Full-length Nucleotide Sequence Research Articles

Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype.

Analysis for complete genomic sequence of HLA-B and HLA-C alleles in the Chinese Han population

In the present study, we have determined the complete genomic sequence and analysed the intron polymorphism of partial HLA-B and HLA-C alleles in the Chinese Han population. Over 3.0 kb DNA fragments of HLA-B and HLA-C loci were amplified by polymerase chain reaction from partial 5' untranslated region to 3' noncoding region respectively, and then the amplified products were sequenced. Full-length nucleotide sequences of 14 HLA-B alleles and 10 HLA-C alleles were obtained and have been submitted to GenBank and IMGT/HLA database. Two novel alleles of HLA-B*52:01:01:02 and HLA-B*59:01:01:02 were identified, and the complete genomic sequence of HLA-B*52:01:01:01 was firstly reported. Totally 157 and 167 polymorphism positions were found in the full-length genomic sequence of HLA-B and HLA-C loci respectively. Our results suggested that many single nucleotide polymorphisms existed in the exon and intron regions, and the data can provide useful information for understanding the evolution of HLA-B and HLA-C alleles.

Reliability of nucleotide sequence information of full-length flagellin A gene (flaA) and flaA short variable region (SVR) for molecular discrimination of Campylobacter lari organisms

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

Analysis and characterization of the complete genome of a member of a new species of kobuvirus associated with swine

A virus belonging to a new species in the genus Kobuvirus, family Picornaviridae, was first isolated in 2008 from apparently healthy pigs in Hungary and China. We report the complete genome sequence and the genetic organization of the novel porcine kobuvirus strain Y-1-CHI, which was identified in China. The RNA genome of strain Y-1-CHI contains 8210 nucleotides (nt) and has an organization similar to that of other picornaviruses. The full-length nucleotide sequence of Y-1-CHI was 88.62%, 58.66%, and 48.86% identical to those of S-1-HUN, U-1, and Aichi virus, respectively. No positive results were found in 454 stool samples from children with acute gastroenteritis. Dendrograms indicated that Y-1-CHI and S-1-HUN are most closely related to each other and belong to the same species. Our results suggest that members of this novel species have the typical genome characteristics of members of the genus Kobuvirus and may be distributed globally in swine.

Complete coding sequence, sequence analysis and transmembrane topology modelling of Trypanosoma brucei rhodesiense Putative Oligosaccharyl transferase (TbOST II)

The partial nucleotide sequence of putative Trypanosoma brucei rhodesiense oligosaccharyl transferase gene was previously reported. Here, we describe the determination of its full-length nucleotide sequence by Inverse PCR (IPCR), subsequent biological sequence analysis and transmembrane topology modelling. The full-length DNA sequence has an Open Reading Frame (ORF) of 2406 bp and encodes a polypeptide of 801 amino acid residues. Protein and DNA sequence analyses revealed that homologues within the genome of other kinetoplastid and various origins exist. Protein topology analysis predicted that Trypanosoma brucei rhodesiense putative oligosaccharyl transferase clone II (TbOST II) is a transmembrane protein with transmembrane helices in probably an N(cytosol)-C(cytosol) orientation. Data from the GenBank database assembly and sequence analyses in general clearly state that TbOST II is the STT3 subunit of OST in T.b. rhodesiense that necessitates further characterisation and functional studies with RNAi. TbOST II sequence had been deposited in the GenBank (accession number GU245937).

Full genomic analysis of a G8P[1] rotavirus strain isolated from an asymptomatic infant in Kenya provides evidence for an artiodactyl‐to‐human interspecies transmission event

Group A rotavirus (GAR) G8P[1] strains, found sometimes in cattle, have been reported rarely from humans. Therefore, analysis of the full genomes of human G8P[1] strains are of significance in the context of studies on interspecies transmission of rotaviruses. However, to date, only partial-length nucleotide sequences are available for the 11 genes of a single human G8P[1] strain, while the partial sequences of two other strains have been reported. The present study reports the first complete genome sequence of a human G8P[1] strain, B12, detected from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the full-length nucleotide sequences of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain B12 were assigned to G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotypes, respectively. Each of the 11 genes of strain B12 appeared to be more related to cognate genes of artiodactyl (ruminant and/or camelid) and/or artiodactyl-derived human GAR strains than those of most other rotaviruses. Strain B12 exhibited low levels of genetic relatedness to canonical human GAR strains, such as Wa and DS-1, ruling out the possibility of its origin from reassortment events between artiodactyl-like human and true human strains. These observations suggest that strain B12 might have been directly transmitted from artiodactyls to humans. Unhygienic conditions and close proximity of humans to livestock at the sampling site might have facilitated this rare event. This is the first report on a full genomic analysis of a rotavirus strain from Kenya. To our knowledge, strain B12 might be the oldest G8 strain characterized molecularly from the Africa continent.

Cloning and expression of isoenzymes of superoxide dismutase in Haematococcus pluvialis (Chlorophyceae) under oxidative stress

Increasing evidence indicates that the biosynthesis and accumulation of the keto-carotenoid astaxanthin represents a chronic (in days or weeks) molecular defense mechanism in the green alga, Haematococcus pluvialis, to protect cells from abiotic stress. However, information with regard to the acute (in minutes or hours) response of the cells to stress is scarce. In this study, two cDNAs encoding manganese superoxide dismutase (MnSOD) were cloned and their full length nucleotide sequences were obtained. Quantitative real-time RT-PCR analysis revealed a general increase in MnSOD transcriptions in response to various abiotic stressors, e.g., high light, excessive amounts of iron or sodium acetate, either singly or in combination. Meanwhile, four isoforms of MnSOD were detected by a nondenaturing polyacrylamide gel electrophoresis approach. In addition, an iron-containing superoxide dismutase (FeSOD) was detected in the cells using the same technique. Differential expressions of SOD isoenzymes under different abiotic stressors were also investigated and discussed, at both mRNA and protein levels.

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%-5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.

Canine Distemper Epizootic among Red Foxes, Italy, 2009

Canine Distemper Epizootic among Red Foxes, Italy, 2009

First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB

A hepatitis A virus (HAV) recovered in Argentina from a stool sample of a sick child in the year 2006 (HAV-Arg/06) was entirely sequenced. Phylogenetic analysis included the HAV-Arg/06 sequence in subgenotype IA, either considering the usual VP1-2A variable junction fragment or the full length nucleotide sequence. Interestingly, a recombination event with subgenotype IB, involving a portion of the 2C–3A nonstructural proteins coding region (nucleotides 4961–5140) was detected using specific software. Only subgenotype IA strains have been detected in Argentina or Uruguay, whereas subgenotype IA and IB strains have been reported to circulate in Brazil. Although recombination has been given an important role in the evolution of picornaviruses, there have been only a few reports of its involvement in the evolution of HAV, probably due to the limited number of complete HAV sequences available. This study constitutes the first report of a full-length HAV sequence in Argentina and the third in South America, after the sequence of the IA isolate HAV5 from Uruguay and the IB isolate HAF-203 from Brazil. The availability of new sequence data covering the complete HAV genome will help establish a more consistent genetic relatedness among HAV isolates and the role of recombination in its evolution.

Alterations of the pilQ gene in Neisseria gonorrhoeae are unlikely contributors to decreased susceptibility to ceftriaxone and cefixime in clinical gonococcal strains

Gonorrhoea remains a global public health problem and the treatment options are diminishing through the emergence of gonococci resistant to most antimicrobials. Previous in vitro studies have indicated a role for Neisseria gonorrhoeae pilQ alterations in conferring resistance to antimicrobials, including penicillin. In this study, we investigated whether pilQ polymorphisms were associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs) in clinical gonococcal strains. Full-length pilQ nucleotide and PilQ amino acid sequences from geographically and temporally diverse gonococcal clinical isolates (n = 63), including the 2008 WHO reference strains, representing a range of ceftriaxone and cefixime MICs (≤0.008-0.25 and <0.016-0.5 mg/L, respectively) and 38 N. gonorrhoeae multiantigen sequence types, were examined. Previously described alterations associated with decreased ESC susceptibility (mosaic penA, mtrR and penB alterations) were also examined. Fifteen different pilQ nucleotide sequence types and nine different PilQ amino acid sequence types were observed, with two PilQ types accounting for 53 (84%) of the isolates. Independent of other genetic resistance determinants (penA mosaic, mtrR promoter deletion and penB), only one pilQ alteration, a D526N substitution, provided a statistically significant association with ceftriaxone (P < 0.01) and cefixime (P < 0.05) MICs. However, the two isolates exhibiting D526N lacked all three previously described alterations associated with decreased ESC susceptibility, thereby providing an alternative basis for the low MICs (≤0.008 mg/L) observed for these strains. The previously described E666K (pilQ2) and F595L (pilQ1) mutations were absent in all 63 isolates. pilQ polymorphisms are unlikely contributors to decreased susceptibility to ESCs in clinical gonococcal strains.

Comparisons of the genetic and neutralization properties of HIV-1 subtype C and CRF07/08_BC env molecular clones isolated from infections in China

Subtype C is the most prevalent HIV-1 subtype globally and a common circulating recombinant form virus, CRF_BC, is predominant in China. It is unclear whether subtype C-based vaccines are capable of neutralizing CRF_BC viruses or can be evaluated using a neutralization assay based on CRF_BC pseudoviruses. Phylogenetic analysis of full-length gp160 nucleotide sequences showed that 37 functional env clones formed two unique clusters. The 30 clones belonged to 07_BC and seven clones 08_BC. Increased length in gp120 was found in 07_BC clones when compared to subtype C and 08_BC clones. The subtype BC clones had more PNLG sites on gp160, and 08_BC had more PNLG sites in the V5 region than subtype C. However, the number of PNLG sites in the C4 region of 07_BC was less than subtype C. The neutralization properties of Env-pseudovirus were analyzed against HIV-positive plasma panels belonging to three subtypes: BC, B and AE. Subtype BC pseudoviruses had higher sensitivities to all the three plasma panels than subtype C viruses. CRF07_BC clones were the most sensitive to subtype BC antibodies, regardless of whether the CRF07_plasma panel or CRF08_BC were used. Heatmap analyses of inhibition ratios showed that clones in this study were antigenically diverse despite being genetic similarity. Thus, genetic and neutralization properties of HIV CRF07/08_BC Env isolates predominating in China show different properties than those of subtype C. These findings are likely to provide useful information for the design and evaluation of HIV-1 neutralizing antibody-based vaccine development in China.

Expression of Ethylene Biosynthesis and Signaling Genes during Differential Abscission Responses of Sweet Orange Leaves and Mature Fruit

When applying abscission agents to tree fruit to facilitate harvest, it is desirable to loosen fruit and not leaves or other organs, but mechanisms controlling leaf and fruit drop are not fully understood. The effect of 450 μL·L−1 ethephon (ethylene-releasing agent) alone or in combination with 1-methylcyclopropene [1-MCP (ethylene perception inhibitor)] on leaf and mature fruit abscission of ‘Valencia’ sweet orange (Citrus sinensis) was studied. Leaf abscission increased and fruit detachment force (FDF) decreased significantly especially 4 days after ethephon treatment. Leaf drop rose to over 80% 7 days after application, whereas FDF was only 30% less than untreated control fruit. When 1-MCP was combined with ethephon and applied to ‘Valencia’ sweet orange canopies, leaf abscission was greatly reduced, but reduction in FDF proceeded unabated. We hypothesized that differential response of ‘Valencia’ sweet orange fruit and leaves to 1-MCP was correlated with expression of ethylene biosynthetic and signaling genes and their downstream action. Partial or full-length nucleotide sequences were obtained for ‘Valencia’ sweet orange homologs of 1-amino-cyclopropane-1-carboxylate synthase-1 (CsACS1), 2 (CsACS2), 1-amino-cyclopropane-1-carboxylate oxidase (CsACO), ethylene response sensor-1 (CsERS1), ethylene response-1 (CsETR1), 2 (CsETR2), 3 (CsETR3), constitutive triple response-1 (CsCTR1), ethylene insensitive-2 (CsEIN2), and ethylene insensitive 3-like-1 (CsEIL1) and 2 (CsEIL2). Ethephon application increased expression of biosynthesis genes CsACS1 and CsACO and receptors CsERS1 and CsETR2 in the abscission zones of leaves and mature fruit. Ethephon-induced increase in gene expression was completely suppressed by 1-MCP application in all but CsACS1 and CsACO in fruit abscission zones. Although gene expression was suppressed initially, CsACS1 and CsACO expression in fruit abscission zones treated with 1-MCP in the presence or absence of ethephon increased over the 7-day measurement period, suggesting that CsACS1 and CsACO expression were negatively regulated by basal ethylene production in this tissue. However, 1-MCP treatment alone did not loosen fruit, indicating that CsACS1 and CsACO played minor roles in fruit abscission. To determine if the difference in ethylene sensitivity was the basis of differential response to ethylene within the same organ, potted ‘Valencia’ sweet orange plants were treated with ethylene, and rates of blade and petiole drop and detachment forces at the laminar and petiolar abscission zones were studied. Although leaf blades abscised earlier than petioles, the force of detachment was similar, indicating no differences in ethylene sensitivity. Overall, the most significant difference between fruit and leaf abscission zones was seen in the expression of CsACS1 and CsACO genes, but the expression pattern was poorly correlated with abscission.

Molecular analysis of lipoxygenase ( LOX) genes in common wheat and phylogenetic investigation of LOX proteins from model and crop plants

Lipoxygenase (LOX) proteins constitute an important class of lipid-hydrolyzing enzymes in cellular organisms. Here, we report a molecular analysis of LOX genes in common wheat ( Triticum aestivum L.) and a more comprehensive phylogenetic investigation of the LOX proteins from higher plants. The full-length nucleotide sequences of two LOX genes ( TaLOX1 and TaLOX2) from common wheat were isolated. TaLOX1 and TaLOX2 were assigned to the short arm of chromosome 4D and the long arm of chromosome 5D, respectively. TaLOX1 and TaLOX2 were both expressed in the developing grains of two common wheat varieties, indicating that they may contribute to the total LOX activity in common wheat seeds. A more extensive phylogenetic analysis conducted with 143 unique LOXs from model and crop plants revealed that plant LOXs could be classified into two subfamilies, which correlated with the absence (subfamily I) or presence (subfamily II) of a predicted chloroplast targeting peptide in the deduced LOX proteins. Our work provides new information on the LOX genes in common wheat and the phylogenetic relationships of higher plant LOXs, which may aid further functional and evolutionary studies of plant LOXs.

Tomato yellow leaf curl viruses: ménage à trois between the virus complex, the plant and the whitefly vector.

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. The tomato yellow leaf curl virus-like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full-length DNA-A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petuniaxhybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.

Identification of a Novel Second-Generation Circulating Recombinant Form (CRF48_01B) in Malaysia: A Descendant of the Previously Identified CRF33_01B

A molecular epidemiological investigation conducted among injecting drug users in eastern Peninsular Malaysia in 2007 identified a cluster of sequences (n = 3) located outside any known HIV-1 genotype. Analyses of near full-length nucleotide sequences of these strains from individuals with no recognizable linkage revealed that they have an identical subtype structure comprised of CRF01_AE and subtype B', distinct from any known circulating recombinant forms (CRFs). This novel CRF, designated CRF48_01B, is closely related to CRF33_01B, previously identified in Kuala Lumpur. Phylogenetic analysis of multiple CRF48_01B genome regions showed that CRF48_01B forms a monophyletic cluster within CRF33_01B, suggesting that this new recombinant is very likely a descendant of CRF33_01B. CRF48_01B thus represents one of the first examples of a "second-generation" CRF, generated by additional crossover with pre-existing CRFs. Corroborating these results, Bayesian molecular clock analyses indicated that CRF48_01B emerged in approximately 2001, approximately approximately 8 years after the emergence of CRF33_01B.

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478 bp with a G + C content of 37.2%. Ab initio annotation indicated four putative open reading frames ( orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter ‘wild’ isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria.

Phylogenetic analysis and prokaryotic expression of NMAAP1 derived from BCG-activated murine macrophages

BCG-activated macrophages exerted anti-tumor activities. Cell surface molecules play an important role in mediating endocytosis by macrophages. In the previous study, we identified a group of 454 membrane proteins specifically expressed on BCG-activated mouse macrophages, including a protein named NMAAP1 (novel macrophage activated associated protein). In this study, we aligned the full-length nucleotide sequences of NMAAP1 and its homologous sequences to construct its phylogenetic tree, and cloned the NMAAP1 cDNA from BCG-activated macrophages to generated NMAAP1 fusion protein in Escherichia coli. Purified the fusion protein were applied for generation of polyclonal antibodies. Western-blotting detection showed that the polyclonal antibodies have high specificities to recognize target protein.

Full genomic analysis of a porcine–bovine reassortant G4P[6] rotavirus strain R479 isolated from an infant in China

During the 2004 surveillance of rotaviruses in Wuhan, China, a G4P[6] rotavirus strain R479 was isolated from a stool specimen collected from a 2-year-old child with diarrhea. The strain R479 had an uncommon subgroup specificity I + II, and analysis of the VP6 gene suggested that it was related to porcine rotaviruses. In the present study, full-length nucleotide sequences of all the RNA segments of R479 were determined and analyzed phylogenetically to identify the origin of individual RNA segments. According to the rotavirus genotyping system based on 11 RNA segments, the genotype of R479 was expressed as G4-P[6]-I5-R1-C1-M1-A1-N1-T7-E1-H1. This genotype includes the porcine-like VP6 genotype (I5) and bovine-like NSP3 genotype (T7). Phylogenetic analysis revealed that R479 genes encoding VP1, VP2, VP3, VP6, VP7, VP8*, NSP1, NSP4, and NSP5 were more closely related to those of porcine rotaviruses than human or other animal rotaviruses. In contrast, it was remarkable that the NSP3 gene of R479 was genetically closely related to only a bovine rotavirus strain UK. The NSP2 gene of R479 was also unique and clustered with only the G5P[8] human strain IAL28 and G3P[24] simian strain TUCH. These results suggested that R479 may be a reassortant virus having the NSP3 gene from a bovine rotavirus in the genetic background of a porcine rotavirus, with an NSP2 gene related to the porcine-human reassortant strain IAL28. To our knowledge, R479 is the first porcine-bovine reassortant rotavirus isolated from a human.

Multiple Introduction of Asian H5N1 Avian Influenza Virus in Croatia by Wild Birds During 2005–2006 and Isolation of the Virus from Apparently Healthy Black-Headed Gulls (Larus ridibundus)

This study describes the introduction and spread of avian influenza A (H5N1) subtype in Croatia. Seventeen isolates were identified during the period from October 2005 to March 2006, all originating from wild birds. The full-length nucleotide sequence analysis of the hemagglutinin (HA) gene of seven representative isolates revealed that three distinct genetic strains involved in the outbreaks, implicating at least three independent introductions of the virus into Croatia during a relatively short period of time. All three genetic strains belonged to clade 2.2 (Qinghai-like viruses) and each strain displayed significant similarity to concurrent H5N1 viruses from other European countries. The dominant strain of the virus was present in all four affected areas and in all three bird species (mute swan, mallard, and black-headed gull), indicating cross-species transmission of the virus. Two other genetic strains were found, together with the dominant strain, only in a marsh at the Adriatic coast during late February and early March 2006, which could be associated with frozen water surfaces in the continental part of Croatia as well as in Eastern Europe in early 2006 and the movement of birds toward warmer areas. This is also the first isolation of highly pathogenic avian influenza virus of H5N1 subtype from apparently healthy black-headed gulls.