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Fulltext Characterization of Plaque Variants and the Involvement of Quasi-Species in a Population of EV-A71

Mandary MB, Masomian M, Ong SK, Poh CL

Viruses, 2020 Jun 17;12(6).
PMID: 32560288 DOI: 10.3390/v12060651

Viral plaque morphologies in human cell lines are markers for growth capability and they have been used to assess the viral fitness and selection of attenuated mutants for live-attenuated vaccine development. In this study, we investigate whether the naturally occurring plaque size variation reflects the virulence of the variants of EV-A71. Variants of two different plaque sizes (big and small) from EV-A71 sub-genotype B4 strain 41 were characterized. The plaque variants displayed different in vitro growth kinetics compared to the parental wild type. The plaque variants showed specific mutations being present in each variant strain. The big plaque variants showed four mutations I97L, N104S, S246P and N282D in the VP1 while the small plaque variants showed I97T, N237T and T292A in the VP1. No other mutations were detected in the whole genome of the two variants. The variants showed stable homogenous small plaques and big plaques, respectively, when re-infected in rhabdomyosarcoma (RD) and Vero cells. The parental strain showed faster growth kinetics and had higher viral RNA copy number than both the big and small plaque variants. Homology modelling shows that both plaque variants have differences in the structure of the VP1 protein due to the presence of unique spontaneous mutations found in each plaque variant This study suggests that the EV-A71 sub-genotype B4 strain 41 has at least two variants with different plaque morphologies. These differences were likely due to the presence of spontaneous mutations that are unique to each of the plaque variants. The ability to maintain the respective plaque morphology upon passaging indicates the presence of quasi-species in the parental population.

Matched MeSH terms: Capsid Proteins/genetics
Genetic variability and evolutionary analyses of the coat protein gene of Tomato mosaic virus

Rangel EA, Alfaro-Fernández A, Font-San-Ambrosio MI, Luis-Arteaga M, Rubio L

Virus Genes, 2011 Dec;43(3):435-8.
PMID: 21881940 DOI: 10.1007/s11262-011-0651-3

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.

Matched MeSH terms: Capsid Proteins/genetics*
Molecular characterization of two distinct begomoviruses from Papaya in China

Wang X, Xie Y, Zhou X

Virus Genes, 2004 Dec;29(3):303-9.
PMID: 15550769

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.

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Genetic characterization of porcine circovirus 2 found in Malaysia

Jaganathan S, Toung OP, Yee PL, Yew TD, Yoon CP, Keong LB

Virol J, 2011;8:437.
PMID: 21914166 DOI: 10.1186/1743-422X-8-437

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.

Matched MeSH terms: Capsid Proteins/genetics*
Fulltext Improving the potency of DNA vaccine against chicken anemia virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) type-1 VP22 protein

Moeini H, Omar AR, Rahim RA, Yusoff K

Virol J, 2011;8:119.
PMID: 21401953 DOI: 10.1186/1743-422X-8-119

Studies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV).

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Genetic characterization of Enterovirus 71 strains circulating in Vietnam in 2012

Donato C, Hoi le T, Hoa NT, Hoa TM, Van Duyet L, Dieu Ngan TT, et al.

Virology, 2016 08;495:1-9.
PMID: 27148893 DOI: 10.1016/j.virol.2016.04.026

BACKGROUND: Enterovirus 71 subgenogroup C4 caused the largest outbreak of Hand, Foot and Mouth Disease (HFMD) in Vietnam during 2011-2012, resulting in over 200,000 hospitalisations and 207 fatalities.
METHODS: A total of 1917 samples with adequate volume for RT-PCR analysis were collected from patients hospitalised with HFMD throughout Vietnam and 637 were positive for EV71. VP1 gene (n=87) and complete genome (n=9) sequencing was performed. Maximum-likelihood phylogenetic analysis was performed to characterise the B5, C4 and C5 strains detected.
RESULTS: Sequence analyses revealed that the dominant subgenogroup associated with the 2012 outbreak was C4, with B5 and C5 strains representing a small proportion of these cases.
CONCLUSIONS: Numerous countries in the region including Malaysia, Taiwan and China have a large influence on strain diversity in Vietnam and understanding the transmission of EV71 throughout Southeast Asia is vital to inform preventative public health measures and vaccine development efforts.

Matched MeSH terms: Capsid Proteins/genetics
Mass mortality of hatchery-produced larvae of Asian seabass, Lates calcarifer (Bloch), associated with viral nervous necrosis in Sabah, Malaysia

Ransangan J, Manin BO

Vet Microbiol, 2010 Sep 28;145(1-2):153-7.
PMID: 20427132 DOI: 10.1016/j.vetmic.2010.03.016

Culture of Asian seabass, Lates calcarifer (Bloch) is a popular aquaculture activity in Malaysia. This fish is in high demand and fetches a good price in the local market. The seed for this fish is commercially produced by induced spawning in hatcheries. However, the seed supply is affected by frequent mass mortality of larvae aged between 15 and 60 dph. The clinical signs shown by the affected larvae include lethargy, loss of appetite, uncoordinated swimming, unusual spiral movement pattern and dark coloration. Histological examination of brain and eye of the affected specimens revealed extensive cell vacuolation in larvae aged 15-25 dph. Partial nucleotide sequence of the nervous necrosis virus coat protein gene of the affected larvae showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia and Australia. This study provides scientific evidence based on molecular technique that many episodes of mass mortality in seabass larvae in Sabah is associated with the viral nervous necrosis. Because no effective treatment has been reported for this infection, stringent biosecurity measures must be adopted for exclusion of the pathogen from the culture system.

Matched MeSH terms: Capsid Proteins/genetics
First report of the infectious spleen and kidney necrosis virus (ISKNV) infection in ornamental fishes in India

Girisha SK, Kushala KB, Nithin MS, Puneeth TG, Naveen Kumar BT, Vinay TN, et al.

Transbound Emerg Dis, 2021 Mar;68(2):964-972.
PMID: 33448668 DOI: 10.1111/tbed.13793

Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018-19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42%) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76% to 100%. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV-positive fish species, three species were reported positive to ISKNV for the first time in the world. Further, the in vitro experiment showed the growth of virus in Asian sea bass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures need to be taken to control these emerging pathogens.

Matched MeSH terms: Capsid Proteins/genetics
Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in the 1997, 2000 and 2005 outbreaks, Peninsular Malaysia

Chua KB, Chua BH, Lee CS, Chem YK, Ismail N, Kiyu A, et al.

Malays J Pathol, 2007 Dec;29(2):69-78.
PMID: 19108398

All known field isolates of enterovirus 71 (EV71) can be divided into three distinct genogroups (A, B, C) and 10 subgenogroups (A, B1-5, C1-4) based on VP1 gene sequences. We examined VP1 gene sequences of 10, 12 and 11 EV71 strains isolated in peninsular Malaysia during the outbreaks of hand, foot and mouth disease in 1997, 2000 and 2005 respectively. Four EV71 strains isolated in the hand, foot and mouth disease outbreak of 2006 in Sarawak (Malaysian Borneo) were included to describe their genetic relationship. Four subgenogroups (C1, C2, B3 and B4) of EV71 co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two subgenogroups (C1 and B4) were noted to cause the outbreak in 2000. In the 2005 outbreak, besides EV71 strains of subgenogroup C1, EV71 strains belonged to subgenogroup B5 were isolated but formed a cluster which was distinct from EV71 strains of the subgenogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to subgenogroup B5. Phylogenetic analysis of the VP1 gene sequences showed that the four Sarawak EV71 isolates belonged to the same cluster as the EV71 strains that were isolated in peninsular Malaysia as early as May 2005. The data suggested that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.

Matched MeSH terms: Capsid Proteins/genetics
RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein

Goh ZH, Mohd NAS, Tan SG, Bhassu S, Tan WS

J Gen Virol, 2014 Sep;95(Pt 9):1919-1928.
PMID: 24878641 DOI: 10.1099/vir.0.064014-0

White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

Matched MeSH terms: Capsid Proteins/genetics*
Mutagenesis of the nucleocapsid protein of Nipah virus involved in capsid assembly

Ong ST, Yusoff K, Kho CL, Abdullah JO, Tan WS

J Gen Virol, 2009 Feb;90(Pt 2):392-397.
PMID: 19141448 DOI: 10.1099/vir.0.005710-0

The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30-32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.

Matched MeSH terms: Nucleocapsid Proteins/genetics*; Capsid Proteins/genetics
Fulltext Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Tan CW, Chan YF, Sim KM, Tan EL, Poh CL

PLoS One, 2012;7(5):e34589.
PMID: 22563456 DOI: 10.1371/journal.pone.0034589

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Evolution and phylogenetic analysis of full-length VP3 genes of Eastern Mediterranean bluetongue virus isolates

Nomikou K, Dovas CI, Maan S, Anthony SJ, Samuel AR, Papanastassopoulou M, et al.

PLoS One, 2009;4(7):e6437.
PMID: 19649272 DOI: 10.1371/journal.pone.0006437

Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.

Matched MeSH terms: Capsid Proteins/genetics*
Fulltext Worldwide increased prevalence of human adenovirus type 3 (HAdV-3) respiratory infections is well correlated with heterogeneous hypervariable regions (HVRs) of hexon

Haque E, Banik U, Monwar T, Anthony L, Adhikary AK

PLoS One, 2018;13(3):e0194516.
PMID: 29590206 DOI: 10.1371/journal.pone.0194516

Human adenovirus type 3 (HAdV-3) respiratory infections occurs worldwide in both children and adults, leading to severe morbidity and mortality, particularly in the paediatric age group and especially in neonates. During HAdV infection, neutralizing antibodies are formed against the epitopes located in the hyper variable regions (HVRs) of the hexon protein. These neutralizing antibodies provide protection against reinfection by viruses of the same type. Therefore it is reasonable to speculate that variations of HAdV-3 in the HVRs could impair the immunity acquired by previous infection with a different strain with variation in its HVRs. HAdV-3 has recently become the major agent of acute respiratory infection worldwide, being responsible for 15% to 87% of all adenoviral respiratory infections. However, despite the increased prevalence of HAdV-3 as respiratory pathogen, the diversity of hexon proteins in circulating strains remains unexplored. This study was designed to explore the variation in HVRs of hexon among globally distributed strains of HAdV-3 as well as to discover possible relationship among them, thus possibly shedding light on the cause for the increased prevalence of HAdV-3. In this study, for the first time we analysed the hexon proteins of all 248 available strains of HAdV-3 from the NCBI database and compared them with those of the HAdV-3 prototype (GB stain). We found that the HVRs of HAdV-3 strains circulating worldwide were highly heterogeneous and have been mutating continuously since -their original isolation. Based on their immense heterogeneity, the strains can be categorized into 25 hexon variants (3Hv-1 to 3Hv-25), 4 of which (3Hv-1 to 3Hv-4) comprises 80% of the strains. This heterogeneity may explain why HAdV-3 has become the most prevalent HAdVs type worldwide. The heterogeneity of hexon proteins also shows that the development of a vaccine against HAdV-3 might be challenging. The data on hexon variants provided here may be useful for the future epidemiological study of HAdV-3 infection.

Matched MeSH terms: Capsid Proteins/genetics*
Fulltext Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018-2019

Amit LN, Mori D, John JL, Chin AZ, Mosiun AK, Jeffree MS, et al.

PLoS One, 2021;16(7):e0254784.
PMID: 34320003 DOI: 10.1371/journal.pone.0254784

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia's national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12-23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Phylogeography and phylogeny of Rhinoviruses collected from Severe Acute Respiratory Infection (SARI) cases over successive epidemic periods in Tunisia

Haddad-Boubaker S, Ben Hamda C, Ghedira K, Mefteh K, Bouafsoun A, Boutiba-Ben Boubaker I, et al.

PLoS One, 2021;16(11):e0259859.
PMID: 34807924 DOI: 10.1371/journal.pone.0259859

Rhinoviruses (RV) are a major cause of Severe Acute Respiratory Infection (SARI) in children, with high genotypic diversity in different regions. However, RV type diversity remains unknown in several regions of the world. In this study, the genetic variability of the frequently circulating RV types in Northern Tunisia was investigated, using phylogenetic and phylogeographic analyses with a specific focus on the most frequent RV types: RV-A101 and RV-C45. This study concerned 13 RV types frequently circulating in Northern Tunisia. They were obtained from respiratory samples collected in 271 pediatric SARI cases, between September 2015 and November 2017. A total of 37 RV VP4-VP2 sequences, selected among a total of 49 generated sequences, was compared to 359 sequences from different regions of the world. Evolutionary analysis of RV-A101 and RV-C45 showed high genetic relationship between different Tunisian strains and Malaysian strains. RV-A101 and C45 progenitor viruses' dates were estimated in 1981 and 1995, respectively. Since the early 2000s, the two types had a wide spread throughout the world. Phylogenetic analyses of other frequently circulating strains showed significant homology of Tunisian strains from the same epidemic period, in contrast with earlier strains. The genetic relatedness of RV-A101 and RV-C45 might result from an introduction of viruses from different clades followed by local dissemination rather than a local persistence of an endemic clades along seasons. International traffic may play a key role in the spread of RV-A101, RV-C45, and other RVs.

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive enterovirus A71 virulence in mice

Tee HK, Tan CW, Yogarajah T, Lee MHP, Chai HJ, Hanapi NA, et al.

PLoS Pathog, 2019 11;15(11):e1007863.
PMID: 31730673 DOI: 10.1371/journal.ppat.1007863

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.

Matched MeSH terms: Capsid Proteins/genetics
Fulltext Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?

Ho KL, Gabrielsen M, Beh PL, Kueh CL, Thong QX, Streetley J, et al.

PLoS Biol, 2018 Oct;16(10):e3000038.
PMID: 30346944 DOI: 10.1371/journal.pbio.3000038

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.

Matched MeSH terms: Capsid Proteins/genetics
Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli

Yee SF, Chu CH, Poili E, Sum MSH

J Virol Methods, 2017 02;240:69-72.
PMID: 27923590 DOI: 10.1016/j.jviromet.2016.12.001

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

Matched MeSH terms: Capsid Proteins/genetics*
Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein

Kaku Y, Park ES, Noguchi A, Inoue S, Lunt R, Malbas FF, et al.

J Virol Methods, 2019 07;269:83-87.
PMID: 30954461 DOI: 10.1016/j.jviromet.2019.03.009

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.

Matched MeSH terms: Capsid Proteins/genetics*

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