Correction to: Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Unfortunately, one of the affiliations of author "A. E. Gorbalenya" was missed in original version. The affiliation is updated here.

Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Enteroviruses (EVs) and rhinoviruses (RVs) are significant pathogens of humans and are the subject of intensive clinical and epidemiological research and public health measures, notably in the eradication of poliovirus and in the investigation and control of emerging pathogenic EV types worldwide. EVs and RVs are highly diverse in their antigenic properties, tissue tropism, disease associations and evolutionary relationships, but the latter often conflict with previously developed biologically defined terms, such as "coxsackieviruses", "polioviruses" and "echoviruses", which were used before their genetic interrelationships were understood. This has created widespread formatting problems and inconsistencies in the nomenclature for EV and RV types and species in the literature and public databases. As members of the International Committee for Taxonomy of Viruses (ICTV) Picornaviridae Study Group, we describe the correct use of taxon names for these viruses and have produced a series of recommendations for the nomenclature of EV and RV types and their abbreviations. We believe their adoption will promote greater clarity and consistency in the terminology used in the scientific and medical literature. The recommendations will additionally provide a useful reference guide for journals, other publications and public databases seeking to use standardised terms for the growing multitude of enteroviruses and rhinoviruses described worldwide.

Comparative genomics of the coxsackie B viruses and related enteroviruses.

Genomic analysis of the group B coxsackieviruses (CVB) has improved our understanding of CVB evolution, epidemiology, and pathogenesis. Comparison of capsid sequence alignments and virion structures allows correlation of capsid diversity with surface features, such as loops, the receptor canyon, and antigenic sites. Pairwise sequence comparisons and phylogenetic analyses can be used to rapidly identify and classify enteroviruses. Enteroviruses are monophyletic by type only within the capsid region. The CVBs as a group are monophyletic in the capsid region, probably due to their shared use of the coxsackievirus-adenovirus receptor (other members of HEV-B use different receptors). Outside the capsid region, enteroviruses are monophyletic only by species (not by type), reflecting a high frequency of intertypic recombination within a species. Further genomic studies, accompanied by well-characterized clinical outcome/disease data, will facilitate fine-scale mapping of genetic determinants that contribute to virulence.

Molecular phylogeny of all human enterovirus serotypes based on comparison of sequences at the 5' end of the region encoding VP2.

Sixty-six human enterovirus serotypes have been described using antibody neutralization, with antigenic variants defined within several serotypes. Despite the availability of sequence data for numerous enteroviruses, the molecular basis of serotype is unknown. Previous studies by others have identified four major phylogenetic groups within the human enteroviruses, but there has been no complete database of homologous sequences for all human enterovirus serotypes. We have determined the homologous partial VP2 sequences for the 12 prototype strains for which VP2 sequence was unavailable and for eight well-characterized antigenic variants. Phylogenetic analysis of all prototype strains produced four major groups, consistent with published enterovirus phylogenies. Many antigenic variants, however, failed to cluster with their respective prototype strains, suggesting that this portion of VP2 may be inappropriate for consistent molecular inference of serotype and for detailed study of enterovirus evolution.

Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses.

To define the relationship between echovirus 23 (E23) and other human enteroviruses, we have determined the complete nucleotide sequence of a strain of E23 isolated from a child with high fever in Connecticut in 1986 and compared the nucleotide and deduced amino acid sequences with those of other enteroviruses representing each of the major enterovirus phylogenetic groups, poliovirus type 1, coxsackievirus A16, coxsackievirus B3, echovirus 22 (E22), and enterovirus 70. The genome of E23 (strain CT86-6760) was 7352 nucleotides in length, exclusive of the poly(A) tail, and the genome organization was typical of the picornaviruses. The nucleotide sequence and deduced amino acid sequences were most related to those of E22, a virus with which E23 shares many biological properties, and was quite divergent from the sequences of other enteroviruses (< 20% average amino sequence identity). These data lend further support to the suggestion that E22 and E23 are distinct from members of the Enterovirus genus and that they should be reclassified in a separate genus within the Picornaviridae.

Serotype-specific identification of enterovirus 71 by PCR.

BACKGROUND: Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. OBJECTIVES: Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. STUDY DESIGN: Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. RESULTS: Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. CONCLUSIONS: Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

Identification and genetic analysis of Panama-genotype Venezuelan equine encephalitis virus subtype ID in Peru.

Venezuelan equine encephalitis (VEE) virus was isolated in 1993, 1994, and 1995 from human cases of acute, undifferentiated, febrile illness in the Peruvian Amazon Basin. Two virus isolates were recovered in 1994 from Peruvian soldiers at a jungle outpost near Pantoja in northern Peru, and 10 isolates were obtained from military personnel and civilians in 1993-1995 in Iquitos, an urban center in northeastern Peru. The genetic relationship of these isolates to other VEE virus strains was determined by sequencing 856-867 nucleotide reverse transcription-polymerase chain reaction fragments derived from the PE2 glycoprotein gene. The sequences were compared with those of other VEE virus strains, including representatives of the IAB, IC, ID, IE, II, and IIIC subtypes. The two Pantoja isolates were most closely related to subtype IC and ID viruses previously isolated in Colombia and Venezuela, and to the ID viruses isolated during the 1970s in Iquitos. All of the recent Iquitos isolates were similar to one another, but they were more closely related to Panamanian ID strains than to isolates previously obtained in Iquitos, Peru, or in Colombia and Venezuela. The recent Iquitos VEE viral isolates were the first Panama-genotype VEE ID virus strains identified outside of the Republic of Panama.

Geographic distribution of Venezuelan equine encephalitis virus subtype IE genotypes in Central America and Mexico.

Phylogenetic analysis of 20 strains of Venezuelan equine encephalitis (VEE) virus subtype IE isolated from 1961 to 1996 in Mexico and throughout Central America showed that VEE virus subtype IE was monophyletic with respect to other VEE virus subtypes. Nonetheless, there were at least three distinct geographically separated VEE virus IE genotypes: northwestern Panama, Pacific coast (Mexico/Guatemala), and Gulf/Caribbean coast (Mexico/Belize). Strains from the Caribbean coast of Guatemala, Honduras, and Nicaragua may cluster with the Gulf/Caribbean genotype, but additional isolates from the region between Guatemala and Panama will be required to firmly establish their phylogenetic position. Viruses associated with two separate equine epizootics in Mexico in the 1990s were phylogenetically related to nonepizootic viruses from neighboring Guatemala and may represent the emergence or re-emergence of equine-virulent VEE virus subtype IE in Middle America.

Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region.

A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.

Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico.

Two outbreaks of encephalitis consistent with an etiology of Venezuelan equine encephalitis (VEE) virus occurred in equines on the Pacific coast of southern Mexico in 1993 (Chiapas State) and in 1996 (Oaxaca State). In Chiapas, there were 125 cases, of which 63 were fatal and in Oaxaca, there were 32 cases and 12 fatalities. Virus was isolated from two horses from each outbreak, including three brain isolates and one from blood. Virus isolates (93-42124, ISET-Chi93, Oax131, and Oax142) were shown by indirect immunofluorescence, hemagglutination inhibition, monoclonal antibody ELISA, and nucleotide sequencing to be VEE virus, subtype IE, a type previously thought to be equine-avirulent. Genetic characterization and phylogenetic analysis indicated that the outbreak viruses were identical or nearly identical to one another and that they were closely related to equine-avirulent IE strains from Guatemala and the Gulf coast of Mexico. In a plaque-reduction neutralization test, sera collected from healthy horses in Chiapas and Oaxaca reacted significantly better with isolate 93-42124 than with Guatemala IE isolate 68U201, suggesting that subtle genetic changes may have resulted in alteration of neutralization domains. It is not clear whether these differences may also influence equine virulence. However, renewed VEE virus subtype IE activity in Mexico, and its apparent conversion to equine virulence, underscores the need for increased surveillance, additional laboratory and epidemiologic studies in VEE-endemic regions, and possibly new vaccines.

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

An outbreak of acute haemorrhagic conjunctivitis in Melaka, Malaysia.

This paper reports a second outbreak of acute haemorrhagic conjunctivitis due to coxsackievirus A24 in peninsular Malaysia. Between June 2002 and early October 2003, 10,327 patients, comprising 3,261 children and 7,066 adults, were treated for acute conjunctivitis in 11 government health clinics in the Melaka Tengah district of the state of Melaka. The figure grossly underestimates the size of the outbreak; as no patients treated in private clinics in the same district were included. Institution and household surveillance showed that the commonest presenting clinical feature of the illness was eye-discharge (91.2%), followed by foreign body sensation (81.8%), pain (78.3%) and subconjunctival haemorrhage (74.4%). The mean duration of illness was 6.5 and five days for patients with and without subconjunctival haemorrhage respectively.

Seven Strains of Enterovirus D68 Detected in the United States during the 2014 Severe Respiratory Disease Outbreak.

Clusters of severe respiratory disease in the United States were reported to the CDC beginning in August 2014. Enterovirus D68 (EV-D68) was identified from 83% (30/36) of initial severe cases. Investigations in August and September found severe EV-D68 cases to be widespread across the United States. We report seven EV-D68 genomes from the outbreak.

An automated genotyping tool for enteroviruses and noroviruses.

BACKGROUND: Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. OBJECTIVE: To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. STUDY DESIGN: An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. RESULTS: Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. DISCUSSION AND CONCLUSIONS: The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons.

High degree of genetic diversity of non-polio enteroviruses identified in Georgia by environmental and clinical surveillance, 2002-2005.

Enterovirus surveillance data are useful for establishing temporal and geographical patterns of circulation and for virus characterization to determine phylogenetic relationships between strains. Almost no information is available on circulating enteroviruses in Georgia and the surrounding region. To describe enterovirus circulation in Georgia, determine relationships with previously characterized strains and assess the role of environmental and clinical enterovirus surveillance, this study analysed a total of 112 non-polio enterovirus isolates identified during 2002-2005 from sewage and human stool samples. Viruses were isolated in cell culture using standard methods and typed by partial sequencing of the VP1 gene. A total of 20 different non-polio enterovirus serotypes were identified over the 4-year period. The most commonly detected enteroviruses included echovirus (E) 6 (21 isolates; 18.8 %), E20, E3 and E7 (11 isolates each; 9.8 %), E11, coxsackievirus (CV) B4 and CVB5 (seven isolates each; 6.3 %), and E13, E19 and E30 (six isolates each; 5.4 %). Phylogenetic analysis showed that many serotypes were represented by more than one genetic lineage. The present study showed a very high degree of enterovirus diversity in Georgia and demonstrated the added value of environmental enterovirus surveillance, particularly in settings with limited clinical surveillance. Several serotypes would not have been detected without having both clinical and environmental surveillance in place. Several serotypes detected in Georgia were among those rarely reported in the USA and Europe (e.g. E3, E20 and E19). As the emergence of new genetic lineages of enterovirus in a particular area is often associated with large-scale outbreaks, continued monitoring of enterovirus strains by both environmental and clinical surveillance and genetic characterization should be encouraged.

Enteroviruses as agents of emerging infectious diseases.

Although the enteroviruses as a group are ubiquitous and not normally considered as "emerging pathogens," the many different serotypes circulate at different frequencies in any given year and the prevalence of a given serotype may fluctuate wildly from year to year. As a result, several enterovirus serotypes have been associated with the emergence of specific diseases (for example, pandemic acute hemorrhagic conjunctivitis) and specific serotypes have emerged to cause outbreaks of major public health concern. Enterovirus 71 is a recognized cause of epidemic severe central nervous system disease in Southeast Asia. Acute hemorrhagic conjunctivitis was a newly described disease in the 1970s associated with emergence of enterovirus 70 and coxsackievirus A24 variant. In addition, the impending eradication of poliovirus and some of the challenges currently faced by the eradication program present the possibility that poliomyelitis could emerge in the posteradication era. These links between enterovirus infections and emerging diseases are reviewed.

Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay.

The binding of the purified poliovirus RNA-dependent RNA polymerase to viral and nonviral RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. Optimal conditions for the binding of purified polymerase (fraction 5-PAS) to 32P-labeled poliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) much much greater than poly(U) greater than poly(C) greater than poly(A). In competitive binding studies, the polymerase bound with equal efficiency to virion RNA and to a subgenomic transcript which contained the 3' end of the genome. The polymerase bound to 18S ribosomal RNA and to globin mRNA equally well, but with a five-fold lower affinity than to virus-specific RNAs. The results suggest that the polymerase exhibits sequence specificity in binding and that polymerase binding sites in poliovirus RNA may contain (G- and/or U)-rich sequences.

Conservation of amino-acid sequence motifs in lentivirus Vif proteins.

The nonstructural/regulatory genes of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are believed to play an important role in the replication and pathogenesis of these viruses. In HIV-1 and other lentiviruses, the vif (viral infectivity factor) open reading frame (ORF) (also termed sor or Q in some lentivirus genomes) is located in the central region, overlapping the 3' end of the pol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks a vif ORF. The predicted Vif protein sequences from 38 lentiviruses were analyzed for the presence of global and local sequence similarity. The Vif proteins of closely related lentiviruses are highly conserved (HIV-1HXB2:HIV-1mn = 91% identity), while those of more distantly related lentirviruses have diverged significantly (HIV-1HXB2:simian immunodeficiency virusmax = 30% identity). A search for local sequence similarity revealed that a unifying feature of predicted lentivirus Vif proteins is the presence of at least one of two short, highly conserved sequence motifs, SL(I/V)X4YX9Y and SLQXLA. SLQXLA was present in 34 of 38 lentiviruses examined, while the remaining four lentiviruses had one (three viruses) or two (one virus) substitutions in this motif (of five total substitutions, three were conservative changes). The SL(I/V)X4YX9Y motif was found only in primate lentiviruses and in bovine immunodeficiency-like virus. Based on these findings, we suggest that the locus designation vif be used to denote all lentivirus ORFs previously called vif, Q, or sor.

Complete sequence of Venezuelan equine encephalitis virus subtype IE reveals conserved and hypervariable domains within the C terminus of nsP3.

The complete nuleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97--100% identity) and more distantly related to VEE strains of other antigenic varieties (83--93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94--95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1--329), which is highly conserved among all alphaviruses, was 93--94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330--563) was only 59--61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts.

Analysis of the transcription pattern and mapping of the putative rev and env splice junctions of bovine immunodeficiency-like virus.

The bovine immunodeficiency-like virus (BIV) genome contains the obligatory structural genes of all retroviruses and, in addition, the complex central region of lentiviruses; this novel region may code for at least five nonstructural/regulatory genes in BIV (K.J.Garvey, M.S. Oberste, J.E. Elser, M.J. Braun, and M.A. Gonda, Virology 175:391-409, 1990). As a prelude to determining the function of these novel open reading frames, the transcriptional pattern of BIV was studied by Northern analysis of RNA from BIV-infected cells. Five size classes of BIV-specific RNAs of 8.5, 4.1, 3.8, 1.7, and 1.4 kb were detected. The 8.5-kb RNA contains sequences from all regions of the genome; it is the virion RNA and probably serves as the gag-pol transcript as well. By using gene-specific probes, subgenomic viral RNAs of 3.8, 1.7, and 1.4 kb were tentatively identified as the env, tat, and rev spliced messages, respectively. The 4.1-kb RNA could not be unambiguously identified but may encode vif. The hybridization patterns of the putative tat and rev mRNAs suggest that they are the products of multiple splicing events. Discrete transcripts for the BIV W and Y central region open reading frames were not defined. The characterization of partial cDNA clones has permitted the mapping of the env and putative rev splice junctions.