GPCR Gene Research Articles

A FMRFamide-like neuropeptide FLP-12 signaling regulates head locomotive behaviors in C. elegans

Neuropeptides play a critical role in regulating behaviors across organisms, but the precise mechanisms by which neuropeptides orchestrate complex behavioral programs are not fully understood. Here, we show that the FMRFamide-like neuropeptide FLP-12 signaling from the SMB head motor neurons, modulates head locomotive behaviors, including stomatal oscillation in C. elegans. lim-4 mutants, in which the SMB neurons are not properly specified, exhibited various head and body locomotive defects, including stomatal oscillation. Chronic activation or inhibition of neuropeptidergic signaling in the SMB motor neurons resulted in a decrease or increase in stomatal oscillation, respectively. The flp-12 neuropeptide gene is expressed and acts in the SMB neurons to regulate head and body locomotion, including stomatal oscillation. Moreover, the frpr-8 GPCR and gpa-7 Gα genes are expressed in the AVD command interneurons to relay the FLP-12 signal to mediate stomatal oscillation. Finally, heterologous expression of FRPR-8 either Xenopus oocytes or HEK293T cells conferred FLP-12 induced responses. Taken together, these results indicate that the C. elegans FMRFamide neuropeptide FLP-12 acts as a modulator of stomatal oscillation via the FRPR-8 GPCR and the GPA-7 G-protein.

The repertoire of G‐protein‐coupled receptor variations in the Japanese population 54KJPN

AbstractG‐protein‐coupled receptors (GPCRs) are the largest superfamily in the human genome and the major targets for the market drugs. Recent massive genomics studies revealed numerous natural variations in the general population. 54KJPN is the most extensive Japanese population genomics study, curating the whole genome sequences from about 54,000 individuals. Here, by analyzing 390 non‐olfactory GPCR genes in the 54KJPN dataset, we annotated 25,443 missense single‐nucleotide variations. Among them, we found 120 major variations that appear with an allele frequency greater than 0.5, including variations that occurred on posttranslational modification sites. Structural alignment of GPCRs using the generic numbering system in the GPCRdb reveals enrichment of alterations in the conserved arginine residue within the DRY motif, which contributes to downstream G‐protein signaling. A comparison with the worldwide 1000 Genomes Project (1KGP) dataset found 23 variations that were present exclusively in the 54KJPN dataset. This study will be the basis for future pharmacogenomics studies for the Japanese population.

Deciphering the genetic landscape of lumpy skin disease: Unraveling variable virulence through comprehensive genome sequence analysis in India

Lumpy Skin Disease (LSD), a poxvirus disease affecting cattle, emerged in India in 2019 and intensified in 2022, resulting in significant economic losses for dairy farmers. There was unusual shift in mortality and morbidity patterns during the second wave. A comprehensive genetic study conducted, analyzing samples from 2019 to 2022 revealed circulation of two distinct subclades (subclade 1.2a and 1.2b) in India, with the latter showing a different pattern in morbidity and mortality. Notably, the Ankyrin repeats gene-based analysis could differentiate animals with varying clinical scores. Genetic variations were significant, with unique deletions identified, including a 12-nucleotide deletion in the GPCR gene in virus isolates collected during 2022 outbreaks, not reported earlier in Indian LSDV strains. A crucial finding was a significant 95-nucleotide deletion in the Functional Resolution Sequence (FRS) repeats of LSDV genomes from 2022 outbreaks, absent in 2019 samples. These deletions may have influenced the virus's virulence in India.

Molecular characterization of lumpy skin disease virus in North Central Vietnam during 2021 and early 2022.

In October 2020, the first outbreaks of lumpy skin disease (LSD) in Lang Son Province, Vietnam were reported by our laboratory. The disease had rapidly spread to the South, and it was reported in 55 of 63 provinces and cities of Vietnam by the end of 2021. The most economic loss caused by this disease occurred in the north-central region in 2021 where approximately 46,788 LSD virus (LSDV) infected cattle and buffaloes have been reported and 8,976 animals have been culled. However, the information on this pathogen circulating in this region is missing. Here, we describe the molecular characterization of LSDV circulating in north-central Vietnam in 2021 and early 2022. In total, 155 LSDV samples were collected during this period and three of these samples from each province were further characterized by Sanger sequencing analysis based on three key maker genes (GPCR, RPO30, and p32). Sequence comparison and phylogenetic analysis based on GPCR, RPO30, and p32 genes indicated that LSDV strains circulating in north-central Vietnam are closely related to previously reported strains in Vietnam regions which bordered China and all LSDV strains were 100% identical. These results show the importance of continuous monitoring and characterization of circulating LSDV strains and are important for vaccine development for the control and eradication of LSD in Vietnam.

Emergence of lumpy skin disease virus (LSDV) infection in domestic Himalayan yaks (Bos grunniens) in Himachal Pradesh, India.

In this study, we investigated and confirmed natural lumpy skin disease virus (LSDV) infection in Himalayan yaks (Bos grunniens) in Himachal Pradesh, India, based on clinical manifestations and results of genome detection, antibody detection, virus isolation, and nucleotide sequencing. Subsequent phylogenetic analysis based on complete GPCR, RPO30, and EEV gene sequences revealed that the LSDV isolates from these yaks and local cattle belonged to LSDV subcluster 1.2.1 rather than the dominant subcluster 1.2.2, which is currently circulating in India, suggesting a separate recent introduction. This is the first report of natural LSDV infection in yaks in India, expanding the known host range of LSDV. Further investigations are needed to assess the impact of LSDV infection in yaks.

Isolation and molecular characterization of lumpy skin disease virus from Tamil Nadu, India during the outbreaks from 2020 to 2022.

Lumpy skin disease (LSD) caused by LSD virus is a WOAH notifiable, high-impact, transboundary poxviral disease of bovines. The first official report of LSDV in India is from Odisha state during August 2019. Since then, cases have been reported from many states including Tamil Nadu, a Southern state of India. The present study deals with isolation and molecular characterization of LSDV from Tamil Nadu during the period August 2020 to July 2022. LSDV was isolated in embryonated chicken eggs (ECE) and BHK 21 cells and was characterized based on P32, RPO30, and GPCR genes. The phylogenetic analysis revealed that Tamil Nadu isolates from India are closely related to other Indian strains, Kenyan strains and strains from neighboring countries such as Bangladesh, Nepal, and Myanmar confirming the common exotic source for the transboundary spread across borders. The presence of unique signature of amino acid (aa) at specific positions (A11, T12, T34, S99, and P199) in the GPCR sequence confirmed the identity of LSDV. A twelve nucleotide (nt94-105) insertion and corresponding aa (TILS) at 30-33 position was found in GPCR sequence and characteristic amino acid proline at 98 position (P98) in the RPO30 gene sequence of our isolates was similar to strains from Bangladesh, Nepal, and Myanmar. Further, dissimilarity of our isolates from Neethling like vaccine strains confirms the circulation of virulent filed strains responsible for the outbreaks.

G-Protein Coupled Receptor Gpr-1 Is Important for the Growth and Nutritional Metabolism of an Invasive Bark Beetle Symbiont Fungi Leptographium procerum.

Leptographium procerum has been demonstrated to play important roles in the invasive success of red turpentine beetle (RTB), one of the most destructive invasive pests in China. Our previous studies found that bacterial volatile ammonia plays an important role in the maintenance of the RTB-L. procerum invasive complex. In this study, we found a GPCR gene Gpr-1 that was a response to ammonia but not involved in the ammonia-induced carbohydrate metabolism. Deletion of Gpr-1 significantly inhibited the growth and pathogenicity but thickened the cell wall of L. procerum, resulting in more resistance to cell wall-perturbing agents. Further analyses suggested that Gpr-1 deletion caused growth defects that might be due to the dysregulation of the amino acid and lipid metabolisms. The thicker cell wall in the ΔGpr-1 mutant was induced through the cell wall remodeling process. Our results indicated that Gpr-1 is essential for the growth of L. procerum by regulating the nutritional metabolism, which can be further explored for potential applications in the management of RTB.

Characterization and molecular identification of the lumpy skin disease virus in cattle in the Mekong Delta of Vietnam.

Lumpy skin disease (LSD) is caused by a virus belonging to the genus Capripoxvirus, exhibiting clinical symptoms ranging from mild signs to the development of nodules. LSD emerged in Asia and Southeast Asia, including Vietnam, in October 2020 and has since spread throughout the region, resulting in productivity and economic losses. This study aimed to investigate the virus-causing papular dermatitis in cattle from the Mekong Delta region of Vietnam by analyzing its GPCR gene and assessing its evolutionary relationship with sequences in the GenBank database. Blood samples (n = 180) were collected from cattle farms in Ben Tre, Tien Giang, and Tra Vinh provinces. PCR targeting the P32 antigen gene was utilized to detect LSDV presence, and GPCR gene amplification was performed to assess genetic variability. LSDV was detected in 8.33% (15/180) of the samples using PCR targeting the P32 antigen gene. Each sample that tested positive for LSDV demonstrated complete amplification of the GPCR gene. Sequence alignments and phylogenetic analyses of the GPCR gene revealed that Mekong Delta LSDV isolates shared genetic similarities and possessed a 12-nucleotide insertion comparable to strains from China in 2019 and Northern Vietnam in 2020. This study provides preliminary insights into the molecular characteristics of LSDV in cattle from the Mekong Delta region of Vietnam. The observed genetic relatedness to other LSDV sequences from Asia and Southeast Asia underscores the importance of regional surveillance and control measures. These findings contribute to the development of effective strategies for LSDV control and prevention.

Emergence of Lumpy Skin Disease Virus Infection in Yaks, Cattle‐Yaks, and Cattle on the Qinghai–Xizang Plateau of China

Lumpy skin disease (LSD) is a viral disease caused by lumpy skin disease virus (LSDV), which mainly infects cattle and can cause huge economic losses. In May 2023, yaks, cattle‐yaks, and cattle in Tibet (Xizang), China, developed fever, skin nodules, and severe discharges and were suspected to be cases of LSD. Samples from these animals were analyzed using molecular biology and serological methods. The RPO30, P32, and GPCR genes were amplified by PCR and sequenced, and the whole genome of the virus was determined using viral metagenomics technology. Sequencing results showed that it was indeed an LSDV infection, and enzyme‐linked immunosorbent assay results confirmed the presence of LSDV antibodies. The whole genome phylogenetic tree shows that LSDV/CHINA/Tibet/2023 is different from the previous epidemic strains in China, but clusters with India 2022 strain. This is the first report of LSD in yaks, cattle‐yaks, and cattle on the highest altitude plateau in the world.

A new variant of lumpy skin disease virus circulating in Vietnam based on sequencing analysis of GPCR gene.

In 2021, Vietnam experienced an outbreak of Lumpy skin disease (LSD), which infected 207,687 cattle and buffaloes, as officially reported, and resulted in the culling of 29,182 animals. In this study, samples from cattle that died and showed typical signs of LSD in the Ha Tinh province of Vietnam were confirmed by three World Organization for Animal Health (WOAH)-recommended methods and further studied to compare the Vietnam and China reference strains to the new clinical cases. Three methods recommended by WOAH for agent detection (PCR, virus isolation, and transmission electron microscopy) were used to confirm this clinical LSD case. The sequence analysis of three well-known markers (P32, RPO30, and GPCR genes) has been utilized in Vietnam to understand this circulating pathogen better. Our findings showed that the CX01 LSDV strain is 100% identical to the Vietnam reference strain HL01 and China reference strains based on P32 and RPO30 genes. Interestingly, analysis of the nucleotide sequence of the GPCR gene showed that the CX01 strain belongs to the same cluster as the reference strains, but it has branches different from those of both the HL01 and China LSDV strains. The nucleotide identification between the CX01 strain and these reference virus strains ranked 99.65%-99.91%, suggesting that it is a new variant of LSDV. This finding is new and indicates that at least two variants of the LSD virus were circulating in Vietnam based on analysis of the GPCR gene. Additionally, these results suggest that the sequence analysis of the GPCR gene is a great tool for subgrouping LSDV circulating in Vietnam.

Lumpy Skin Disease (LSD) in Yak (Bos grunniens): An Evidence of Species Spillover from Cattle in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus (LSDV), is a global concern that affects cattle and buffalo. Recently, the disease has been reported in new species such as the Indian Gazelle, Camel, Banteng, Gaur, and Giraffe from various parts of the world. This report provides an insight into the occurrence of LSD in Yak from Sikkim, a North-Eastern state of India. During the investigation, both cattle and yak exhibited typical clinical signs of LSD, including skin nodular lesions. The morbidity, mortality, and case fatality rates for cattle were 9.08%, 1.84%, and 20.24%, respectively. Similarly, the morbidity, mortality, and case fatality rates in yak were 7.57%, 1.24%, and 16.33%, respectively. The virus isolation and amplification of LSDV-specific genes confirmed the presence of LSDV in cattle, yak, and vectors. Further, demonstrated antibodies in randomly collected sera from naïve and unvaccinated cattle and yak using indirect Enzyme Linked Immuno-sorbent Assay (iELISA) and Serum Neutralisation test (SNT) from this region. Sequencing and phylogenetic analysis of P32, GPCR, and RPO30 genes revealed that the virus isolated from both species was 100% identical to each other and also closely related to the field LSDV isolates circulating in the Indian subcontinent. The study highlighted the emergence of LSDV in unconventional hosts and underscored the need to include other bovine species in national disease control programs, encompassing disease surveillance initiatives.

Molecular detection and phylogenetic analysis of recent isolate of lumpy skin disease virus from clinical cases in Menofia governorate between 2018 to 2020 based on GPCR gene.

Molecular detection and phylogenetic analysis of recent isolate of lumpy skin disease virus from clinical cases in Menofia governorate between 2018 to 2020 based on GPCR gene.

Genetic relatedness between recent field isolate strain of LSDV and other field and vaccinal strains based on GPCR, ORF095 and ORF103 genes in Egypt 2020

Genetic relatedness between recent field isolate strain of LSDV and other field and vaccinal strains based on GPCR, ORF095 and ORF103 genes in Egypt 2020

Identification and expression analysis of G protein-coupled receptors in the cotton aphid, Aphis gossypii Glover

G protein-coupled receptors play important roles in mediating signal transformation and physiological processes. As a new type of insecticide target, GPCRs have attracted much attention in recent years. However, GPCRs have not yet been identified in Aphis gossypii. In the present study, a total of 87 GPCRs were identified from A. gossypii, including 65 Family A, 12 Family B, 7 Family C, and 3 Family F receptors. Most of the GPCRs in A. gossypii showed considerable sequence identity, and all of them have conserved transformmembrane domains. Newly identified GPCR genes were differentially expressed in different developmental stages and tissues. Moreover, we found that 34 GPCR genes were highly overexpressed in a sulfoxaflor-resistant strain, 4 and 10 of them were highly induced by LC15 and LC50 of sulfoxaflor, respectively. Furthermore, silencing of two highly overexpressed GPCRs by RNAi indicated that suppression the expression of AgoGPCR48 and AgoGPCR53 significantly increased the susceptibility of A. gossypii to sulfoxaflor, suggesting that these GPCR genes may be associated with sulfoxaflor resistance in A. gossypii. Our results imply that the overexpression of GPCR genes contribute to the sulfoxaflor resistance development in A. gossypii and provide useful targets for developing novel insecticides to manage this pest.

Rapid Spread and Genetic Characterisation of a Recently Emerged Recombinant Lumpy Skin Disease Virus in Thailand.

Simple SummaryLumpy skin disease (LSD) is an economically important disease of cattle caused by LSD virus (LSDV), a member of poxviruses. It had never been found in Thailand before March 2021, but has since spread broadly to various provinces. Regional veterinarians have collected samples from the LSD cattle and submitted them for diagnosis as a part of disease surveillance during the outbreaks. Our study aimed to monitor the distribution of the outbreaks by recording the LSD cases based on clinical signs and laboratory tests up to June 2022, and characterise the causative agent virologically and genetically. Outbreak maps were created to illustrate the rapid temporal distribution of the LSD index cases in each province of Thailand. We detected two distant origins of the outbreaks. LSDV DNA was confirmed in blood, milk, and skin samples collected from sick animals by real-time PCR. LSDV was proven to be the causative virus based on serological, virological, and pathological diagnoses. By genetic analysis, the Thai LSDV is a recombinant virus derived from a vaccine strain previously appearing in China and Vietnam. Its genetic material is a mosaic hybrid genome containing the vaccine virus DNA as the backbone interspersed with DNA fragments of a field strain.The emergence of the lumpy skin disease virus (LSDV) was first detected in north-eastern Thailand in March 2021. Since then, the abrupt increase of LSD cases was observed throughout the country as outbreaks have spread rapidly to 64 out of a total of 77 provinces within four months. Blood, milk, and nodular skin samples collected from affected animals have been diagnosed by real-time PCR targeting the p32 gene. LSDV was isolated by primary lamb testis (PLT) cells, followed by Madin-Darby bovine kidney (MDBK) cells, and confirmed by immunoperoxidase monolayer assay (IPMA). Histopathology and immunohistochemistry (IHC) of a skin lesion showed inclusion bodies in keratinocytes and skin epithelial cells. Phylogenetic analyses of RPO30 and GPCR genes, and the whole genome revealed that Thai viruses were closely related to the vaccine-derived recombinant LSDV strains found previously in China and Vietnam. Recombination analysis confirmed that the Thai LSDV possesses a mosaic hybrid genome containing the vaccine virus DNA as the backbone and a field strain DNA as the minor donor. This is an inclusive report on the disease distributions, complete diagnoses, and genetic characterisation of LSDV during the first wave of LSD outbreaks in Thailand.

Molecular characterization of a novel subgenotype of lumpy skin disease virus strain isolated in Inner Mongolia of China

BackgroundThe outbreak of Lumpy skin disease (LSD) in cattle caused by LSD virus (LSDV) was first reported in August 2019 in China. Since then, several LSD outbreaks have been reported in seven different provinces of China. Until now, several Lumpy skin disease virus (LSDV) strains from China have been reported and sequenced including LSDV/Xinjiang/2019 (MN598005.1), China/GD01/2020 (MW355944.1), and LSDV/Hongkong/2021 (MW732649.1). In October 2020, more than 1,700 cattle imported from Chile arrived in Xilingol, Inner Mongolia, and were diagnosed with LSD. Currently, limited data on the origin of the virus is available.MethodsNucleotide sequences of the ORF11, ORF36, ORF74, ORF117, ORF126 genes and the complete genome of LSDV strains and isolates were downloaded from NCBI database. MEGA7.0 was used to perform phylogenetic analysis with Neighbor-Joining (NJ). DNASTAR software is used to analyze homologous comparison analysis with related genes of reference strains included in Genbank.ResultsCompared with other strains isolated from China, the results of full genome sequence analysis showed the LSDV/NMG/2020 strain belonged to the recombinant strains. The LSDV/NMG/2020 strain is different from the current LSDV field isolates in Africa, the Middle East, Europe, and the newly emerged LSDV Russia variants. Based on the identities of P32, RPO30, EEV, GPCR and LSDV117 genes (99.8%, 99%, 99.8%, 99% and 98.7%), the sub-cluster recombinant containing LSDV/NMG/2020 strain is phylogenetically closer to the Russia strain (Saratov/2017).ConclusionsIn this study, we reported a new isolated LSDV strain named LSDV/NMG/2020. The results of genomic characterization and phylogenetic analysis demonstrated that the LSDV/NMG/2020 isolate was a vaccine-like recombinant strain.

Phylogenetic analysis of lumpy skin disease virus isolates in Russia in 2019-2021

Lumpy skin disease continues to pose a threat to countries in the East and Asia-Pacific regions. Although only occasional LSDV outbreaks have been reported recently in Russia, these have been mainly restricted to the Far East region of the country. An increase in the number of outbreaks in South East Asia has been attributed to recombinant vaccine-like LSDV strains. In this scenario, it is epidemiologically important to perform phylogenetic analysis to track the distribution of LSDV worldwide at the genetic level to understand routes of migration and molecular evolution patterns. In this study, we investigated the RPO30 and GPCR gene regions of LSDV isolates associated with outbreaks in 2019-2021 in Siberia and the Far East region of Russia. The inferred phylogeny confirms the recombinant origin of these sequenced isolates. Based on sequences of these selected loci, the isolates from 2019 differed from isolates detected in Russia in the past and from isolates from Asian countries, while the isolates from 2020 and 2021 exhibited a high degree of similarity to the Asian isolates. These findings indicate that recombinant LSDV strains continue to persist and additionally point to the establishment of a specific lineage of recombinant isolates in the region. Full genome sequencing is still needed to gain more information about how the circulating isolates are related to each other.

Different responses to risperidone treatment in Schizophrenia: a multicenter genome-wide association and whole exome sequencing joint study

Risperidone is routinely used in the clinical management of schizophrenia, but the treatment response is highly variable among different patients. The genetic underpinnings of the treatment response are not well understood. We performed a pharmacogenomic study of the treatment response to risperidone in patients with schizophrenia by using a SNP microarray -based genome-wide association study (GWAS) and whole exome sequencing (WES)-based GWAS. DNA samples were collected from 189 patients for the GWAS and from 222 patients for the WES after quality control in multiple centers of China. Antipsychotic response phenotypes of patients who received eight weeks of risperidone treatment were quantified with percentage change on the Positive and Negative Syndrome Scale (PANSS). The GWAS revealed a significant association between several SNPs and treatment response, such as three GRM7 SNPs (rs141134664, rs57521140, and rs73809055). Gene-based analysis in WES revealed 13 genes that were associated with antipsychotic response, such as GPR12 and MAP2K3. We did not identify shared loci or genes between GWAS and WES, but association signals tended to cluster into the GPCR gene family and GPCR signaling pathway, which may play an important role in the treatment response etiology. This study may provide a research paradigm for pharmacogenomic research, and these data provide a promising illustration of our potential to identify genetic variants underlying antipsychotic responses and may ultimately facilitate precision medicine in schizophrenia.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

Genomic characterization of lumpy skin disease virus in southern China.

Lumpy skin disease virus (LSDV) is of high economic importance and has spread rapidly to many European and Asian countries in recent years. LSDV was introduced to China in 2019 and have caused severe outbreaks in several provinces. Here, we detected an LSDV strain (GD01/2020) from a cattle farm with typical LSD symptoms in Guangdong, southern China using a novel quantitative real-time PCR assay targeting the viral GPCR gene. We obtained the whole genomic sequence of GD01/2020 through metagenomic analysis. The GD01/2020 was highly homologous to the LSDVs isolated in Xinjiang, China in 2019, and distinct from all the LSDVs identified in other countries, in their sequences of GPCR and RPO30 genes. The GD01/2020 was a vaccine-recombinant strain, but distinct from two recombinant LSDVs identified in Russia. At least 25 putative recombination events between a vaccine strain and a field strain were identified in the genome of GD01/2020, which could affect the virulence and transmissibility of the virus. These results suggested that a virulent vaccine-recombinant LSDV from an unknown origin was introduced into Xinjiang, China in 2019 and spread to Guangdong, China in 2020.