 from Guangxi province, while isolate GD2 from Guangdong province belongs to Group II. The complete DNA-A sequence of G2 and GD2 were characterized. Sequence comparisons showed that the DNA-A of G2 and GD2 were most closely related to that of Ageratum yellow vein China virus- [Hn2] and Ageratum yellow vein virus , respectively, with 83.4 and 75.2% nucleotide sequence identity, while DNA-A sequence between G2 and GD2 had only 73.4% sequence identity. The molecular data suggests that G2 and GD2 are two distinct begomoviruses, for which the name Papaya leaf curl China virus (PaLCuCNV) for G2 and Papaya leaf curl Guangdong virus (PaLCuGDV) for GD2 are proposed. Comparison of individual encoded proteins showed the coat protein of G2 and GD2 shared highest amino acid sequence identity (97.7 and 94.2%, respectively) with that of Pepper leaf curl virus -[Malaysia] (PepLCV-[MY]), suggesting the CP of these viruses may have identical ancestor.
Newcastle disease virus (NDV) strain AF2240 is a viscerotropic velogenic strain that is used as a vaccine challenge virus in Malaysia. The identification of the full length genome will be a crucial platform for further studies of this isolate. In this study, we fully sequenced the genome of a derivative of this strain named AF2240-I. The 15,192 nt long genome contains a 55-nt leader sequence at the 3' whereas the trailer region consists of 114 nt at the 5'. The intergenic sequences between the NP-P, P-M, M-F, F-HN, and HN-L genes comprise 1, 1, 1, 31, and 47 nt, respectively. The acknowledged cleavage site of fusion protein showed amino acid sequence of 112-R-R-Q-K-R-F-117, which corresponds to those of virulent NDV strains. Phylogenetic analysis of the whole virus genome shows that the strain AF2240-I belongs to genotype VIII and is more closely related to velogenic strains QH1, QH4, Fontana, Largo, and Italienas compared to other strains of NDV. Differences are noticed in the hemagglutinin-neuraminidase (HN) and matrix (M) gene between AF2240 and its derivative AF2240-I. This is the first report of a complete genome sequence of an NDV strain isolated in Malaysia.
Boid inclusion body disease (BIBD) is a viral disease of boids caused by reptarenavirus. In this study, tissue from naturally infected boid snakes were homogenized and propagated in African Monkey kidney (Vero) and rat embryonic fibroblast (REF) cells. Virus replication was determined by the presence of cytopathic effect, while viral morphology was observed using transmission electron microscopy. Viral RNA was amplified using RT-PCR with primers specific for the L-segment of reptarenavirus; similarly, quantification of viral replication was done using qPCR at 24-144 h postinfection. Viral cytopathology was characterized by cell rounding and detachment in both Vero and REF cells. The viral morphology showed round-to-pleomorphic particles ranging from 105 to 150 nm which had sand-like granules. Sanger sequencing identified four closely associated reptarenavirus species from 15 (37.5 %) of the total samples tested, and these were named as follows: reptarenavirus UPM-MY 01, 02, 03, and 04. These isolates were phylogenetically closely related to the University Helsinki virus (UHV), Boa Arenavirus NL (ROUTV; BAV), and unidentified reptarenavirus L20 (URAV-L20). Comparison of deduced amino acid sequences further confirmed identities to L-protein of UHV, L-polymerase of BAV and RNA-dependent RNA polymerase of URAV-L20. Viral replication in Vero cells increased steadily from 24 to 72 h and peaked at 144 h. This is the first study in South East Asia to isolate and characterize reptarenavirus in boid snakes with BIBD.
This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.
Coat protein genes CP1, CP2 and CP3 of an isolate (MaP1) of rice tungro spherical virus (RTSV) from Malaysia were isolated, cloned and sequenced. Comparative analysis indicated that MaP1 isolate is closely related to the Philippine isolate.
Base usage and dinucleotide frequency have been extensively studied in many eukaryotic organisms and bacteria, but not for viruses. In this paper, a comprehensive analysis of these aspects for infectious bursal disease virus (IBDV) was presented. The analysis of base usage indicated that all of the IBDV genes possess equivalent overall nucleotide distributions. However when the base usage at each codon positions was analysed by using cluster analysis, the VP5 open reading frame (ORF) formed a different cluster isolated from the other genes. The unusual base usage of VP5 ORF may indicate that the gene was originated by the virus "overprinting strategy", a strategy in which virus may create novel gene by utilizing the unused reading frames of its existing genes. Meanwhile, the GC content of the IBDV genes and the chicken's coding sequences was comparable; suggesting the virus imitation of the host to increase its translational efficiency. The analysis of dinucleotide frequency indicated that IBDV genome had dinucleotide bias: the frequencies of CpG and TpA were lower and the TpG was higher than the expected. Classical methylation pathway, a process where CpG converted to TpG, may explain the significant correlation between the CpG deficiency and TpG abundance. "Principal component analysis of the dinucleotide frequencies" (DF-PCA) was used to analyse the overall dinucleotide frequencies of IBDV genome. DF-PCA on the hypervariable region and polyprotein (VPX-VP4-VP3) gene showed that the very virulent IBDV (vvIBDV) was segregated from other strains; which meant vvIBDV had a unique dinucleotide pattern. In summary, the study of base usage and dinucleotide frequency had unravelled many overlooked genomic properties of the virus.
In addition to polioviruses, non-polio enteroviruses (NPEVs) are frequently isolated from patients with acute flaccid paralysis (AFP) worldwide. In polio-free countries, there have been expectations that with disappearing wild poliovirus from the community, the rate of AFP would decrease, but the increasing number of AFP cases proved this notion to be wrong. There are speculations that NPEVs might be the cause of increasing AFP rate. The aim of this study was to investigate frequency, genetic diversity, circulation patterns of NPEVs isolated from AFP cases in Iran from 2015 to 2018. Fifty-three NPEVs were isolated from stool specimens of AFP cases during four years of AFP surveillance. Nested PCR and VP1 sequencing revealed 20 NPEV types in which Echovirus 3 (13.2%), Echovirus 6 (13.2%), Echovirus 7 (7.5%), Echovirus 13 (7.5%) and Echovirus 21 (7.5%) were the most frequent. Coxsackie B viruses were isolated for the first time in AFP cases in Iran. The phylogenetic analysis of Echovirus 3 and Echovirus 6 revealed that Iranian echovirus strains belonged to the same cluster, indicating these viruses have been circulating in Iran for a long time. Compared to global Echovirus 3 and Echovirus 6 references, Echovirus 3 and Echovirus 6 strains detected in this study were closely related to Indian and Malaysia strains, respectively. The results of this study demonstrated a wide variety of NPEV types in Iranian patients, some of which had not been reported in previous studies. Moreover, this study highlights the need for NPEV surveillance in AFP cases.
Human bocavirus (HBoV) is a single-stranded DNA virus in Parvoviridae family, causing respiratory diseases in human. The recent identifications of genomic recombination among the four human bocavirus genotypes and related non-human primate bocaviruses have shed lights into the evolutionary processes underpinning the diversity of primate bocavirus. Among these reports, however, we found inconsistency and possible alternative interpretations of the recombination events. In this study, these recombination events were reviewed, and the related genome sequences were re-analysed, aiming to inform the research community of bocavirus with more consistent knowledge and comprehensive interpretations on the recombination history of primate bocavirus.
The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.
Influenza A (IAV) is a major human respiratory pathogen that contributes to a significant threat to health security, worldwide. Despite vaccinations and previous immunisations through infections, humans can still be infected with influenza several times throughout their lives. This phenomenon is attributed to the antigenic changes of hemagglutinin (HA) and neuraminidase (NA) proteins in IAV via genetic mutation and reassortment, conferring antigenic drift and antigenic shift, respectively. Numerous findings indicate that slow antigenic drift and reassortment-derived antigenic shift exhibited by IAV are key processes that allow IAVs to overcome the previously acquired host immunity, which eventually leads to the annual re-emergence of seasonal influenza and even pandemic influenza, in rare occasions. As a result, current therapeutic options hit a brick wall quickly. As IAV remains a constant threat for new outbreaks worldwide, the underlying processes of genetic changes and alternative antiviral approaches for IAV should be further explored to improve disease management. In the light of the above, this review discusses the characteristics and mechanisms of mutations and reassortments that contribute to IAV's evolution. We also discuss several alternative RNA-targeting antiviral approaches, namely the CRISPR/Cas13 systems, RNA interference (RNAi), and antisense oligonucleotides (ASO) as potential antiviral approaches against IAV.
The Coronavirus Disease 2019 (COVID-19), a pneumonic disease caused by the SARS Coronavirus 2 (SARS-CoV-2), is the 7th Coronavirus to have successfully infected and caused an outbreak in humans. Genome comparisons have shown that previous isolates, the SARS-related coronavirus (SARSr-CoV), including the SARS-CoV are closely related, yet different in disease manifestation. Several explanations were suggested for the undetermined origin of SARS-CoV-2, in particular, bats, avian and Malayan pangolins as reservoir hosts, owing to the high genetic similarity. The general morphology and structure of all these viral isolates overlap with analogous disease symptoms such as fever, dry cough, fatigue, dyspnoea and headache, very similar to the current SARS-CoV-2. Chest CT scans for SARS-CoV-2, SARS-CoV and MERS-CoV reveal pulmonary lesions, bilateral ground-glass opacities, and segmental consolidation in the lungs, a common pathological trait. With greatly overlapping similarities among the previous coronavirus, the SARS-CoV, it becomes interesting to observe marked differences in disease severity of the SARS-CoV-2 thereby imparting it the ability to rapidly transmit, exhibit greater stability, bypass innate host defences, and increasingly adapt to their new host thereby resulting in the current pandemic. The most recent B.1.1.7, B.1.351 and P.1 variants of SARS-CoV-2, highlight the fact that changes in amino acids in the Spike protein can contribute to enhanced infection and transmission efficiency. This review covers a comparative analysis of previous coronavirus outbreaks and highlights the differences and similarities among different coronaviruses, including the most recent isolates that have evolved to become easily transmissible with higher replication efficiency in humans.
The feline leukemia virus (FeLV) belongs to the Retroviridae family and Gammaretrovirus genus, and causes a variety of neoplastic and non-neoplastic diseases in domestic cats (Felis catus), such as thymic and multicentric lymphomas, myelodysplastic syndromes, acute myeloid leukemia, aplastic anemia, and immunodeficiency. The aim of the present study was to carry out the molecular characterization of FeLV-positive samples and determine the circulating viral subtype in the city of São Luís, Maranhão, Brazil, as well as identify its phylogenetic relationship and genetic diversity. The FIV Ac/FeLV Ag Test Kit (Alere™) and the commercial immunoenzymatic assay kit (Alere™) were used to detect the positive samples, which were subsequently confirmed by ELISA (ELISA - SNAP® Combo FeLV/FIV). To confirm the presence of proviral DNA, a polymerase chain reaction (PCR) was performed to amplify the target fragments of 450, 235, and 166 bp of the FeLV gag gene. For the detection of FeLV subtypes, nested PCR was performed for FeLV-A, B, and C, with amplification of 2350-, 1072-, 866-, and 1755-bp fragments for the FeLV env gene. The results obtained by nested PCR showed that the four positive samples amplified the A and B subtypes. The C subtype was not amplified. There was an AB combination but no ABC combination. Phylogenetic analysis revealed similarities (78% bootstrap) between the subtype circulating in Brazil and FeLV-AB and with the subtypes of Eastern Asia (Japan) and Southeast Asia (Malaysia), demonstrating that this subtype possesses high genetic variability and a differentiated genotype.