Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype

Summary. Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004–2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region. First three authors contributed equally to this work.     

Isolation and identification of a non-haemagglutinating strain of rabbit hemorrhagic disease virus from China and sequence analysis for the VP60 Gene

A variant strain of rabbit hemorrhagic disease virus, designated “whn-1”, was isolated and identified in China. The virus lacked haemagglutinating activity at 25, 37 and 4°C, respectively, and gave negative results in the HAT after two passages in experimentally infected rabbits, but gave positive results in Agar Diffusion Reaction (ADR) and Counter Immunoelectrophoresis (CIE). Using electron microscopy, negatively stained particles of the RHDV isolate showed that the virions was approximately 35 nm in diameter. The capsid protein VP60 gene of whn-1 strain was cloned into pMD18-T vector by RT-PCR assays and sequenced. The obtained VP60 gene sequence has been submitted to GenBank with the accession number: DQ069280. The whole VP60 gene of whn-1 was 1740 nt in size and encodes 579 aa. Alignment with other 16 strains of RHDV in the world, including such “RHDVa” strains as France 99-05, France-Reu-00, Germany-Triptis and ChinaTP, in addition to RCV and EBHSV, showed that the homology of RHDV strains were 90.0–98.0% for nucleotide sequence, 94.3–99.0% for amino acid sequence, respectively. The results indicated that the sequences of VP60 gene of different RHDV isolates, including non-haemagglutinating whn-1 strain and low-haemagglutinating Rainham strain, were relatively highly homologous, and the major variant amino acid were located within region C (301–328 aa) and region E(344–434 aa), which were specific to “RHDVa” strains. Moreover, the molecular characterisation of VP60 protein of RHDV whn-1 strain, such as Hydrophilicity plot, Flexible regions, Antigenic index, etc., were compared with reference RHDV strains of Spanish-AST/89, France-99-5 and UK-Rainham in this article. From the experiment, it’s concluded that, the “whn-1” strain is probably an antigenic variant of “RHDVa”, and the 3 amino acids of Phe (304), Ala (305), Ser (309), and 5 amino acids of Gly (359), Asn (365), Ala (369), Ala (370), Asn (386), located in P2 region in the VP60 protein, probably played an important role in the haemagglutination activity.     

Characterization of VP6 genes from rotavirus strains collected in the United States from 1996–2002

We sequenced 22 VP6 genes from common rotavirus strains P[8], G1; P[4], G2; P[8], G3; P[8], G4 and P[8], G9 and uncommon type P[6], G9 collected in the US over a 6-year period. All strains defined as members of VP6 antigenic subgroup (SG) I according to reactivity patterns with monoclonal antibodies formed a genetic cluster (Genogroup I) with SG I reference strains. Similarly, all strains in antigenic SGII formed a group (Genogroup II) with corresponding standard strains of the same SG. Most US strains of each genogroup had diverged by 10–15% from the VP6 gene sequence of reference strains collected >20 years earlier and some recent isolates from other countries. Evolutionary analysis demonstrated that recently isolated US strains of both genogroups have diverged into 2–3 related clusters consistent with other recent findings. Unexpectedly, some recent isolates from other countries have diverged greatly from both older reference isolates and from the recent US isolates characterized here. This finding suggests that genetic diversity in human rotavirus VP6 genes may be greater than previously recognized. These sequences will help in the construction of a VP6 gene database to aid in the development of broadly reactive molecular assays and permit identification of regions where primers and probes for existing assays may need to be redesigned. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.     

Full genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9–P[6]–I1–R1–C1–M1–A8–N1–T1–E1–H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing countries.     

Genetic characterization and molecular epidemiology of foot-and-mouth disease viruses isolated from Afghanistan in 2003–2005

Foot-and-mouth disease virus (FMDV) isolates collected from various geographic locations in Afghanistan between 2003 and 2005 were genetically characterized, and their phylogeny was reconstructed utilizing nucleotide sequences of the complete VP1 coding region. Three serotypes of FMDV (types A, O, and Asia 1) were identified as causing clinical disease in Afghanistan during this period. Phylogenetic analysis revealed that the type A viruses were most closely related to isolates collected in Iran during 2002–2004. This is the first published report of serotype A in Afghanistan since 1975, therefore indicating the need for inclusion of serotype A in vaccine formulations that will be used to control disease outbreaks in this country. Serotype O virus isolates were closely related to PanAsia strains, including those that originated from Bhutan and Nepal during 2003–2004. The Asia 1 viruses, collected along the northern and eastern borders of Afghanistan, were most closely related to FMDV isolates collected in Pakistan during 2003 and 2004. Data obtained from this study provide valuable information on the FMDV serotypes circulating in Afghanistan and their genetic relationship with strains causing FMD in neighboring countries.     

Phylogenetic and serological characterization of echovirus 11 and echovirus 19 strains causing uveitis

Summary.  The strains of echovirus 19 (EV19) and echovirus 11 (EV11), isolated from infants with similar clinical symptoms of acute enterovirus uveitis (EU) in Russia (Siberia) in 1980–1989, were investigated phylogenetically (nucleotide sequence of a 300 nt fragment in 5′ NTR and VP4 junction) and serologically. The result confirmed that viruses belong to the Enterovirus genus, with 58–80% nt sequence homology with previously sequenced enteroviruses, and showed the genetical identity between the strains isolated during each of five outbreaks of the EU. The results also demonstrated that isolates from the last three outbreaks of EU belong to the same phylogenetic group despite the remarkable spatial and temporal distance between the outbreaks. The results confirm the role of these echoviruses in the etiology of the EU. Based on phylogenetic and serological comparisons the studied strains were divided into three distinct groups: group I, EV19/K (Krasnoyarsk, 1980–1981), group II, EV11/A (Krasnoyarsk, 1982), group III, EV11/B (Krasnoyarsk, 1986; Omsk, 1987–1988; Irkutsk, 1989). Minor details of the epidemiology of the outbreaks were also revealed. Received February 16, 2001 Accepted July 13, 2001     

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain

Summary.  The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1–4, and 6–11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype. Received September 2, 1997 Accepted January 8, 1998     

Molecular Characterization of Foot-and-mouth Disease Virus in Hong Kong During 2001–2002

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, The nucleotide sequences of the VP1 coding region of FMDV type O strains O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02, isolated from the disease outbreak that occurred in Hong Kong Special Administrative Region (Hong Kong SAR) of China during 2001–2002, were determined and compared with the sequences of other FMDVs. The results revealed that the VP1 gene of the five isolates had the same nucleotide (nt) sequences (639 nt), coding for 213 amino acids, and no changes were found either at the critical amino acid sites 144 (Val), 148 (Leu), 154 (Lys) and 208 (Pro) within the VP1 protein epitope (amino acids 140–160, 200–213), or in the amino acids 145–147 comprising the arginine–glycine–aspartic acid (RGD) sequence that is involved in the adsorption of virus to host cell. Analysis of the VP1 gene nucleotide sequence revealed that the five isolates examined were most closely related to FMDVs found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Furthermore, although the critical amino acids on the antigen epitope of the prevalent Hong Kong isolates and the serotype O vaccine strain, O1/Manisa/Turkey/69, showed relative conservativeness, they were distantly related genetically, which showed that there existed variation between the prevalent Hong Kong FMDV strains and the vaccine strain.     

A large Finnish echovirus 30 outbreak was preceded by silent circulation of the same genotype

An outbreak of echovirus 30 (E-30) in 2009 was confirmed by both frequent isolation of the virus from sewage as well as from patient samples in Finland. Over the last 10 years E-30 had only been isolated sporadically in Finland. We here study the phylogenetic relationships of the strains from the outbreak in the context of E-30 circulation over the last 20 years. The analyzed region comprised 276 nucleotides in the 5′ end of VP1 (nucleotides 132–407 in the VP1 of the E-30 Bastianni strain). The Finnish strains were clustered into at least four distinct genogroups, with seven clusters exceeding the genotype demarcation of 12% and the 2009 epidemic strains forming the largest genogroup VII. Moreover, we detected largely divergent genotypes in 2007 and 2009. Interestingly, close genetic relatives of the epidemic strains had already been isolated a few years before the outbreak. Phylodynamic analysis estimated 8.9 years (95% highest posterior density intervals 7.0–11.0) as the age of genogroup VII, indicating a probable origin and evolutionary history prior to its introduction and epidemic expansion in Finland. Finally, the most recent common ancestor for the current E-30 diversity dates back to 1939 (95% highest posterior density intervals 1913–1956).     

Molecular identification of coxsackievirus A24 variant,isolated from an outbreak of acute hemorrhagic conjunctivitis in Singapore in 2005

Summary An outbreak of acute hemorrhagic conjunctivitis (AHC) was reported in Singapore military camps in the year 2005. A total of 103 conjunctival swab specimens were collected from military personnel diagnosed clinically with AHC. PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by virus isolation. Molecular typing using a partial VP1 gene confirmed a variant of coxsackievirus A24 (CA24v) as the most likely etiological agent for the outbreak. Full-length genome sequencing was carried out on 2 selected virus strains, DSO-26SIN05 and DSO-52SIN05. Sequence comparison and phylogenetic analyses of the VP4, VP1 and 3Cpro gene regions were performed, clustering the Singapore CA24v strains with viruses originating from Asia in the post-2000 era. In addition, we report evolution rates of 4.2 × 10⁻³ and 1.0 × 10⁻³ nucleotide/year, respectively, for the VP4 capsid and 3Cpro gene regions. Our result shows a focal evolutionary point around 1965–1966, suggesting that the CA24v virus has been evolving constantly since its emergence in Singapore, nearly 40 years ago.     

Molecular analysis of the VP7 gene of pheasant rotaviruses identifies a new genotype,designated G23

Rotavirus-associated enteritis has been reported in pheasants, but there is no information on the genetic/antigenic features of pheasant rotaviruses. In this study, we sequenced the VP7-encoding genome segment of three pheasant rotavirus strains detected during 2008 in Hungary. The full-length genome segment was 1,070 bp long, while the open reading frame was predicted to encode a 330-aa-long protein. The nucleotide sequence identities among the three pheasant rotavirus strains were high (≥94%), whereas the range of nucleotide sequence identities to other avian and mammalian rotavirus VP7 genes fell between 68 and 73% and between 60 and 66%, respectively. Our findings indicate that these Hungarian pheasant rotaviruses need to be considered representatives of a new VP7 genotype specificity, designated G23.     

Molecular epidemiologic analysis of group A rotaviruses in adults and children with diarrhea in Wuhan city,China, 2000–2006

Summary To compare epidemiologic features and genetic characteristics of group A rotaviruses causing diarrhea in children and adults, a survey was conducted in Wuhan, China, during the period of Dec. 2000–May 2006. A total of 3839 stool specimens from diarrheal patients from eight hospitals were analyzed. Winter seasonality was observed for rotavirus diarrhea in both adults and children, showing overall rotavirus-positive rates of 9.0 and 23.9%, respectively. Throughout the study period, G3 was the most frequent G serotype in both adults and children (detection rates 86.2 and 87.8%, respectively), and was mostly associated with VP4 genotype P[8], VP 6 genotype II (subgroup II), and NSP4 genotype B. G3 rotaviruses were differentiated into eight electropherotypes, among which seven types were found in specimens from both adults and children. VP7 gene sequences of G3 rotaviruses from adults and children (6 and 4 strains, respectively), detected in different years and different hospitals, showed extremely high sequence identities (99–100%) to each other and to a few G3 rotavirus strains reported in Asia. However, lower sequence identities (82–96%) were observed to most of the human and animal G3 rotaviruses reported so far, including some Chinese strains. These findings indicate that in Wuhan, China, epidemic and genetic features of rotaviruses are similar in adults and children, and it has been suggested that G3 rotaviruses that might have originated from the same rotavirus were circulating among children and adults as prevailing viruses. In this study, two rotavirus strains, G9P[8] strain L169, derived from an adult, and G4P[6] strain R479, derived from a child, were isolated and genetically analyzed. The VP7 gene of L169 belongs to a major lineage of G9 rotaviruses that are globally widespread, but is distinct from G9 rotaviruses reported previously in China. The strain R479 had a VP7 gene which was divergent from most G4 human rotaviruses and showed an unusual dual subgroup specificity, I + II. The R479 VP6 gene does not belong to the main clusters of subgroup I and II rotaviruses phylogenetically, but is related to those of the porcine rotaviruses and some unusual human rotaviruses represented by the RMC321 strain isolated in eastern India.     

Characterization of full-length VP4 genes of OP354-like P[8] human rotavirus strains detected in Bangladesh representing a novel P[8] subtype

The G1 and G9 rotavirus strains MMC71 and MMC38 (subgroup II, NSP4 genogroup B), respectively, isolated from children in Bangladesh, were analyzed genetically. Full-length VP4 genes of these strains had 98.9% identity to each other and showed 83.9–89.4% identity to those of the P[4] and P[8] rotaviruses. Phylogenetic analysis of VP4 nucleotide sequences revealed that strains MMC38 and MMC71 were located in a lineage of P[8] strains. However, the cluster was highly divergent from the previously established P[8] strains. The VP8* portions of strains MMC38 and MMC71 showed more than 93.9% nucleotide sequence identity to OP354-like P[8] strains, and these strains were clustered into the same lineage. These findings indicate that the VP4 of these strains should be classified into a subtype of the P[8] genotype (P[8]b) that is distinct from that of common P[8] rotaviruses (P[8]a).     

Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses

We determined the nucleotide sequences of the outer capsid glycoprotein (VP7) genes of 38 porcine group B rotaviruses (GBRs) from feces of pigs at 27 farms in Japan between 2000 and 2007. Substantial diversity among porcine GBR VP7 genes was observed, with up to 42.4% difference in nucleotides and 49.8% in amino acids. On comparison of VP7 genes, porcine GBRs were clearly distinct from the published corresponding genes from human, bovine and murine GBRs (53.7–70.8% identity in nucleotides and 45.8–73.4% identity in amino acids). Phylogenetic analysis showed that the VP7s of GBRs could be divided into five genotypes: the murine strain was genotype 1, human strains were genotype 2, bovine and some porcine strains were genotype 3, and other porcine strains belonged to genotype 4 or 5. In addition, GBR VP7s in genotypes 3 and 5 were further divided into four and five clusters, respectively. No relationship between VP7 genotype and double-stranded RNA migration patterns of porcine GBRs in polyacrylamide gel electrophoresis were observed. However, an antigen enzyme-linked immunosorbent assay using antiserum to recombinant bovine GBR VP6 did not react with fecal samples containing one cluster of genotype 5 of porcine GBRs. The abundant divergence of porcine GBR VP7 genes suggests that porcine species might be an original natural host of GBR infection and that different serotypes might exist among porcine GBRs. To our knowledge, this is the first report to describe the gene sequences and typing of porcine GBR VP7s.     

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.     

Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus,a member of a new species in the genus <Emphasis Type="Italic">Kobuvirus</Emphasis>, family <Emphasis Type="Italic">Picornaviridae</Emphasis>

Kobuvirus is a new genus in the family Picornaviridae. Two species are currently known: Aichi virus (human kobuvirus) and Bovine kobuvirus (U-1). In this study, the complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus (Kobuvirus/swine/S-1-HUN/2007/Hungary, EU787450) were determined. The structure of the S-1-HUN genome, VPg–5′UTR–leader protein–structural proteins (VP0, VP3, VP1)–non-structural proteins (2A–2C, 3A–3D)–3′UTR–poly(A) tail, was found to be typical of picornavirus. The 8210-nucleotide (nt)-long RNA genome contains a large open reading frame (7467 nt) encoding a potential polyprotein precursor of 2488 amino acids (aa) that has 57/56% and 63/64% nt/aa identity with Aichi virus and U-1, respectively. The 5′UTR contains a hepacivirus/pestivirus-like internal ribosomal entry site (IRES type IV group-B-like) with conserved pseudoknot, II and IIIa–f domains. A tandem repeat (a 30-amino-acid-long motif) was detected in 2B. Thirty-nine (65%) of the 60 fecal samples from pigs under the age of 6 months at the tested farm were positive (the incidence was 90% under the age of 3 weeks). Porcine kobuvirus belongs to a potential new species—the third—in the genus Kobuvirus. Nucleotide sequence data reported are available in the GenBank database under accession number EU787450.     

Survey of Human Group C Rotaviruses in Japan during the Winter of 1992 to 1993

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.