Capsid Region Research Articles

Mechanism of Host Cell MAPK/ERK-2 Incorporation into Lentivirus Particles: Characterization of the Interaction between MAPK/ERK-2 and Proline-Rich-Domain Containing Capsid Region of Structural Protein Gag

The characteristic event that follows infection of a cell by retroviruses Including human immunodeficiency virus (HIV)/ simian immunodeficiency virus (SIV) is the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Nuclear transport of newly synthesized viral DNA requires phosphorylation of proteins in the reverse transcription complex by virion-associated cellular kinases. Recently, we demonstrated that disruption of cellular mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 2 (ERK-2) incorporation into SIV virions inhibits virus replication in nonproliferating target cells, indicating that MAPK/ERK-2 plays an important role in HIV /SIV replication. The mechanism of incorporation of MAPK/ERK-2 into virus particles is not defined. In this regard, we hypothesized that a likely interaction of MAPK/ERK-2 with Gag p55 may enable its packaging into virus particles. In the present investigation, we provided evidence for the first time that MAPK/ERK-2 interacts with the structural Gag polyprotein p55 using a combination of mutagenesis and protein–protein interaction analysis. We further show that MAPK/ERK-2 interacts specifically with the poly-proline motif present in the capsid region of Gag p55. Utilizing virus-like particles directed by Gag, we have shown that the exchange of conserved proline residues within capsid of Gag p55 resulted in impaired incorporation of MAPK/ERK-2. In addition, the deletion of a domain comprising amino acids 201 to 255 within host cell MAPK/ERK-2 abrogates its interaction with Gag p55. The relevance of the poly-proline motif is further evident by its conservation in diverse retroviruses, as noted from the sequence analysis and structural modeling studies of predicted amino acid sequences of the corresponding Gag proteins. Collectively, these data suggest that the interaction of MAPK/ERK-2 with Gag polyprotein results in its incorporation into virus particles and may be essential for retroviral replication.

부산지역에서 분리된 norovirus 유전자형 연구

2008년부터 2010년까지 최근 3년 동안 부산지역에서 산발적으로 발생한 급성 위장관염 환자를 대상으로 유전자를 검사한 결과 4,101건 중 426건(10.4%)에서 노로바이러스를 확인하였다. 연도별 검출현황은 2008년에 14.7%(222/1,506), 2009년에 6.9%(95/1,384), 2010년에 9.0%(109/1,211)로 나타났다. 월별 분석 결과는 2008년에는 3월에 35.7%(50/140)로 가장 높은 검출율을 보였고, 2009년 역시 3월에 21.9%(23/105)로 높게 나타났으며, 2010년에는 1월에 23.8%(29/122)로 높은 검출율을 보여 겨울절기에 노로바이러스가 유행하는 것을 알 수 있었다. 반면 매해 7-8월 여름절기에는 노로바이러스가 거의 분리되지 않았다. 연령별 로는 1세 영아군과 13-19세 중등학생군에서 각각 20.9%로 가장 높은 검출율을 나타내었으며, 2-6세 소아군에서 17.5%, 20-29세군에서 13.4%, 7-12세 초등학생군에서 12.7%, 30-39세군에서 9.1%, 0세 신생아군에서 8.7%, 50-59세군에서 7.2%, 60-69세군과 70세이상군에서 각각 6.7%, 40-49세 4.5%로 확인되었다. 노로바이러스 양성 검체 340건에서 유전자형을 분석한 결과 GI군 7종류, GII군 13종류로 총 20종류가 검출되어 다양한 유전자형의 노로바이러스들이 유행함을 알 수 있었다. 연구 결과 부산지역에서는 GI군이 21.8%(76/348), GII군이 78.2%(272/348)로 GII군이 우세하여 유행하였고, 유전자형 총 20종 중 GII.4형이 49.1%로 가장 많이 검출되었다. Norovirus (NoV) causes major acute non-bacterial gastroenteritis in humans. NoV genus is a member of the family Caliciviridae, which is transmitted by contaminated food and water or from human to human. Many genotypes of genogroups I and II have been reported because of their high genetic diversity. To obtain molecular epidemiological information on gastroenteritis sporadic cases in Busan, Korea, we analyzed the nucleotide sequences of NoV strains detected during 2008~2010. We performed one step RT-PCR amplifying the open reading frame (ORF) 2 (capsid region) followed by semi-nested PCR. Fecal samples were collected from 4,071 acute gastroenteritis, and genotypes of the 421 positive samples were determined by sequence analysis. Based on partial sequence of capsid region, 7 NoV were categorized into genogroup I and 13 into genogroup II. Prevalent genotypes among gastroenteritis patients within Busan were GII.4, GI.6, GII.5 in 2008~2010. The results of this study will contribute to the currently available epidemiological data and improve public health and hygiene via development of diagnostic methods and sustainable surveillance.

Paralytic Poliomyelitis Associated With Sabin Monovalent and Bivalent Oral Polio Vaccines in Hungary

Historical records of patients with vaccine-associated paralytic poliomyelitis (VAPP) in Hungary during 1961-1981 were reviewed to assess the risk of VAPP after oral polio vaccine (OPV) administration. A confirmed VAPP case was defined as a diagnosis of paralytic poliomyelitis and residual paralysis at 60 days in a patient with an epidemiologic link to the vaccine. Archived poliovirus isolates were retested using polymerase chain reaction and sequencing of the viral protein 1 capsid region. This review confirmed 46 of 47 cases previously reported as VAPP. Three cases originally linked to monovalent OPV (mOPV) 3 and one case linked to mOPV1 presented after administration of bivalent OPV 1 + 3 (bOPV). The adjusted VAPP risk per million doses administered was 0.18 for mOPV1 (2 cases/11.13 million doses), 2.96 for mOPV3 (32 cases/10.81 million doses), and 12.82 for bOPV (5 cases/390,000 doses). Absence of protection from immunization with inactivated poliovirus vaccine or exposure to OPV virus from routine immunization and recent injections could explain the higher relative risk of VAPP in Hungarian children. In polio-endemic areas in which mOPV3 and bOPV are needed to achieve eradication, the higher risk of VAPP would be offset by the high risk of paralysis due to wild poliovirus and higher per-dose efficacy of mOPV3 and bOPV compared with trivalent OPV.

Role of TRIM5α RING Domain E3 Ubiquitin Ligase Activity in Capsid Disassembly, Reverse Transcription Blockade, and Restriction of Simian Immunodeficiency Virus

The mammalian tripartite motif protein, TRIM5α, recognizes retroviral capsids entering the cytoplasm and blocks virus infection. Depending on the particular TRIM5α protein and retrovirus, complete disruption of the TRIM5α RING domain decreases virus-restricting activity to various degrees. TRIM5α exhibits RING domain-dependent E3 ubiquitin ligase activity, but the specific role of this activity in viral restriction is unknown. We created a panel of African green monkey TRIM5α (TRIM5α(AGM)) mutants, many of which are specifically altered in RING domain E3 ubiquitin ligase function, and characterized the phenotypes of these mutants with respect to restriction of simian and human immunodeficiency viruses (SIV(mac) and HIV-1, respectively). TRIM5α(AGM) ubiquitin ligase activity was essential for both the accelerated disassembly of SIV(mac) capsids and the disruption of reverse transcription. The levels of SIV(mac) particulate capsids in the cytosol of target cells expressing the TRIM5α variants strongly correlated with the levels of viral late reverse transcripts. RING-mediated ubiquitylation and B30.2(SPRY) domain-determined capsid binding independently contributed to the potency of SIV(mac) restriction by TRIM5α(AGM). In contrast, TRIM5α proteins attenuated in RING ubiquitin ligase function still accelerated HIV-1 capsid disassembly, inhibited reverse transcription, and blocked infection. Replacement of the helix-4/5 loop in the SIV(mac) capsid with the corresponding region of the HIV-1 capsid diminished the dependence of restriction on TRIM5α RING function. Thus, ubiquitylation mediated by the RING domain of TRIM5α(AGM) is essential for blocking SIV(mac) infection at the stage of capsid uncoating.

Capsid protein sequence diversity of avian nephritis virus

The capsid gene sequences of 25 avian nephritis viruses (ANVs), collected in the UK, Germany and Belgium from the 1980s to 2008, were determined and compared with those of serotype 1 (ANV-1) and serotype 2 (ANV-2) ANV isolates. Amino acid identities as low as 51% were determined. Pairwise comparisons supported by phylogenetic analysis identified six ANVs, including ANV-1 and ANV-2, which shared<80% amino acid identities with one another, and which were selected to be representative of six groups. The ANVs were not distributed according to geographical location or year of sampling, and the detection of ANVs from five different groups in 11 samples sourced from six flocks belonging to the same UK organization within a 4-month period indicated that sequence-diverse ANVs were co-circulating. Amino acid alignments demonstrated the existence of variable regions throughout the capsid protein, nine of which were selected for detailed comparisons. With most ANVs, the variable region sequences were similar to those of one of the six representative ANVs, but some ANV capsids displayed novel variable region profiles, in which variable regions that were characteristic of more than one representative ANV were present. Phylogenetic analysis based on C-terminal sequences of approximately 260 amino acids and SimPlot analysis provided evidence that RNA recombination events located in the 1250 to 1350 nucleotide region resulted in new combinations of the N-terminal and C-terminal capsid regions. The high level of capsid sequence diversity observed in the present study has important implications for both the control and diagnosis of ANV infections.

A Potential Role of Distinctively Delayed Blood Clearance of Recombinant Adeno-associated Virus Serotype 9 in Robust Cardiac Transduction

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

Standardized positive controls for detection of norovirus by reverse transcription PCR.

BackgroundNorovirus is one of the most common causes of nonbacterial gastroenteritis in humans. Rapid spread by contaminated food and person-to-person transmission through the fecal-oral route are characteristics of norovirus epidemiology and result in high morbidity in vulnerable patient populations. Therefore, detection of norovirus is a major public health concern. Currently, the most common method for detecting and differentiating among norovirus strains in clinical and environmental samples is reverse transcription PCR (RT-PCR). Standardized positive controls used in RT-PCR assays to detect norovirus are designed to overcome the problem of false-negative results due to PCR inhibitors and suboptimal reaction conditions.ResultsIn the current study, four types of RNA transcripts were produced from plasmids: norovirus GI-5 and GII-4 capsid regions with human rotavirus (VP7 gene derived) fragment insertions, and norovirus GI-6 and GII-4 capsid regions with hepatitis A virus (VP1/P2A gene derived) fragment insertions. These size-distinguishable products were used as positive controls under the RT-PCR assay conditions used to detect NoV in stool and groundwater samples. Their reliability and reproducibility was confirmed by multiple sets of experiments.ConclusionsThese standardized products may contribute to the reliable and accurate diagnosis by RT-PCR of norovirus outbreaks, when conducted by laboratories located in different regions.

Complete Genome Analysis of Coxsackievirus A2, A4, A5, and A10 Strains Isolated from Hand, Foot, and Mouth Disease Patients in China Revealing Frequent Recombination of Human Enterovirus A

Coxsackievirus (CV) strains CVA2, CVA4, CVA5, and CVA10 were isolated from patients with hand, foot, and mouth disease during a 2009 outbreak in China. Full genome sequences for four representative strains, CVA2/SD/CHN/09 (A2SD09), CVA4/SZ/CHN/09 (A4SZ09), CVA5/SD/CHN/09 (A5SD09), and CVA10/SD/CHN/09 (A10SD09), were determined. Phylogenetic and recombination analyses of the isolates by comparison with human enterovirus A prototype strains revealed that genetic recombination occurred during cocirculation of the viruses. The A2SD09 and A4SZ09 strains were most closely related to their corresponding prototype strains in the capsid region but shared noncapsid sequences with each other. Similarly, strains A5SD09 and A10SD09 had serotype-specific homology for the capsid proteins but shared noncapsid sequences with each other. Phylogenetic analyses of the four isolates with homotypic strains showed that CVA2 strains were divided into five genotypes. The A2SD09 strain clustered with Mongolia strains isolated in 2003, forming genotype V. The A4SZ09 strain and other isolates from mainland China and Taiwan clustered with genotype III strains and are likely related to strains that circulated in Europe and Mongolia. The A5SD09 strain is closely related to other Chinese strains isolated in 2008. The A10SD09 isolate, together with other Chinese strains isolated since 2004, formed a distinct lineage that was likely imported from Japan and South Korea. This study shows that natural recombination is a frequent event in human enterovirus A evolution. More comprehensive surveillance of enteroviruses that focus not only on EV71 or CVA16 is needed for us to understand the molecular epidemiology of enteroviruses and to track recombination events which may ultimately affect the virulence of viruses during outbreaks.

Clinical and Molecular epidemiologic analyses of norovirus‐associated sporadic gastroenteritis in adults from Beijing, China

Noroviruses (NoVs) are a major cause of nonbacterial acute gastroenteritis. The molecular epidemiology of NoV infections in China has not been well characterized. To study the incidence of NoV infections and the nature of the circulating NoV genotypes, 403 specimens were collected from adult outpatients with acute gastroenteritis in Beijing, China, between October 2007 and September 2008. Samples were examined for NoV by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequences corresponding to both the NoV polymerase and capsid regions were characterized. Among the 403 specimens, 48 (11.9%) were positive for NoV and 1/3 NoV-associated gastroenteritis cases occurred during the colder months (November and December). Based on polymerase region sequences, 6 NoV genotypes (GII-4, GII-b, GII-6, GI-2, GI-3, and GI-4) were identified with GII-4 2006b being the most predominant genotype (37/48, 77.1%). Eleven distinct genotype sequences in polymerase and capsid regions were identified, indicating a genetic diversity among NoV isolates. This study suggested NoVs were an important pathogen responsible for sporadic acute gastroenteritis in adults in Beijing, China.

Development and validation of a microarray for the confirmation and typing of norovirus RT-PCR products

Noroviruses are implicated in many worldwide institutional, food and waterborne outbreaks each year. Genetic typing of isolates is valuable for monitoring outbreak spread as well as variation in circulating strains. Microarrays have the potential to provide rapid genotype information for norovirus samples. The NoroChip v3.0 provides an oligonucleotide hybridization platform to screen for over 600 potential interactions in each experiment. The NoroChip v3.0 was developed at Health Canada and validated in seven international partner laboratories. Each laboratory validated the NoroChip v3.0 using norovirus amplicons routinely characterized in their testing protocols. Fragments from the capsid region (region C) and a 2.4kb amplicon spanning polymerase and capsid sequences (region AD) were validated in six of the partner laboratories and provided correct genogroup typing information (GI or GII) when hybridized to the NoroChip v3.0. Results indicate that the current limiting factor for implementing the NoroChip v3.0 as a strain typing tool is the difficulty obtaining a long, specific amplicon from all circulating norovirus strains. Data obtained with the longer region AD amplicon provided the best discrimination between norovirus strains.

First complete genomic characterization of two tick-borne encephalitis virus isolates obtained from wild rodents in South Korea

We determined for the first time the complete genome sequences of two Korean strains of the tick-borne encephalitis virus (TBEV), designated KrM 93 and KrM 213, isolated from the lung tissues of wild rodents in 2006. The genomes are 11,097 nucleotides (nt) in length and consist of a 132 nt 5'-noncoding region (NCR), a 10,245nt open reading frame (ORF) containing 10 viral protein-coding regions (3,415 amino acids), and a 720nt 3'-NCR. Compared with the 31 fully sequenced TBEV strains currently available, KrM 93 and KrM 213 show genomic nucleotide (and deduced amino acid) sequence divergences ranging from 1.8 (0.7) to 19.2 (26.6)% and 1.9 (0.8) to 19.3 (26.7)%, respectively. Phylogenetic and recombination analyses based on the complete genome sequence were performed to identify genetic variations and relationships between the TBEV strains. These showed that the Korean TBEV strains clustered with the Western subtype rather than with Far-Eastern or Siberian subtypes, and phylogenetic trees derived from capsid (C), envelope (E), nonstructural (NS) 4B and NS5 regions represented the same branching pattern shown by the complete genome-based tree. Although no recombination events were identified in these two Korean strains, 11 putative recombination events were identified within the NS5 regions or in the 3'-NCRs of TBEV strains in general. The results provide insight into the genetics of TBEV strains to understand the molecular epidemiology, genetic diversity, and evolution of TBEV.

Identification of mixed equine rhinitis B virus infections leading to further insight on the relationship between genotype, serotype and acid stability phenotype

Equine rhinitis B virus (ERBV) is the single species in the genus Erbovirus, family Picornaviridae. Equine rhinitis B viruses exist in three serotypes and are associated with respiratory disease in horses. Members of the species vary in stability at acid pH. To date there has been discordance in genotype, serotype and acid stability phenotype groupings. To identify capsid regions associated with acid stability, two viruses were serially treated at pH 3.3 to isolate acid-stable mutants. An acid-stable mutant of the prototype acid-labile serotype 1 virus contained a single amino acid change in the C-terminus of VP1. Similar treatment with a separate isolate identified a multiple ERBV serotype infection with acid-labile serotype 1 and acid-stable serotype 3. Dual infections were subsequently identified in original swabs taken from the infected horse and from two further cell culture passaged viruses originally isolated in Switzerland. Serotype specific rat antisera were produced and used to examine a collection of isolates from a range of genotypes, acid stability phenotypes and serotypes. In contrast to previous reports, we showed viruses previously classified as acid-stable serotype 1 are in fact serotype 3 and that there is a clear association of serotype with genotype and acid stability phenotype in ERBVs. Additionally, we have shown that ERBV capsids dissociate into pentamers in acidic conditions below their threshold of stability, similarly to closely related viruses in the same family.

Full genome sequence analysis of parechoviruses from Brazil reveals geographical patterns in the evolution of non-structural genes and intratypic recombination in the capsid region

Due to high genome plasticity, the evolutionary fate and geographical history of picornaviruses is hard to follow. Here, we determined the complete coding sequences of eight human parechoviruses (HPeV) of types 1, 5 and 6 directly from clinical samples from Brazil. The capsid genes of these strains were not remarkably different from European, North American and Japanese HPeV. Full genome analysis revealed frequent intertypic recombination in the non-structural genome region. In addition, evidence of recombination between viruses of the same type in the capsid-encoding genome region among HPeV1 and HPeV4 was obtained. Bayesian phylogenetic analysis indicated that strains without evidence of recombination with each other in any genome region were separated by no more than 35 years of circulation. Interestingly, in the 3C gene, all Brazilian parechoviruses grouped together regardless of serotype. The most recent common ancestor of these strains dated back 108 years, suggesting long-term endemicity of this particular P3 genome lineage in South America. Our results support the idea that picornavirus replicative genes acquire capsid proteins introduced by new strains. Under certain epidemiological conditions, replicative genes may be maintained in circumscript geographical regions.

Molecular epidemiology of noroviruses detected in seasonal outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, from 1996–1997 to 2008–2009

In seasons from 1996-1997 through 2008-2009, noroviruses (NoVs) were detected in 505 outbreaks (71%) of nonbacterial gastroenteritis in Osaka City, Japan using molecular diagnosis with reverse transcription (RT)-PCR or real-time RT-PCR. The occurrences of NoV-associated outbreaks were related with the cold season during November-March (85.3%), and occasionally small epidemics of NoVs occurring during April-June were observed. Oyster-associated outbreaks were dominant transmission modes (25-61.1%) before the 2003-2004 season, and decreased (5-20.5%) from the 2003-2004 season, although outbreaks attributable to food-borne transmission (except for oysters) and person-to-person contact increased from the 2003-2004 season. The NoV strains were characterized into genotypes based on sequence analysis of partial capsid regions. Genotyping analyses identified at least 30 genotypes (12 in genogroup I [GI] and 18 in genogroup II [GII]) of NoV. The most common genotype was GII.4 (44.6%), followed in order by GII.3, GII.6, GII.2, and GII.5. The number of GII.4 NoVs increased greatly from the 2003-2004 season, eventually comprising a large share among the NoV- associated outbreaks (97.4%) of the 2006-2007 season. Occasional increased prevalence of genotypes other than GII.4 was observed during this study period. This study showed the appearance, spread, and disappearance of various genotypes and the change of NoV epidemic in a limited geographic region. Continuous NoV molecular surveillance is important for understanding NoV infections and for improving measures for their control and prevention.

Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.

Simian immunodeficiency virus-specific CD4+ T cells from successful vaccinees target the SIV Gag capsid

We recently demonstrated that vaccinated rhesus macaques controlled viral replication of a heterologous SIV challenge. Here, we analyzed anamnestic SIV-specific CD4+ T-cell responses expanding immediately after challenge and show that successful vaccinees consistently targeted a short region of the Gag-p27 Capsid (amino acids 249-291). We have also defined the major histocompatibility complex class II (MHC-II) restricting alleles for several of these responses and show that DQ-restricted CD4+ T-cells depend on unique combinations of both the DQA and DQB alleles. Analysis of SIV-specific CD4+ T-cell responses elicited by a successful vaccine may have important implications in the understanding of vaccine design.

TRIMming SIV Transmission between Species

TRIMming SIV Transmission between Species

Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination

BackgroundFoot-and-mouth disease (FMD) is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced.ResultsPhylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L), the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965) and serotype O viral isolate (K/117/1999) revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O.ConclusionsSequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2), a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A and O has been obtained. In addition to characterization using the VP1 coding region, analyses involving the non-structural protein coding regions should be performed in order to identify evolutionary processes shaping FMD viral populations.

Evolutionary trajectory of the VP1 gene of human enterovirus 71 genogroup B and C viruses

From 1963 to 1986, human enterovirus 71 (HEV71) infections in the Netherlands were successively caused by viruses of subgenogroups B0, B1 and B2. A genogroup shift occurred in 1987, after which viruses of subgenogroups C1 and C2 were detected exclusively. This is in line with HEV71 typing in Australia, Europe and the USA, but is distinct from that in the Asian Pacific region, where HEV71 subgenogroups B3-B5 and C4-C5 have caused large outbreaks since 1997. To understand these observations in HEV71 epidemiology, the VP1-encoding regions of 199 HEV71 strains isolated in the Netherlands between 1963 and 2008 were used to study the detailed evolutionary trajectory and population dynamics of HEV71. Genogroup B viruses showed an epochal evolution, whereas genogroup C viruses evolved independently, which is in line with the co-circulation of C1 and C2 viruses in the Netherlands since 1997. Considering that strains from the Netherlands are interspersed phylogenetically with GenBank reference strains, the evolution of B1-B2, C1-C2 viruses has a global nature. Phylodynamic analysis confirmed that increased reporting of HEV71 infections in 1986 and 2007 reflected true epidemics of B2 and C2 viruses, respectively. Sequence analysis of the complete capsid region of a subset of isolates revealed several (sub)genogroup-specific residues. Subgenogroup B2-specific rabbit antiserum showed cross-neutralization of B0, B1 and B2 viruses, but not of subgenogroup C1 or C2 viruses, probably explaining the global shift to genogroup C in 1987 following a B2 epidemic. Anti-C1 rabbit serum neutralized both genogroup B and C viruses. Global herd immunity against C1 and C2 viruses possibly explains why epidemics with subgenogroups B4 and C4 are restricted to the Asian Pacific region.

Genogroup IIb Norovirus Infections and Association with Enteric Symptoms in a Neonatal Nursery in Southern India

Noroviruses (NoVs) are increasingly recognized as an important cause of acute gastroenteritis in children worldwide. However, there are limited data on the role of NoVs in neonatal infections and disease. The objectives of the present study were to determine the prevalence of NoVs in neonates with gastrointestinal disease using a case-control study design and to characterize the NoV strains infecting neonates. A total of 309 fecal samples from 161 neonates with gastrointestinal symptoms and 148 asymptomatic controls were screened for genogroup II (GII) NoV using reverse transcription-PCR. A subset of PCR-positive amplicons for the polymerase and capsid regions was sequenced. NoV was detected in 26.2% of samples, with the rate of detection being significantly higher among symptomatic neonates (60/161, 37.2%) than asymptomatic neonates (24/148, 14.1%) (P < 0.001). On the basis of sequencing of 29 strains, a single NoV strain, GIIb, was identified to be the predominant (27/29, 93.1%) cause of neonatal infections. Coinfection with rotavirus was seen in nearly one-third of symptomatic neonates. The study demonstrates a high prevalence of NoV infections in neonates and indicates that coinfection with rotavirus may result in significantly more gastrointestinal disease in this population.