Pan-Enterovirus Amplicon-Based High-Throughput Sequencing Detects the Complete Capsid of a EVA71 Genotype C1 Variant via Wastewater-Based Epidemiology in Arizona

We describe the complete capsid of a genotype C1-like Enterovirus A71 variant recovered from wastewater in a neighborhood in the greater Tempe, Arizona area (Southwest United States) in May 2020 using a pan-enterovirus amplicon-based high-throughput sequencing strategy. The variant seems to have been circulating for over two years, but its sequence has not been documented in that period. As the SARS-CoV-2 pandemic has resulted in changes in health-seeking behavior and overwhelmed pathogen diagnostics, our findings highlight the importance of wastewater-based epidemiology (WBE ) as an early warning system for virus surveillance.

Infectious Chikungunya Virus (CHIKV) with a Complete Capsid Deletion: a New Approach for a CHIKV Vaccine

ABSTRACTChikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemics of debilitating disease worldwide. Currently, there are no licensed vaccines or antivirals available against CHIKV infection. In this study, we generated a novel live attenuated vaccine (LAV) candidate for CHIKV with a complete deficiency of capsid (ΔC-CHIKV). It could propagate in BHK-21 cells, and had antigenic properties similar to those of native CHIKV. Vaccination of either immunocompromised IFNAR−/−mice or immunocompetent C57BL/6 mice with a single dose of ΔC-CHIKV conferred complete protection upon challenge with wild-type (WT) CHIKV. Taken together, this vaccine candidate appeared to be safe and efficacious, representing a novel strategy for CHIKV vaccine design.IMPORTANCECurrently, there is no licensed vaccine against CHIKV infection. An ideal CHIKV vaccine should generate an optimal balance between efficacy and safety. Live attenuated vaccines that can elicit strong immune responses often involve a trade-off of reduced safety. Here, a novel live attenuated vaccine candidate for CHIKV lacking the entire capsid gene, ΔC-CHIKV, was developed. It was demonstrated to be genetically stable, highly attenuated, immunogenic, and able to confer complete protection against lethal CHIKV challenge after a single dose of immunization. Such an infectious vaccine candidate devoid of capsid provides a novel strategy for the development of a live attenuated CHIKV vaccine.

Emergence of a new GII.17 norovirus variant in patients with acute gastroenteritis in Jiangsu, China, September 2014 to March 2015

From September 2014 to March 2015, 23 outbreaks of norovirus (NoV) acute gastroenteritis occurred in Jiangsu, China. Partial sequencing of the NoV capsid gene suggested that 16 of the 23 outbreaks were related to a new GII.17 variant. This variant was first detected in sporadic specimens in October 2014, and became predominant in February 2015. Analysis of the RNA-dependent RNA polymerase (RdRp), and complete capsid including the protruding domain P2 sequences confirmed this GII.17 variant as distinct from previously identified GII variants.

Emerging novel porcine parvoviruses in Europe: origin, evolution, phylodynamics and phylogeography

To elucidate the spatiotemporal phylodynamics, dispersion and evolutionary processes underlying the emergence of novel porcine parvovirus 2 (PPV2), PPV3 and PPV4 species, we analysed all available complete capsid genes, together with ours, obtained in Europe. Bayesian phylogeography indicates that Romania (PPV2 and PPV4) and Croatia (PPV3) are the most likely ancestral areas from which PPVs have subsequently spread to other European countries and regions. The timescale of our reconstruction supported a relatively recent history of the currently circulating novel PPV species (1920s to 1980s) in the domestic or sylvatic host. While PPV2 strains exhibited a large genetic exchange characterized by significant recombination and gene flow between distinct regions and hosts, PPV3 and PPV4 showed a diversification reflected by the accumulation of geographically structured polymorphisms. The RNA-like evolutionary rates detected inter- and intrahost recombination and the positive selection sites provided evidence that the PPV2–4 capsid gene plays a prominent role in host adaptation.

Genetic Analysis of the Capsid Gene of Genotype GII.2 Noroviruses

ABSTRACT Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank (n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands (n = 10) and in Osaka City, Japan (n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.

VP1 sequences of German porcine parvovirus isolates define two genetic lineages

In order to evaluate the genetic variability of Porcine parvovirus (PPV), the complete capsid protein sequences (VP1/VP2) from seven recent field isolates from Germany, one isolate from the UK and one German vaccine strain were sequenced and analysed, along with two American (NADL-2 and Kresse), three Asian and 22 Brazilian partial PPV sequences retrieved from GenBank. The analysis revealed a high degree of diversity: 1·2–2·6 % at the nucleotide level and 1·2–6·8 % at the amino acid level. Phylogenetic analysis defined two German clusters: one formed by four German isolates and the English, Asian and American sequences; and the second, distinct cluster formed by the other three of the seven German isolates examined. The latter cluster was still observed when the 22 partial sequences (853 nt of the 3′ terminus of the VP2 gene) from the Brazilian isolates were included in the analyses, indicating that the VP2 sequence determines the phylogeny.

Evidence of Recombination in the Norovirus Capsid Gene

ABSTRACT Noroviruses are single-stranded RNA viruses with high genomic variability. They have emerged in the last decade as a major cause of acute gastroenteritis. It remains so far unclear whether norovirus evolution is driven by sequence mutation and/or recombination. In this study, we have assessed the occurrence of recombination in the norovirus capsid gene. For this purpose, 69 complete capsid sequences of norovirus strains accessible in GenBank as well as 25 complete capsid sequences generated from norovirus-positive clinical samples were examined. Unreported recombination was detected in about 8% of norovirus strains belonging to genetic clusters I/1 (n = 1), II/1 (n = 1), II/3 (n = 1), II/4 (n = 3), and II/5 (n = 1). Recombination breakpoints were mainly located at the interface of the putative P1-1 and P2 domains of the capsid protein and/or within the P2 domain. The recombination region displayed features such as length, sequence composition (upstream and downstream GC- and AU-rich sequences, respectively), and predicted RNA secondary structure that are characteristic of homologous recombination activators. Our results suggest that recombination in the norovirus capsid gene may naturally occur, involving capsid domains presumably exposed to immunological pressure.

Enteroviruses 76, 89, 90 and 91 represent a novel group within the species Human enterovirus A

Molecular methods have enabled the rapid identification of new enterovirus (EV) serotypes that would have been untypable using existing neutralizing antisera. Nineteen strains of four new EV types termed EV76 (11 isolates), EV89 (two isolates), EV90 (four isolates) and EV91 (two isolates), isolated from clinical specimens from patients in France (one isolate) and Bangladesh (18 isolates), are described. Nucleotide sequences encoding the VP1 capsid protein (882–888 nt) are less than 65 % identical to the homologous sequences of the recognized human EV serotypes, but within each group the sequences are more than 78 % identical. The deduced amino acid sequences of the complete capsid (P1) region are more than 94 % identical within type but less than 76 % identical to those of the recognized serotypes. For both VP1 and P1, the 19 isolates are monophyletic by type with respect to all other EV serotypes. Using the proposed molecular typing scheme, these data support their identification as four new types within the species Human enterovirus A (HEV-A). In almost all cases, the VP1 sequences were more similar to those of some simian EVs than to the human EVs. Partial 3D sequences of all 19 isolates also clustered within HEV-A; they were monophyletic as a group, but not by type, suggesting that recombination has occurred among viruses of these four types. Partial 3D sequences were more closely related to those of simian EVs than to human viruses in HEV-A. These results suggest that the four new types may represent a new subgroup within HEV-A, in addition to the existing human and simian subgroups.

Molecular identification and characterization of two proposed new enterovirus serotypes, EV74 and EV75

Sequencing of the gene that encodes the capsid protein VP1 has been used as a surrogate for antigenic typing in order to distinguish enterovirus serotypes; three new serotypes were identified recently by this method. In this study, 14 enterovirus isolates from six countries were characterized as members of two new types within the species Human enterovirus B, based on sequencing of the complete capsid-encoding (P1) region. Isolates within each of these two types differed significantly from one another and from all other known enterovirus serotypes on the basis of sequences that encode either VP1 alone or the entire P1 region. Members of each type were ⩾77·2 % identical to one another (89·5 % amino acid identity) in VP1, but members of the two different types differed from one another and from other enteroviruses by ⩾31 % in nucleotide sequence (25 % amino acid sequence difference), indicating that the two groups represent separate new candidate enterovirus types. The complete P1 sequences differed from those of all other enterovirus serotypes by ⩾31 % (26 % amino acid sequence difference), but were highly conserved within a serotype (<8 % amino acid sequence difference). Phylogenetic analyses demonstrated that isolates of the same serotype were monophyletic in both VP1 and the capsid as a whole, as shown previously for other enterovirus serotypes. This paper proposes that these 14 isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).