Partial VP1 Sequences Research Articles

Co-existence of multiple strains of two novel porcine bocaviruses in the same pig, a previously undescribed phenomenon in members of the family Parvoviridae, and evidence for inter- and intra-host genetic diversity and recombination

Despite the recent discovery of novel bocaviruses from porcine samples, their genetic evolution and diversity are poorly understood. This study reports the identification and complete genome characterization of two novel parvoviruses, porcine bocavirus 3 (PBoV3) and porcine bocavirus 4 (PBoV4), from various porcine tissues/samples, displaying marked intra- and inter-host genetic diversity, with recombination events. Bocaviruses were detected by PCR among 16.5 % (55/333) of porcine samples (lymph nodes, serum, nasopharyngeal and faecal samples) from healthy, sick or deceased pigs from farms and a slaughterhouse in Hong Kong. As marked nucleotide polymorphisms were observed in the partial VP1 sequences, complete VP1 genes from one nasopharyngeal and three faecal specimens were cloned and sequenced, which suggested the presence of two different bocaviruses and demonstrated significant intra- and inter-host genetic diversity. Complete genome sequences revealed the presence of two bocaviruses, PBoV3 and PBoV4, in a faecal and nasopharyngeal specimen, respectively, with two genotypes, PBoV4-1 and PBoV4-2, in the latter. Their genomes encoded three ORFs, characteristic of bocaviruses. Phylogenetic analysis showed that they were distantly related to other bocaviruses, forming a distinct cluster within the genus. Recombination analysis showed possible recombination events among VP1 sequences of PBoV4 strains from a faecal specimen, with two breakpoints identified (with a 68 and 71 bp region), suggesting that different strains/variants within the same host could have arisen from recombination. This is the first report describing marked sequence diversity and the co-existence of two viruses of the family Parvoviridae within the same host, which may have originated from and, in turn, facilitated recombination.

Saffold Cardioviruses in Children with Diarrhea, Thailand

To the Editor: Cardioviruses currently consist of at least 3 viruses: Theiler murine encephalomyocarditis virus, encephalomyocarditis virus, and Saffold virus (SAFV) (1–4). Saffold cardiovirus in the family Picornaviridae was isolated and identified from fecal specimens of a child with fever of unknown origin in the United States (3). Several reports have documented the presence of SAFV in fecal samples and respiratory secretions (5–10). However, it is not clear whether SAFV is associated with any disease, including gastroenteritis in humans, and epidemiologic data for SAFV are limited. We report an epidemiologic survey of SAFV in children hospitalized with diarrhea in Chiang Mai, Thailand. A total of 150 fecal specimens were obtained from children hospitalized with acute gastroenteritis in Chiang Mai during January–December 2007. Patient ages ranged from >1 to 5 years. SAFV in fecal specimens was detected by using a nested PCR and primers specific for the virus 5′ untranslated region (7). A negative control was also included to monitor any contamination that might have occurred during the PCR. SAFVs detected were further analyzed by amplification of the viral protein (VP) 1 gene (6,9,10) and direct sequencing of the VP1 PCR amplicon by using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). VP1 sequence was compared with VP1 sequences of reference strains available in the National Center for Biotechnology Information (Bethesda, MD, USA). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA4 (www.megasoftware.net). Nucleotide sequences of SAFV strains described were deposited in GenBank under accession nos. {type:entrez-nucleotide-range,attrs:{text:HQ668170-HQ668173,start_term:HQ668170,end_term:HQ668173,start_term_id:335347130,end_term_id:335347136}}HQ668170-HQ668173. Four (2.7%) of 150 specimens were positive for SAFV (CMH023/2007, CMH038/2007, CMH045/2007, and CMH143/2007). Two of these specimens (CMH023/2007 and CMH038/2007) were obtained in February 2007, one (CMH045/2007) in March 2007, and 1 (CMH143/2007) in November 2007. Co-infections with other viruses were detected in all 4 samples. Two specimens (CMH023/2007and CMH045/2007), were co-infected with noroviruses GII/16 and GII/4 genotypes, respectively. One SAFV-positive sample (CMH038/2007) was co-infected with a group A rotavirus G1P[8] genotype, and another (CMH143/2007) was co-infected with human parechovirus. All SAFV-positive specimens were further amplified for the VP1 gene to determine their phylogenetic lineages and genetic relationships with other SAFV reference strains. When we used 3 sets of primers used in other studies (6,9,10) for amplification of the VP1 gene, this gene was amplified only by the primer set reported by Itagaki et al. (10). Analysis of partial VP1 sequences (369 nt) of 4 SAFV strains showed that strains CMH023/2007 and CMH143/2007 were highly conserved (nt sequence identities >97%). These 2 SAFV strains were most closely related to the prototype strain of SAFV1 ({type:entrez-nucleotide,attrs:{text:EF165067,term_id:172050184,term_text:EF165067}}EF165067) isolated in the United States (nt sequence identity range 87.6%–88.9%) and SAFV strains from China (LZ50419, BCH895, GL311, and GL377) (Figure). In addition, the other 2 SAFVs identified in the present study (CMH038/2007 and CMH045/2007) were identical to each other and closely related to SAFV2 strains from China (BCHU79, BCHU353) and Finland (Finland 2008, FIN08–13B) (nt sequence identity range 94.8%–95.6%). Phylogenetic analysis showed that CMH038/2007 and CMH045/2007 were clustered within the SAFV2 lineage (Figure). Figure Phylogenetic analysis of the partial nucleotide sequence (369 nt) encoding the viral protein 1 gene of Saffold virus (SAFV) isolated in this study and other reference strains. The tree was generated by using the neighbor-joining method and MEGA4 (www.megasoftware.net ... The 4 strains of SAFV were isolated from children with acute gastroenteritis who were co-infected with other viral pathogens (norovirus, group A rotavirus, and human parechovirus). Therefore, we could not determine whether SAFVs identified in this study were associated with acute gastroenteritis. The detection rate for SAFV in children with acute gastroenteritis (2.7%) in our study was consistent with that in a study in Beijing, People’s Republic of China (3.2%) (9). Phylogenetic analysis of the VP1 region demonstrated that 2 SAFV lineages (SAFV1 and SAFV2) were circulating in Chiang Mai, Thailand. Further extensive epidemiologic surveillance of SAFV in other areas may provide a better understanding of the distribution, heterogeneity, and association of SAFV with enteric diseases in humans.

Characterization of a novel porcine enterovirus in domestic pig in Hungary

Porcine enteroviruses (PEVs) of genus Enterovirus are small, non-enveloped viruses with single-stranded, positive sense genomic RNA, belonging to the family Picornaviridae. The discovery of two distinct serotypes (PEV9 and 10) was first reported in 1979. Despite the sporadic detection and partial genome sequences of these viruses our knowledge about the prevalence and molecular epidemiology of PEV types in domestic pigs is very deficient. In this study, we identified a novel PEV from fecal samples of clinically healthy pigs ( Sus scrofa domestica) in Hungary by RT-PCR using human enterovirus generic primer pairs for 5′UTR region, with subsequent partial VP1 and complete genome sequencing and phylogenetic analysis. Among 45 fecal and blood sample pairs collected at the same farm from domestic pigs divided into three age groups (10 days, 4 weeks, and 3 months of age, N = 15 each group) six (40%) of the 15 fecal samples of 10-day-old pigs were enterovirus-positive. PEV was not detected in serum samples. Sequence- and phylogenetic analysis of the complete genome of swine/K23/2008/HUN (HQ702854) show relationship to PEV strains but it is separated from the PEV9 and 10, especially in structural regions. Swine/K23/2008/HUN has average of 77 and 75% amino acid identity in the P1 region, and only 61% in VP1 region to PEV9 and 10, respectively. The partial VP1 sequences of the Hungarian PEV strains show 99% nucleotide identity compared to each other. PEVs could be capable of at least local endemic spread among newborn piglets and cause no clinical symptoms or viraemia. Sequence data indicates that the Hungarian PEV strain belongs to a novel PEV. To clarify the taxonomic confusion related to PEV – as a consequence of recent extensive taxonomic changes among porcine enteric picornaviruses – we propose that PEV9 and PEV10 should be reclassified as PEV1 and PEV2. In this classification swine/K23/2008/HUN represents PEV3.

Coxsackievirus B3‐associated aseptic meningitis: An emerging infection in Hong Kong

Enterovirus (EV) infection is a common disease of childhood and associated not uncommonly with aseptic meningitis. In the summer of 2008, laboratory surveillance has detected increased number of coxsackievirus B3 (CVB3) associated aseptic meningitis in Hong Kong, constituting 11.6% of those infected. This study analyzed the epidemiology, circulating pattern, and clinical presentations of CVB3 in Hong Kong over the last 10 years with reference to the circulation of EV in the locality. Enteroviruses (EV) were isolated from respiratory, cerebrospinal fluid (CSF), stool, and vesicular samples using human rhabdomyosarcoma, human laryngeal carcinoma (HEp2-C), human lung fibroblast (MRC-5), and African green monkey kidney (Vero) cell lines. Virus isolates were identified and characterized by indirect immunofluorescence (IF) using monoclonal antibodies (mAB), neutralization test as well as partial VP1 sequencing. Different from previous years, IF test result showed that majority of the isolates from 2008 were untypeable by the mAB suggesting antigenic change. Sequence analysis revealed that these isolates were clustered with recent isolate from Fuyang, China. Review of data from 1999 to 2008 showed increased activity of CVB3 in the years 2005 and 2008, and isolates in these 2 years displayed an amino acid change from threonine to alanine at codon 277 of the VP1 gene, which may be associated with central nervous system (CNS) disease.

Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland

Background A nationwide outbreak of hand, foot and mouth disease (HFMD) occurred in Finland in autumn 2008. The outbreak was untypical since a considerable number of clinically diagnosed patients were adults. Furthermore, many of the patients suffered from onychomadesis several weeks after the acute phase of HFMD. Objectives Detection, identification and phylogenetic analysis of human enteroviruses (HEV) that caused the outbreak. Study design A total of 420 clinical specimens were obtained from 317 HFMD cases all over the country. The presence of HEV in the specimens was analysed by virus isolation and/or direct real-time RT-PCR; selected HEV strains were further typed by molecular methods. The genetic similarities of HEV strains were assessed by phylogenetic analyses on partial VP1 sequences. Results HEV were detected in 212 HFMD cases, including both children and adults, throughout Finland. Two HEV types, coxsackieviruses A6 (CV-A6) and A10 (CV-A10), were identified as the causative agents of the outbreak. One genetic variant of CV-A6 predominated, but, additionally, three other genetically distinct CV-A6 strains were found. All CV-A10 strains segregated into one genetic cluster distinct from previously reported CV-A10 sequences. Conclusions The Finnish 2008 HFMD outbreak was caused by two infrequently detected, co-circulating, coxsackie A viruses. Our data suggest endemic circulation of both CV-A types in Northern Europe and that the outbreak was due to the emergence of new genetic variants of these viruses.

Identification and phylogenic analysis of Coxsackie-virus B5 during an outbreak of aseptic meningitis in Shandong

To identify the pathogen that caused an outbreak of aseptic meningitis in Shandong province in 2005. Phylogenic analysis was carried out on Coxsackie-virus B5 (CVB5) which was isolated during this outbreak. 78 stool and 58 cerebrospinal fluids (CSF) specimens were collected from some inpatients during this outbreak. Virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) was then performed. Phylo-genetic trees based on entire and partial VP1 sequences were constructed among CVB5 isolates and others published in GenBank. The isolation rates of stool and CSF specimens were 38.5% (30/78) and 48.3% (28/58) respectively. Among the results of serotype identification and molecular typing of 58 positive isolates, 54 were identified as CVB5, 2 as ECHO24, 1 as CVB3 and 1 as CVA9. Results from viral investigation showed that CVB5 was the main pathogen causing this outbreak. Data from homological comparisons indicated that Shandong strains had the highest nucleotide acid identity with the Zhejiang/12/02 strain (97.5% - 97.8%), and lower identity (78.3% - 78.6%) with the prototype strain (Faulkner strain). Phylogenic tree in VP1 region showed that CVB5 could be separated into four genotypes. Isolates of this outbreak belonged to genotype D. CVB5 was the major etiological agent correlated with this outbreak. The shift of predominant genotype might serve as one of the causes that associated with the outbreaks of aseptic meningitis.

Molecular and Epidemiological Characterization of Enteroviruses Isolated in Chungnam, Korea from 2005 to 2006

Enteroviruses were identified and characterized from patients with aseptic meningitis and other enterovirusrelated diseases in Chungnam, Korea from 2005 to 2006. Enteroviruses were isolated from 79 of 519 cases (15.2%) in 2005, and 37 of 386 cases (9.6%) in 2006. Based on partial VP1 sequencing, a total of 116 enterovirus isolates were resolved into 13 types. Prevalent among the Chungnam isolates were echovirus 18 and coxsackievirus B5 in 2005, and echoviruses 5 and 25 in 2006. This is the first time echoviruses 5 and 18 have been identified in Korea since enterovirus surveillance began there in 1993. The temporal distribution of enterovirus epidemics in Chungnam showed a remarkable seasonal pattern, with cases occurring during most of the three months of the summer from June to August. The highest rate of enterovirus-positive cases occurred in patients less than 1 year of age. The ratio of male to female enterovirus-positive patients was approximately 1.8:1. Comparison of the VP1 amino acid sequences of the 15 coxsackievirus B5 isolates with reference strains revealed that all Chungnam isolates are substituted at positions 23 (V23I), 19 (S19G), 75 (Y75F), and 95 (N95S). Upon comparing the nine ECV5 isolates with foreign strains, it was found that only the Chungnam isolates, with the exception of Kor06-ECV5-239cn, have P at position 153 and F at position 146. The three ECV9 isolates from 2006 show alterations at amino acids 36, 148, and 154 outside of the BC-loop and at position 84 in the BC-loop, whereas the seven isolates from 2005 and the other ECV9 strains in the database only show the alteration at position 84 (D, I, N, S). The five ECV25 isolates have an S residue at position 134, whereas most of the foreign strains have an N residue.

Resolving ambiguities in genetic typing of human enterovirus species C clinical isolates and identification of enterovirus 96, 99 and 102

Molecular methods, based on sequencing the region encoding the VP1 major capsid protein, have recently become the gold standard for enterovirus typing. In the most commonly used scheme, sequences more than 75% identical (>85% amino acid identity) in complete or partial VP1 sequence are considered to represent the same type. However, as sequence data have accumulated, it has become clear that the '75%/85% rule' may not be universally applicable. To address this issue, we have determined nucleotide sequences for the complete P1 capsid region of a collection of 53 isolates from the species Human enterovirus C (HEV-C), comparing them with each other and with those of 20 reference strains. Pairwise identities, similarity plots and phylogenetic reconstructions identified three potential new enterovirus types, EV96, EV99 and EV102. When pairwise sequence comparisons were considered in aggregate, there was overlap in percentage identity between comparisons of homotypic strains and heterotypic strains. In particular, the differences between coxsackievirus (CV) A13 and CVA17, CVA24 and EV99, and CVA20 and EV102 were difficult to discern, largely because of intratypic sequence diversity. Closer inspection revealed the minimum intratypic values and maximum intratypic values varied by type, suggesting that the rules were at least consistent within a type. By plotting VP1 amino acid identity vs nucleotide identity for each sequence pair and considering each type separately, members of each type were fully resolved from those of other types. This study suggests that a more stringent value of 88% VP1 amino acid identity is more appropriate for routine typing and that other criteria may need to be applied, on a case by case basis, where lower values are seen.

Multiple outbreaks of acute hemorrhagic conjunctivitis due to a variant of coxsackievirus A24: Guangdong, China, 2007

Acute hemorrhagic conjunctivitis (AHC) is usually caused by enterovirus 70, coxsackievirus A24(CA24v) and adenoviruses. Several outbreaks of AHC caused by a CA24v have occurred since it was imported into China in 1971. Multiple outbreaks of AHC reappeared in 10 cities of Guangdong during June to November in 2007. The epidemic began in the June, and spread extensively, with a peak in the September. A total of 31,659 cases were reported to center for disease control and prevention of Guangdong, it was estimated that the number of actual AHC was >200 thousands. Forty conjunctival swab specimens were collected from the cases diagnosed clinically with AHC. (RT)-PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by 16 isolates. We demonstrated the most likely etiological agent for the multiple outbreaks was a variant of coxsackievirus A24 by molecular typing using a partial VP1 sequence. Sequence comparison and phylogenetic analyses of the VP1 and 3Cpro gene regions were performed by Neighbor-joining method, the strains from different outbreaks and different geographical areas within Guangdong had no sequence divergence in 2007. The representative isolates from mainland of China including Hangzhou, Ningbo, Beijing, Yunnan, Liaoning, and Henan were analyzed in this study. Phylogenetic analysis revealed theses isolates were located in different clusters, a close phylogenetic and chronological relationship with Singaporean, South Korean and Thailand isolates had been observed. This confirms CA24v circulated in China's mainland has not evolved independently, but co-evolved with the isolates of Southeast Asia.

Eight years of experience with molecular identification of human enteroviruses.

We have successfully typed 1,121 human enterovirus (HEV) isolates during the last 8 years by adapting partial VP1 sequencing to routine identification of HEV isolated from diverse clinical and environmental specimens. The isolates include 48 of the 59 traditional nonpoliovirus HEV serotypes and members of 8 newly discovered types, which would have remained untypeable by neutralization using the conventional cross-sectional pools of antisera.

Mosaic genome structure of echovirus type 30 that circulated in Taiwan in 2001

An echovirus type 30 (E30) outbreak occurred in Taiwan in 2001. In this study, one 1998 and nineteen 2001 enterovirus isolates from cerebrospinal fluid (CSF) of children with meningitis were genetically analyzed. Although negative results were obtained using the E30-specific monoclocal antibody in an immunofluorescent assay (IFA) test of all 20 isolates, molecular typing by partial VP1 sequences and subsequent neutralization test identified them as E30. Among those, seven of them were misidentified as echovirus type 4 (E4) when E4-specific monoclonal antibody was used. Complete genome sequences of one E30 isolate (TW-2513) that were IFA-positive to E4 and another (TW-3182) that was IFA-negative to both E30 and E4 were determined and analyzed. The overall percentage nucleotide identity in the structural coding region (P1) between these two isolates is 98.4, while those in the nonstructural regions P2 and P3 are only 83.2 and 84.4, respectively, indicating that the two 2001 Taiwanese E30 strains were probably recombinant. Recombination analysis of these two E30 genomes revealed that their genome structures are mosaic, which might have been formed gradually and frequently over time.

Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Alpha9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002.

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.

RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses.

RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5'-nontranslated region (5'-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, approximately 320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5'-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.

Typing of human enteroviruses by partial sequencing of VP1.

Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an "unknown" may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.