Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).     

TaqMan real-time PCR for detection of hepatopancreatic parvovirus from Australia

Hepatopancreatic parvovirus is an emerging disease in crustacean aquaculture. Consequently, methods of detection are needed that enable the sensitive detection and confirmation of the virus better than currently used methods such as histology and conventional polymerase chain reaction (PCR). A TaqMan based real-time PCR assay was developed for the detection of the Australian isolate of hepatopancreatic parvovirus which is only 85% similar to its nearest known relative. The TaqMan assay was developed within the capsid protein region of the genome and is optimised to detect as little as 10 copies of the targeted sequence per PCR vial. The hepatopancreatic parvovirus primers and probe were HPV140F 5′-CTA CTC CAA TGG AAA CTT CTG AGC-3′, HPV140R 5′-GTG GCG TTG GAA GGC ACT TC-3′ and HPV140probe 5′-FAM TAC CGC CGC ACC GCA GCA GC TAMRA-3′, respectively. The assay was specific for the hepatopancreatic parvovirus strain from Australian Penaeus merguiensis as it did not detect related crustacean and canine parvoviruses from Australia. In addition, the very low homology of the target sequence with published sequences from the Thai and Korean strains of hepatopancreatic parvovirus and other prawn viruses such as WSSV, suggested this assay would be specific for the Australian hepatopancreatic parvovirus isolate. Furthermore, it detected hepatopancreatic parvovirus in 22/22 wild-caught P. merguiensis clinical samples and 473/545 (87%) farmed P. merguiensis. This assay has the potential to be used for diagnostic purposes and in robotic applications, particularly for the detection and quantitation of low-grade infections.     

Molecular isolation and characterization of a novel occlusion body protein gene from Penaeus monodon nucleopolyhedrovirus

The full-length of the occlusion body (OB) protein gene of Penaeus monodon nucleopolyhedrovirus (PemoNPV) was successfully isolated. The OB gene sequence contained an open reading frame (ORF) of 1359 nucleotides encoding a protein of 452 amino acid residues with a predicted molecular mass of 50.6 kDa. A putative late promoter element, TAAG, was identified 72 nt upstream of the translation start site. The amino acid sequences of tryptic digested peptides of PemoNPV OB protein obtained from LC-MS analysis matched quite well with various regions of deduced amino acid sequences. Recombinant PemoNPV OB proteins specifically reacted with monoclonal antibodies to PemoNPV OB protein. After comparison with nucleotide database, the PemoNPV OB ORF demonstrated 67% identity to an uncharacterized ORF of a baculovirus pathogenic for Penaeus vannamei. However, comparison against protein databases revealed no significant homology to other known proteins.To our knowledge, this PemoNPV OB gene is the first isolated and characterized gene of nucleopolyhedrovirus from shrimp.     

Molecular characterisation of Banana bunchy top virus (BBTV) from Pakistan

Banana bunchy top disease is caused by a single-stranded circular DNA virus, banana bunchy top virus (BBTV), which is a member of the genus Babuvirus (family Nanoviridae). We have cloned and sequenced five components (DNA-R, DNA-S, DNA-N, DNA-M and DNA-C) of a BBTV isolate originating from Pakistan. In addition, the DNA-R and several other components of five further isolates, originating from geographically distinct sites across the banana-growing area of Sindh province, Pakistan, were cloned and sequenced. Analysis of the sequences indicates that BBTV present in Pakistan belongs to the “South Pacific” group of isolates and that the genetic diversity of the virus in the country is very low. The virus shows the highest levels of sequence identity to BBTV isolates originating from Egypt, India and Australia. The significance of these results with respect to the possible origin of the virus in Pakistan and the prospects for obtaining genetically engineered resistance to the virus are discussed.     

Molecular diagnosis of Nocardia farcinica from a cerebral abscess

Histopathology and special stains of a brain biopsy specimen from a 42-year-old man revealed numerous gram-positive bacilli arranged in branching filaments, suggesting Nocardia infection. Antibiotic therapy with trimethoprim-sulfamethoxazole markedly decreased the abscess size, and the patient improved. DNA was analyzed from formalin-fixed sections of the cerebral abscess by a 16S ribosomal DNA polymerase chain reaction assay demonstrating the presence of either Nocardia farcinica or N otitidiscaviarum. A species-specific polymerase chain reaction assay confirmed N farcinica as the etiologic agent.     

Molecular characteristics of Chinese patients with Rett syndrome

Objective

Rett syndrome (RTT) is a neurodevelopmental disorder which affects 1/10,000 girls. The aim of this study is to delineate the molecular characteristics of Rett syndrome in China based on the largest group of Chinese patients ever studied.

Methods

In all, 365 Chinese patients with Rett syndrome were recruited. Clinical information including the family reproductive history was collected through interviewing patients and their parents as well as questionnaires. MECP2, CDKL5, FOXG1 mutational analysis was performed using polymerase chain reaction (PCR), direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The parental origin of mutated MECP2 gene, the MECP2 gene mutation rate in the patients' mothers, and the X-chromosome inactivation pattern of the mothers who carry the mutation were also analyzed.

Results

Almost all of the patients were sporadic cases except one pair of twins. The pregnancy loss in probands' mothers and sex ratio of offspring in probands^' generation were available in 352 families and were comparable to the general population. Out of the 365 cases, 315 had MECP2 gene mutations and 3 had de novo CDKL5 gene mutations. No patients had FOXG1 mutation. Among the 315 cases with MECP2 mutations, 274 were typical cases and 41 were atypical cases. All the 3 cases with CDKL5 gene mutations were atypical RTT with early-onset seizures. The analysis of parental origin of mutated MECP2 gene were performed on 139 cases, 90 (64.7%) cases were informative for the study. The result showed 94.4% cases with mutations from paternal origin and 5.6% from maternal origin. Among the cases with paternal mutation, 90.6% had point mutations. C > T was the most common one, accounting for 85.7% of the point mutations. Only one normal phenotype mother (0.41%) carried the same p.R133C mutation of MECP2 gene as her daughter with mild phenotype. The different patterns of X-chromosome inactivation in the mother and the daughter may explain their different phenotypes.

Conclusion

The high rate of paternal origin of the mutated MECP2 gene may explain the high occurrence of RTT in female gender. The family cases of RTT are rare and the recurrence risk of RTT is very low in China. Only 0.41% (1/244) mothers carry the pathogenic gene. FOXG1 mutations were not found in this group of Chinese patients.     

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H) > 2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.     

Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously

Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37 kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei.     

Role of tau kinases (CDK5R1 and GSK3B) in Parkinson's disease: a study from India

Glycogen synthase kinase-3β (GSK3B) and cyclin-dependent kinase 5 (CDK5) are the 2 major protein kinases involved in abnormal phosphorylation of tau. To determine their potential role in the pathogenesis of Parkinson's disease (PD) we analyzed 2 functional single nucleotide polymorphisms (SNPs) of GSK3B (rs334558 and rs6438552) and rs735555 of CDK5 regulatory subunit 1 (CDK5R1) in 373 PD cases and 346 healthy controls of eastern India. The C,C and T,C haplotypes of GSK3B were respectively moderately associated with increased risk and protection for late onset PD (LOPD) (odds ratio [OR], 1.399; 95% confidence interval [CI], 1.069-1.829; p = 0.015, and OR, 0.436; 95% CI, 0.222-0.853; p = 0.016, respectively). Moreover, moderate to significant interaction between different loci were observed for the entire PD cohort or late onset PD only. However, among these interactions, individuals carrying the (C/C) genotype at both loci (rs6438552 and rs735555) had almost twice the risk of developing PD than those without this genotypic combination (OR, 1.871; 95% CI, 1.181-2.964; p = 0.009). Thus, synergistic effect between the 2 major tau kinases, through these SNPs, appears to determine the risk profile for PD.     

Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets

A novel coronavirus, designated as ferret enteric coronavirus (FECV), was identified in feces of domestic ferrets clinically diagnosed with epizootic catarrhal enteritis (ECE). Initially, partial sequences of the polymerase, spike, membrane protein, and nucleocapsid genes were generated using coronavirus consensus PCR assays. Subsequently, the complete sequences of the nucleocapsid gene and the last two open reading frames at the 3' terminus of the FECV genome were obtained. Phylogenetic analyses based on predicted partial amino acid sequences of the polymerase, spike, and membrane proteins, and full sequence of the nucleocapsid protein showed that FECV is genetically most closely related to group 1 coronaviruses. FECV is more similar to feline coronavirus, porcine transmissible gastroenteritis virus, and canine coronavirus than to porcine epidemic diarrhea virus and human coronavirus 229E. Molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with clinical cases of ECE.     

Molecular cloning and expression analysis of major histocompatibility complex class I,IIA and IIB genes of blunt snout bream (Megalobrama amblycephala)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class IIA and IIB genes from blunt snout bream (Megalobrama amblycephala) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC genes in M. amblycephala, tissue distribution and immune response of the MHC genes to bacterial challenge were analyzed. All the characteristic features of MHC class II chain structure could be identified in the deduced amino sequences of MHC IIA and IIB, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. The deduced amino acid sequence of the MHC IIA and IIB molecules shared from 48% to 88% and from 65% to 77% similarity with those of other teleosts, respectively. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC I and II genes were ubiquitously expressed in ten tissues, with high level in immune related tissues, including kidney, intestine, gill and spleen. Challenge of M. amblycephala with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC I, MHC IIA and IIB mRNA within 72 h after infection in gill, kidney, intestine and liver, followed by a recovery to normal level after 120 h. The changes of expression levels for MHC IIA and IIB in most tissues were significantly higher than that of MHC I in the corresponding tissues at most time points (P < 0.05). These results demonstrated the MHC genes played an important role in response to bacterial infection in M. amblycephala; however, MHC class I and II genes showed different functional activity, which need be further investigated in teleost.     

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.     

Molecular characterisation of congenital glaucoma in a consanguineous Canadian community: a step towards preventing glaucoma related blindness

Glaucoma is a leading cause of irreversible blindness in Canada. Congenital glaucoma usually manifests during the first years of life and is characterised by severe visual loss and autosomal recessive inheritance. Two disease loci, on chromosomes 1p36 and 2p21, have been associated with various forms of congenital glaucoma. A branch of a large six generation family from a consanguineous Amish community in south western Ontario was affected with congenital glaucoma and was studied by linkage and mutational analysis to identify the glaucoma related genetic defects. Linkage analysis using the MLINK component of the LINKAGE package (v 5.1) showed evidence of linkage to the 2p21 region (Zmax=3.34, θ=0, D2S1348 and D2S1346). Mutational analysis of the primary candidate gene, CYP1B1, was done by direct cycle sequencing, dideoxy fingerprinting analysis, and fragment analysis. Two different disease causing mutations in exon 3, 1410del13 and 1505G→A, both segregated with the disease phenotype. The two different combinations of these alleles appeared to result in a variable expressivity of the phenotype. The compound heterozygote appeared to have a milder phenotype when compared to the homozygotes for the 13 bp deletion. The congenital glaucoma phenotype for this large inbred Amish family is the result of mutations in CYP1B1 (2p21). The molecular information derived from this study will be used to help identify carriers of the CYP1B1 mutation in this community and optimise the management of those at risk of developing glaucoma.


Keywords: congenital glaucoma; CYP1B1; gene; genetic counselling     

Molecular evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999-2008

West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥ 2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V → A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.