Analysis the molecular similarity of least common amino acid sites in ACE2 receptor to predict the potential susceptible species for SARS-CoV-2.

SARS-CoV-2 infections in animals have been reported globally. However, the understanding of the complete spectrum of animals susceptible to SARS-CoV-2 remains limited. The virus's dynamic nature and its potential to infect a wide range of animals are crucial considerations for a One Health approach that integrates both human and animal health. This study introduces a bioinformatic approach to predict potential susceptibility to SARS-CoV-2 in both domestic and wild animals. By examining genomic sequencing, we establish phylogenetic relationships between the virus and its potential hosts. We focus on the interaction between the SARS-CoV-2 genome sequence and specific regions of the host species' ACE2 receptor. We analyzed and compared ACE2 receptor sequences from 29 species known to be infected, selecting 10 least common amino acid sites (LCAS) from key binding domains based on similarity patterns. Our analysis included 49 species across primates, carnivores, rodents, and artiodactyls, revealing complete consistency in the LCAS and identifying them as potentially susceptible. We employed the LCAS similarity pattern to predict the likelihood of SARS-CoV-2 infection in unexamined species. This method serves as a valuable screening tool for assessing infection risks in domestic and wild animals, aiding in the prevention of disease outbreaks.

Viral metagenomic analysis reveals diverse viruses and a novel bocaparvovirus in the enteric virome of snow leopard (Panthera uncia).

The enteric virome, comprising a complex community of viruses inhabiting the gastrointestinal tract, plays a significant role in health and disease dynamics. In this study, the fecal sample of a wild snow leopard was subjected to viral metagenomic analysis using a double barcode Illumina MiSeq platform. The resulting reads were de novo assembled into contigs with SOAPdenovo2 version r240. Additional bioinformatic analysis of the assembled genome and genome annotation was done using the Geneious prime software (version 2022.0.2). Following viral metagenomic analysis and bioinformatic analysis, a total of 7 viral families and a novel specie of bocaparvovirus tentatively named Panthera uncia bocaparvovirus (PuBOV) with GenBank accession number OQ627713 were identified. The complete genome of PuBOV was predicted to contain 3 open reading frames (ORFs), contains 5433 nucleotides and has a G + C content of 47.40 %. BLASTx analysis and pairwise sequence comparison indicated the novel virus genome was a new species in the genus Bocaparvovirus based on the species demarcation criteria of the International Committee on the Taxonomy of Viruses. This study provides valuable insights into the diversity and composition of the enteric virome in wild endangered snow leopards. The identification and characterization of viruses in wildlife is crucial for developing effective strategies to manage and mitigate potential zoonotic and other viral disease threats to human and animal health.

Gut phageome of the giant panda (Ailuropoda melanoleuca) reveals greater diversity than relative species.

The gut flora is a treasure house of diverse bacteriophages maintaining a harmonious and coexistent relationship with their hosts. The giant panda (Ailuropoda melanoleuca), as a vulnerable endemic species in China, has existed for millions of years and is regarded as a flagship species for biodiversity conservation. And yet, limited studies have analyzed the phage communities in the gut of giant pandas. Using viral metagenomic analysis, the phageomes of giant pandas and other relative species were investigated. Our study explored and compared the composition of phage communities from different animal sources. Giant pandas possessed more diverse and abundant phage communities in the gut compared with other relevant animals. Phylogenetic analyses based on the phage terminase large subunit (TerL) showed that the Caudovirales phages in giant pandas also presented highly genetic diversity. Our study revealed the diversity of phage communities in giant pandas and other relative species, contributing to the health maintenance of giant pandas and laying the groundwork for molecular evolution research of bacteriophages in mammals. IMPORTANCE Gut phageome plays an important role in shaping gut microbiomes by direct interactions with bacteria or indirect influences on the host immune system, potentially regulating host health and disease status. The giant panda (Ailuropoda melanoleuca) is a vulnerable and umbrella species for biodiversity conservation. Our work explored and compared the gut phageome of giant pandas and relative species, contributing to the health maintenance of giant pandas.

The evolutionary dynamics history of canine distemper virus through analysis of the hemagglutinin gene during 1930-2020.

Canine distemper virus (CDV) is a lethal viral disease of carnivores which is considered to be a serious threat to domestic and wild species. Despite the widespread use of vaccines, CDV still occurs in vaccinated animals and current vaccines does not guarantee complete protection. In this study, a total of 286 hemagglutinin (H) gene sequences of the virus isolated in 25 countries during 90 years (1930-2020) were analyzed by Bayesian maximum likelihood analysis to estimate the population dynamics. We identified the most recent common ancestor (TMRCA) of the virus in 1868 in the USA which arrived in continental Europe in 1948, and from there, the virus spread rapidly to other continents. The Canidae family was identified as the original host as well as a source of the subsequent spread. We identified 11 lineages of geographic co-circulating strains globally. The effective population size experienced a two-phase-exponential growth between 2000-2005 and 2010-2012. Our findings provide a novel insight into the epidemic history of canine distemper virus which may facilitate more effective disease management. This study uses a large set of sequencing data on the H gene of CDV to identify distinct lineages of the virus, track its geographic spread over time, analyze its likelihood of transmission within and between animal families, and provide suggestions for improved strategies to combat the virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s10344-023-01685-z.

Modeling the environmental suitability for Bacillus anthracis in the Qinghai Lake Basin, China.

Bacillus anthracis is a gram-positive, rod-shaped and endospore-forming bacterium that causes anthrax, a deadly disease to livestock and, occasionally, to humans. The spores are extremely hardy and may remain viable for many years in soil. Previous studies have identified East Qinghai and neighbouring Gansu in northwest China as a potential source of anthrax infection. This study was carried out to identify conditions and areas in the Qinghai Lake basin that are environmentally suitable for B. anthracis distribution. Anthrax occurrence data from 2005-2016 and environmental variables were spatially modeled by a maximum entropy algorithm to evaluate the contribution of the variables to the distribution of B. anthracis. Principal Component Analysis and Variance Inflation Analysis were adopted to limit the number of environmental variables and minimize multicollinearity. Model performance was evaluated using AUC (area under the curve) ROC (receiver operating characteristics) curves. The three variables that contributed most to the suitability model for B. anthracis are a relatively high annual mean temperature of -2 to 0°C, (53%), soil type classified as; cambisols and kastanozems (35%), and a high human population density of 40 individuals per km2 (12%). The resulting distribution map identifies the permanently inhabited rim of the Qinghai Lake as highly suitable for B. anthracis. Our environmental suitability map and the identified variables provide the nature reserve managers and animal health authorities readily available information to devise both surveillance strategy and control strategy (administration of vaccine to livestock) in B. anthracis suitable regions to abate future epidemics.

Spatial modeling and ecological suitability of monkeypox disease in Southern Nigeria.

The reemergence of monkeypoxvirus (MPXV) in 2017 after about 39 years of no reported cases in Nigeria, and the recent incidence in countries such as the United States of America, United Kingdom, Singapore, and Israel which have been reportedly linked with travelers from Africa, have heightened concern that MPXV may have emerged to occupy the vacant ecological and immunological niche created by the extinct smallpox virus. This study was carried out to identify environmental conditions and areas that are environmentally suitable (risky areas) for MPXV in southern Nigeria. One hundred and sixteen (116) spatially unique MPXV occurrence data from 2017-2021 and corresponding environmental variables were spatially modeled by a maximum entropy algorithm to evaluate the contribution of the variables to the distribution of the viral disease. A variance inflation analysis was adopted to limit the number of environmental variables and minimize multicollinearity. The five variables that contributed to the suitability model for MPXV disease are precipitation of driest quarter (47%), elevation (26%), human population density (17%), minimum temperature in December (7%), and maximum temperature in March (3%). For validation, our model had a high AUC value of 0.92 and standard deviation of 0.009 indicating that it had excellent ability to predict the suitable areas for monkeypox disease. Categorized risk classes across southern states was also identified. A total of eight states were predicted to be at high risk of monkeypox outbreak occurrence. These findings can guide policymakers in resources allocation and distribution to effectively implement targeted control measures for MPXV outbreaks in southern Nigeria.

Virome of Giant Panda-Infesting Ticks Reveals Novel Bunyaviruses and Other Viruses That Are Genetically Close to Those from Giant Pandas.

Tick infestations have been reported as one of the factors threatening the health of giant pandas, but studies of viral pathogens carried by ticks feeding on the blood of giant pandas are limited. To assess whether blood-sucking ticks of giant pandas can carry viral pathogens and if so, whether the viruses in ticks are associated with those previously detected in giant panda hosts, we determined the viromes of ticks detached from giant pandas in a field stocking area in Sichuan Province, southwest China. Using viral metagenomics we identified 32 viral species in ticks, half of which (including anellovirus [n = 9], circovirus [n = 3], and gemycircularvirus [n = 4]) showed homology to viruses carried by giant pandas and their associated host species (such as red pandas and mosquitoes) in the same living domain. Remarkably, several viruses in this study phylogenetically assigned as bunyavirus, hepe-like virus, and circovirus were detected with relatively high abundance, but whether these newly identified tick-associated viruses can replicate in ticks and then transmit to host animals during a blood meal will require further investigation. These findings further expand our understanding of the role of giant panda-infesting ticks in the local ecosystem, especially related to viral acquisition and transmission, and lay a foundation to assess the risk for giant panda exposure to tick-borne viruses. IMPORTANCE Ticks rank only second to mosquitoes as blood-feeding arthropods, capable of spreading pathogens (including viruses, bacteria, and parasites) to hosts during a blood meal. To better understand the relationship between viruses carried by ticks and viruses that have been reported in giant pandas, it is necessary to analyze the viromes of giant panda-parasitic blood-sucking ticks. This study collected 421 ticks on the body surface of giant pandas in Sichuan Province, China. We characterized the extensive genetic diversity of viruses harbored by these ticks and reported frequent communication of viruses between giant pandas and their ticks. While most of the virome discovered here are nonpathogenic viruses from giant pandas and potentially tick-specific viruses, we revealed some possible tick-borne viruses, represented by novel bunyaviruses. This research contributes to the literature because currently there are few studies on the virome of giant panda-infesting ticks.

Prevalence and multilocus genotyping of Giardia duodenalis in zoo animals in three cities in China.

Giardia duodenalis is a flagellated parasitic microorganism that parasitizes in the intestines of humans and animals. Although asymptomatic infections commonly exist in both humans and animals, some enteric symptoms have been reported in immunocompromised individuals, posing a threat to public health. Children could be infected with G. duodenalis through an environment contaminated by infective animals. Thus, the investigation of the prevalence and genotypes of G. duodenalis in zoo animals is important. In this study, 672 fecal samples of 113 species of animals, including non-human primates, artiodactyla, perissodactyla, proboscidian, marsupial, birds, carnivora, and rodents, were collected from three zoos in Hangzhou city, Dalian city, and Suzhou city in China. The samples were screened for the positivity of G. duodenalis by nested PCR based on the ß-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) gene loci. The overall G. duodenalis prevalence was 10.6% (71/672). The prevalence in non-human primates, artiodactyla, perissodactyla, proboscidian, marsupial, birds, carnivora, and rodent was 6.9% (10/144), 9.0% (12/133), 17.1% (6/35), 0% (0/6), 8.7% (2/23), 13.3% (28/211), 6.7% (7/105), and 40.0% (6/15), respectively. The region and category were considered risk factors for G. duodenalis infection in zoo animals (p < 0.001). Additionally, four genotypes of G. duodenalis were identified in zoo animals, including assemblage E (n = 46), assemblage A (n = 18), assemblage B (n = 6), and assemblage D (n = 1). The assemblages A, B, D, and E are also genotypes observed in humans and other animals. Among the sequences obtained in our study, one multilocus genotype (MLG) of the sub-assemblage AI was observed within assemblage A. Furthermore, three MLGs were detected within assemblage B. These findings reveal G. duodenalis genetic variability in zoo animals in three cities in China and suggest that zoo animals could be a potential source of human infection with G. duodenalis.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

Predicting the possibility of African horse sickness (AHS) introduction into China using spatial risk analysis and habitat connectivity of Culicoides.

African horse sickness (AHS) is a devastating equine infectious disease. On 17 March 2020, it first appeared in Thailand and threatened all the South-East Asia equine industry security. Therefore, it is imperative to carry out risk warnings of the AHS in China. The maximum entropy algorithm was used to model AHS and Culicoides separately by using climate and non-climate variables. The least cost path (LCP) method was used to analyze the habitat connectivity of Culicoides with the reclassified land cover and altitude as cost factors. The models showed the mean area under the curve as 0.918 and 0.964 for AHS and Culicoides. The prediction result map shows that there is a high risk area in the southern part of China while the habitats of the Culicoides are connected to each other. Therefore, the risk of introducing AHS into China is high and control of the border area should be strengthened immediately.

Parvovirus dark matter in the cloaca of wild birds.

With the development of viral metagenomics and next-generation sequencing technology, more and more novel parvoviruses have been identified in recent years, including even entirely new lineages. The Parvoviridae family includes a different group of viruses that can infect a wide variety of animals. In this study, systematic analysis was performed to identify the "dark matter" (datasets that cannot be easily attributed to known viruses) of parvoviruses and to explore their genetic diversity from wild birds' cloacal swab samples. We have tentatively defined this parvovirus "dark matter" as a highly divergent lineage in the Parvoviridae family. All parvoviruses showed several characteristics, including 2 major protein-coding genes and similar genome lengths. Moreover, we observed that the novel parvo-like viruses share similar genome organizations to most viruses in Parvoviridae but could not clustered with the established subfamilies in phylogenetic analysis. We also found some new members associated with the Bidnaviridae family, which may be derived from parvovirus. This suggests that systematic analysis of domestic and wild animal samples is necessary to explore the genetic diversity of parvoviruses and to mine for more of this potential dark matter.

Environmental suitability of Yersinia pestis and the spatial dynamics of plague in the Qinghai Lake region, China.

Plague, a highly infectious disease caused by Yersinia pestis, has killed millions of people in history and is still active in the natural foci of the world nowadays. Understanding the spatiotemporal patterns of plague outbreaks in history is critically important, as it may help facilitate the prevention and control for potential future outbreaks. This study's objective was to estimate the effect of the topography, vegetation, climate, and other environmental factors on the Y. pestis ecological niche. A maximum entropy algorithm spatially modelled plague occurrence data from 2004-2018 and the environmental variables to evaluate the contribution of the variables to the distribution of Y. pestis. Our results found that the average minimum temperature in September (-8 °C to +5 °C) and the sheep population density (250 sheep per km2) were influential in characterising the niche. The rim of Qinghai Lake showed more favourable conditions for Y. pestis presence than other areas within the study area. Identifying various factors will assist any future modelling efforts. Our suitability map identifies hotspots and will help public health officials in resource allocation in their quest to abate future plague outbreaks.

A predictive analysis on the risk of peste des petits ruminants in livestock in the Trans-Himalayan region and validation of its transboundary transmission paths.

Although the Trans-Himalayan region (THR) is an important endemic and rendezvous area of peste des petits ruminants (PPR), monitoring and prevention measurements are difficult to execute because of the rough geographical conditions. Besides, a heterogeneous breeding system and the poor veterinary service of susceptible animals compound the existing problems. Here, we propose a forecasting system to define the key points of PPR prevention and aid the countries in saving time, labor, and products to achieve the goal of the global eradication project of PPR. The spatial distribution of PPR was predicted in the THR for the first time using a niche model that was constructed with a combination of eco-geographical, anthropoid, meteorological, and host variables. The transboundary least-cost paths (LCPs) of small ruminants in the THR were also calculated. Our results reveal that the low-elevation area of the THR had a higher PPR risk and was mainly dominated by human variables. The high-elevation area had lower risk and was mainly dominated by natural variables. Eight LCPs representing corridors among India, Nepal, Bhutan, Bangladesh, and China were obtained. This confirmed the potential risk of transboundary communication by relying on PPR contamination on the grasslands for the first time. The predicted potential risk communication between the two livestock systems and landscapes (high and low elevation) might play a role in driving PPR transboundary transmission.

Transboundary spread of peste des petits ruminants virus in western China: A prediction model.

In pan Pamir Plateau countries, Peste des petits ruminants (PPR) has brought huge losses to the livestock industry and threaten the endangered wildlife. In unknown regions, revealing PPRV transmission among countries is the premise of effective prevention and control, therefore calls for quantified monitoring on disease communication among countries. In this paper, a MaxEnt model was built for the first time to predict the PPR risk within the research area. The least cost path (LCP) for PPR transboundary communication were calculated and referred to as the maximum available paths (MAP). The results show that there are many places with high-risk in the research area, and the domestic risk in China is lower than that in foreign countries and is mainly determined by human activities. Five LCPs representing corridors among Kazakhstan, Tajikistan, Pakistan, India and China were obtained. This study proves for the first time that there is the possibility of cross-border transmission of diseases by wild and domestic animals. In the future, it will play an important role in monitoring the PPR epidemic and blocking-up its cross-border transmission.

Prevalence and subtypes of Blastocystis sp. infection in zoo animals in three cities in China.

Blastocystis is a highly prevalent eukaryotic parasite of many animals and humans worldwide. It can compromise the gastrointestinal tract and cause gastrointestinal symptoms, constituting a serious threat to human health and animal growth. Many animals are potential sources of Blastocystis infection in humans. However, limited data are available regarding the prevalence and subtype distribution of Blastocystis infection among zoo animals in China. Therefore, the present study examined the prevalence and subtypes of Blastocystis in zoo animals in Hangzhou, Dalian, and Suzhou cities, China. Of 450 fecal samples from zoo animals, 27 (6.0%) were PCR-positive for Blastocystis, with 7.7% (8/104), 11.3% (7/62), 16.7% (3/18), 1.8% (2/114), 6.3% (1/16), 9.5% (2/21), and 3.6% (4/109) in artiodactyla, aves, rodentia, nonhuman primates, perissodactyla, marsupialia, and carnivora, respectively. Significant differences in the prevalence of Blastocystis were found among different animal groups (P < 0.05). Sequence analysis showed 7 known subtypes (ST2, ST4, ST5, ST7, ST8, ST10, and ST14) of Blastocystis in the present study, with ST10 (10/27) as the predominant subtype in all three of the examined zoos. To our knowledge, this is the first report of Blastocystis infection in Damaliscus dorcas, Cervus elaphus, Macropus rufogriseus, Grus japonensis, Trichoglossus haematodus, Panthera tigris ssp. tigris (white), Panthera tigris ssp. altaica, Lycaon pictus, Suricata suricatta, and Dolichotis patagonum in China. These results demonstrate the presence of Blastocystis infection in zoo animals and provided baseline data for preventing and controlling Blastocystis infection in zoo animals and humans in China.

Molecular detection and identification of tick-borne bacteria and protozoans in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China.

BACKGROUND: Small ruminants are important hosts for various tick species and tick-associated organisms, many of which are zoonotic. The aim of the present study was to determine the presence of tick-borne protozoans and bacteria of public health and veterinary significance in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. METHODS: The occurrence of piroplasms, Anaplasma phagocytophilum, A. bovis, A. marginale, A. capra, A. ovis, Ehrlichia spp. and spotted fever group rickettsiae was molecularly investigated and analyzed in 134 goats and 9 free ranging C. pygargus living in close proximity. RESULTS: Piroplasm DNA was detected in 16 (11.9%) goats and 5 C. pygargus. Sequence analysis of 18S rRNA sequences identified 3 Theileria species (T. luwenshuni, T. capreoli and T. cervi). Four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) were identified in goats and C. pygargus. Anaplasma ovis and A. bovis were detected in 11 (8.2%) and 6 (4.5%) goats, respectively; A. phagocytophilum, A. bovis and A. capra were found in 3, 7 and 3 C. pygargus, respectively. Sequence analysis of 16S rRNA sequences revealed the presence of 5 different genetic variants of A. bovis in goats and C. pygargus, while the analysis of 16S rRNA and gltA sequence data showed that A. capra isolates identified from C. pygargus were closely related to the genotype identified from sheep and Haemaphysalis qinghaiensis, but differed with the genotype from humans. Anaplasma/Theileria mixed infection was observed in 2 (1.5%) goats and 5 C. pygargus, and co-existence involving potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. All samples were negative for A. marginale, Ehrlichia spp. and SFG rickettsiae. CONCLUSIONS: These findings report the tick-borne pathogens in goats and C. pygargus, and a greater diversity of these pathogens were observed in wild animals. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The contact between wild and domestic animals may increase the potential risk of spread and transmission of tick-borne diseases.

A novel picornavirus in feces of a rainbow lorikeet (Trichoglossus moluccanus) shows a close relationship to members of the genus Avihepatovirus.

A novel picornavirus, named "lorikeet picornavirus 1" (LoPV-1), was detected in a fecal sample from rainbow lorikeets using viral metagenomic analysis, and its complete genome sequence was determined and analyzed. The genome of LoPV-1 is 7862 nt long, including a 617-nt 5' UTR, a type IV IRES 5'UTR with an '8-like' motif, a 7032-nt polyprotein ORF, and a 213-nt 3' UTR. Phylogenetic analysis and pairwise asequence comparisons based on the amino acid sequences of P1, P2, and P3 indicated that LoPV-1 showed the closest relationship to two picornaviruses that were isolated recently from red-crowned cranes and clustered together with members of the genus Avihepatovirus.

Asian black bear (Ursus thibetanus) picornavirus related to seal aquamavirus A.

The complete genome of a bear picornavirus 1 (BePV-1) in the viscera of an Asian black bear (Ursus thibetanus) from China was characterized using viral metagenomics and RT-PCR/Sanger sequencing. The genome of BePV1 is 6703 nt long, contains a type-IV IRES 5'UTR with the '8-like' motif, encodes a 2053-aa-long polyprotein showing a 3-4-4 organization pattern and two 2A genes. BePV-1 showed the highest overall genome nucleotide sequence identity of 71.7% to a picornavirus genome from an Arctic ringed seal (Phoca hispida) from Canada, classified as a member of the species Aquamavirus A, currently the only one in the genus Aquamavirus. Phylogenetic and genetic distance analyses of P1 and 3D indicated that Asian bear picornavirus (aquamavirus B) represents the second sequenced member of the genus Aquamavirus.

Analysis of codon usage bias in the VP1 gene of the human norovirus GII.2 genotype.

To investigate the codon usage patterns of all available VP1 gene sequences of the GII.2 genotype, to determine the factors that affect these patterns, and to provide comprehensive details of the characteristics and evolution of the gene. Complete 519 sequences of VP1 gene of the HuNoV GII.2 genotype with known sampling dates and geographic locations from 1971 - 2017 were retrieved from the GenBank nucleotide database of the National Center for Biotechnology Information (NCBI) and analyzed. The percentage composition of T, C, A, and G nucleotides were 24.80 ± 0.30, 26.61 ± 0.31, 25.84 ± 0.13, and 22.75 ± 0.17 %, respectively, with C and A relatively more abundant than T and G, and C the most abundant (p < 0.0001). The values of T3s (34.10 ± 0.90 %) and C3s (33.54 ± 0.90 %) were significantly higher than those of A3s (29.98 ± 0.43 %) and G3s (24.13 ± 0.51 %) (p < 0.0001). While T3s was highest among the four nucleotides, G3s was the lowest. Among the 18 most frequently employed synonymous codons, six optional codons ended with T, five ended with C, five ended with A and two ended with G. Codons ending with T were the most frequently used. The ENC ranged from 51.90 to 54.25 (mean = 52.38 ± 0.43) among the 519 VP1 gene sequences. There were significant correlations between ENC and C % and G % (p < 0.01). Codons containing CpG (1 and 2 or 2 and 3 codon positions) showed the lowest frequencies, while 30, 29, and 2 codons were above, below and on the mean line, respectively. The first four principal components accounted for 69.11 % of the total variation, with the first, second, third, and fourth principal axes contributing 37.90, 14.83, 9.61, and 6.77 %, respectively. The strains were not clustered by country of isolation or year of sampling. Gravy were significantly correlated with T3s, C3s, G3s, GC3s, and ENC (p < 0.01). Mutation pressure and natural selection contributed to the codon usage bias of the VP1 gene of the HuNoV GII.2 genotype. There was a correlation between GC12s and GC3s (R2 = 0.032; p < 0.0001). The relative neutrality was 3.20 %, while natural selection was 96.80 %. The VP1 gene exhibits low codon usage bias which is affected primarily by natural selection, followed by mutation pressure and translational selection.