High divergence of human astrovirus genotypes circulating in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2017–2020

Human astrovirus (HAstV) is one of the common causes of acute gastroenteritis in children. The investigation of molecular epidemiology of HAstV is essential for monitoring the emergence and/or re-emergence of new HAstV genotypes, as well as understanding the evolution of HAstV circulating in children suffering from acute gastroenteritis. The present study aimed to investigate the prevalence and distribution of HAstVs strains circulating in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand during 2017–2020. A total of 1500 fecal specimens collected from children with acute gastroenteritis were screened for HAstV by RT-PCR that targeted the partial RdRp in ORF1b and strains were characterized by sequencing and phylogenetic analysis. Of the 1500 fecal samples, 39 (2.6%) were positive for HAstV. Of these, both classic and novel HAstV genotypes, including classic HAstV1–HAstV5, novel HAstV-MLB1, MLB2, and HAstV-VA2, were detected. The data in this study revealed a high divergence of HAstV genotypes circulating in pediatric patients admitted to the hospitals with acute gastroenteritis in Chiang Mai, Thailand during 2017–2020.

Virological and Epidemiological Features of Norovirus Infections in Brazil, 2017–2018

Noroviruses are considered an important cause of acute gastroenteritis (AGE) across all age groups. Here, we investigated the incidence of norovirus, genotypes circulation, and norovirus shedding in AGE stool samples from outpatients in Brazil. During a two-year period, 1546 AGE stool samples from ten Brazilian states were analyzed by RT-qPCR to detect and quantify GI and GII noroviruses. Positive samples were genotyped by dual sequencing using the ORF1/2 junction region. Overall, we detected norovirus in 32.1% of samples, with a massive predominance of GII viruses (89.1%). We also observed a significant difference between the median viral load of norovirus GI (3.4×105 GC/g of stool) and GII (1.9×107 GC/g). The most affected age group was children aged between 6 and 24 m old, and norovirus infection was detected throughout the year without marked seasonality. Phylogenetic analysis of partial RdRp and VP1 regions identified six and 11 genotype combinations of GI and GII, respectively. GII.4 Sydney[P16] was by far the predominant genotype (47.6%), followed by GII.2[P16], GII.4 Sydney[P31], and GII.6[P7]. We detected, for the first time in Brazil, the intergenogroup recombinant genotype GIX.1[GII.P15]. Our study contributes to the knowledge of norovirus genotypes circulation at the national level, reinforcing the importance of molecular surveillance programs for future vaccine designs.

Astrovirus in Reunion Free-Tailed Bat (Mormopterus francoismoutoui)

Astroviruses (AstVs) are RNA viruses infecting a large diversity of avian and mammalian species, including bats, livestock, and humans. We investigated AstV infection in a free-tailed bat species, Mormopterus francoismoutoui, endemic to Reunion Island. A total of 380 guano samples were collected in a maternity colony during 38 different sampling sessions, from 21 June 2016 to 4 September 2018. Each sample was tested for the presence of the AstV RNA-dependent RNA-polymerase (RdRp) gene using a pan-AstV semi-nested polymerase chain reaction assay. In total, 27 guano samples (7.1%) tested positive, with high genetic diversity of the partial RdRp gene sequences among positive samples. Phylogenetic analysis further revealed that the detected viruses were genetically related to AstVs reported in rats, reptiles, dogs, and pigs, but did not cluster with AstVs commonly found in bats. Although more investigations need to be conducted to assess the prevalence of infected bats in the studied population, our findings show that Reunion free-tailed bats are exposed to AstVs, and suggest that cross-species transmission may occur with other hosts sharing the same habitat.

Molecular Survey on Kobuviruses in Domestic and Wild Ungulates From Northwestern Italian Alps

Since the first identification in 1989 in humans, kobuviruses (KoVs) have been identified from a wide range of animal species including carnivores, rodents, birds, ungulates, rabbits, and bats. Several studies have described the identification of genetically related KoVs in the fecal virome of domestic and wild animals suggesting a mutual exchange of viruses. By screening a total of 231 fecal samples from wild and domestic ungulates, KoVs RNA was detected in wild boars (3.2%; 2/63), chamois (4.6%; 2/43), and goats (2.6%; 2/77). On phylogenetic analysis of the partial RdRp sequence, the wild boar strains clustered within the species Aichivirus C whilst the strains identified in domestic and wild ruminants grouped into the species Aichivirus B. The complete VP1 gene was obtained for chamois and goat KoVs. Interestingly, upon phylogenetic analysis the strains grouped together with a KoV of ovine origin within a distinct genetic type (B3) of the species Aichivirus B.

Major Concerns on the Identification of Bat Coronavirus Strain RaTG13 and Quality of Related Nature Paper

A recent manuscript (Zhou, P. et al. “A pneumonia outbreak associated with a new coronavirus of probable bat origin”, Nature 579, 270–273 (2020). https://doi.org/10.1038/s41586-020-2012-7) from Wuhan Institute of Virology claimed the identification of a bat coronavirus, RaTG13, which showed 96.2% genome homology with SARS-CoV-2. In this paper, we raise the puzzling observations surrounding the identification, characterization, unique genome features of this RaTG13 strain, as well as its 100% nucleotide identity in partial RdRp gene with another bat coronavirus strain BtCoV/4991. And the paper presented premature hypothesis of potential bat origin of SARS-CoV-2 while RaTG13 strain was not successfully isolated. We also present the concerns on the methodology, data quality and experiment procedures described in this paper. We call for the authors to provide additional data, to share related samples to be verified and further characterized by other scientists.

Partial RdRp sequences offer a robust method for Coronavirus subgenus classification

The recent reclassification of the Riboviria, and the introduction of multiple new taxonomic categories including both subfamilies and subgenera for coronaviruses (family Coronaviridae, subfamily Orthocoronavirinae) represents a major shift in how official classifications are used to designate specific viral lineages. While the newly defined subgenera provide much-needed standardisation for commonly cited viruses of public health importance, no method has been proposed for the assignment of subgenus based on partial sequence data, or for sequences that are divergent from the designated holotype reference genomes. Here, we describe the genetic variation of a partial region of the coronavirus RNA-dependent RNA polymerase (RdRp), which is one of the most used partial sequence loci for both detection and classification of coronaviruses in molecular epidemiology. We infer Bayesian phylogenies from more than 7000 publicly available coronavirus sequences and examine clade groupings relative to all subgenus holotype sequences. Our phylogenetic analyses are largely coherent with genome-scale analyses based on designated holotype members for each subgenus. Distance measures between sequences form discrete clusters between taxa, offering logical threshold boundaries that can attribute subgenus or indicate sequences that are likely to belong to unclassified subgenera both accurately and robustly. We thus propose that partial RdRp sequence data of coronaviruses is sufficient for the attribution of subgenus-level taxonomic classifications and we supply the R package, “MyCoV”, which provides a method for attributing subgenus and assessing the reliability of the attribution.Importance StatementThe analysis of polymerase chain reaction amplicons derived from biological samples is the most common modern method for detection and classification of infecting viral agents, such as Coronaviruses. Recent updates to the official standard for taxonomic classification of Coronaviruses, however, may leave researchers unsure as to whether the viral sequences they obtain by these methods can be classified into specific viral taxa due to variations in the sequences when compared to type strains. Here, we present a plausible method for defining genetic dissimilarity cut-offs that will allow researchers to state which taxon their virus belongs to and with what level of certainty. To assist in this, we also provide the R package ‘MyCoV’ which classifies user generated sequences.

Astrovirus in Reunion Free-tailed Bat (Mormopterus francoismoutoui)

Astroviruses (AstVs) are RNA viruses responsible for infection of a large diversity of avian and mammalian species, including bats, livestock, and humans. We investigated AstV infection in a free-tailed bat species, Mormopterus francoismoutoui, endemic to Reunion Island. A total of 190 guano samples were collected in a maternity colony during 19 different sampling sessions, between June 2016 and June 2017. Biological material was tested for the presence of the AstV RNA-dependent RNA-polymerase (RdRp) gene with a pan-AstV semi-nested polymerase chain reaction assay. In total, 15 guano samples (7.9%) tested positive, with high genetic diversity of the partial RdRp gene sequences among positive samples. A phylogenetic analysis further revealed that the detected viruses were genetically related to AstVs reported in reptiles, dogs, and pigs, but did not cluster with AstVs commonly found in bats. Although more investigation need to be conducted to assess the level of infected bats in the studied population, our findings suggest that Reunion free-tailed bats are exposed to AstV, and that cross-species transmission may occur with other hosts sharing the same habitat.

Singleseed detection of an uncharacterised Betaflexiviridae virus in Actinidia

A sensitive conventional RTPCR assay has been developed for an uncharacterised virus within the family Betaflexiviridae prompted by detection of the virus in Actinidia seedlings held in postentry quarantine that had been grown from openpollinated seed Infection is asymptomatic and presently assessed as lowtozero biosecurity risk The assay targets the RNAdependent RNA polymerase (RdRP) of the virus primer sequences (BetaF2 and BetaR2 278bp amplicon) being based on a partial RdRP sequence for the virus The assay detects the nine virus isolates found to date in a range of sample types (fresh leaf stem cambium archived RNA) The assay which includes a multiplexed internal control has been accredited by International Accreditation New Zealand for Actinidia testing To support management of the virus the assay and associated RNA extraction method have been applied to testing dried Actinidia seed directly They have been found sufficiently sensitive to detect the virus in individual seeds Initial results show efficient transmission of the virus from infected pollen to the seed

Molecular Identification and Characterization of Tomato zonate spot virus in Tobacco in Guangxi, China

In Guangxi Province of southwest China, diseases caused by Tospoviruses (family Bunyaviridae) pose a serious threat to tobacco (Nicotiana tobacum) production. During surveys conducted annually at Xinrong Village in Jingxi County from 2008 to 2010, more than 130 ha of fields were found to have 10 to 50% of plants exhibiting symptoms similar to spotted wilt caused by Tomato spotted wilt virus (TSWV). During this period, disease symptoms at similar prevalence and incidence were also found at Fushan, Debao County in most of the cultivars produced in these areas, including Yunyan 85, 87, 92, 97, and K326. Symptoms on tobacco varied but commonly included dwarfing, midrib browning, distorted apical buds, and concentric ringspots that coalesced to form large areas of dead leaf tissue. Mechanical inoculation from diseased tobacco leaves with concentric ringspots back to tobacco cv. Yunyan 85 or 87, resulted in 12 of 16 plants with symptoms similar to those observed in the field. No symptoms on plants developed following inoculation with buffer only. Symptoms found in the field resembled those caused by TSWV. However, testing using TSWV-specific antiserum was shown to be negative by double-antibody sandwich-ELISA (Agdia, Elkhart, IN). Total RNA was extracted from 27 diseased tobacco plants collected from different regions in Guangxi using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA extracts were amplified by reverse transcription (RT)-PCR using the degenerate primers T2740 (ATGGGDATNTTTGATTTCATG) and T3920c (TCATGCTCATSAGRTAAATYTCTCT) designed to target the partial RNA-dependent RNA polymerase (RdRp) sequence of members in the genus Tospovirus (3). Amplification was performed at 42°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 7-min final extension at 72°C. A PCR product of approximately 1.2 kb was amplified from 21 diseased plants. RT-PCR amplicons were cloned into the pUC19-T Simple Vector (TaKaRa, Dalian, China) and sequenced in both directions. Sequences were assembled and analyzed by DNAStar 5.01 (DNASTAR, Madison, WI). Sequences of representative isolates were deposited in GenBank (Accession Nos. JN020022 to JN020027). The 1.2-kb partial RdRp sequences of these isolates were shown to have 94.4 to 95.3% nucleotide identity and 96.5 to 97.5% amino acids identity to Tomato zonate spot virus (TZSV) (GenBank Accession No. NC_010491) (1). Among these TZSV isolates from Guangxi, the partial RdRp sequences have 98.0 to 99.4% nucleotide identity and 98.8 to 100% amino acids identity with each other. The presence of TZSV was further confirmed in diseased tobacco plants by indirect ELISA using antiserum of TZSV (provided by Prof. Zhongkai Zhang, Agricultural Academy of Yunnan, China). TZSV has been characterized as a novel tospovirus on various hosts including tobacco in Yunnan province (1,2). To our knowledge, this is the first report of TZSV-associated disease on tobacco in Guangxi Province, southwest China. Further work is necessary to study the epidemiology and management of the disease. References: (1) J. Dong et al. Arch. Virol. 153:855, 2008. (2) J. Dong et al. J. Insect Sci. 10:166, 2010. (3) Y. Lin. Master Thesis. National Chung Hsing University, Taichung, Taiwan, Republic of China, 2007.