Chinese Strains Research Articles

Polymorphism of P66 in Borrelia Burgdorferi Strains in China

ObjectiveTo study the polymorphism in P66 and its human B-cell epitopes of Borrelia burgdorferi strains in China. MethodsPolymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese B. burgdorferi. Then the sequences were analyzed by MEGA 5.10 software and compared with the human B-cell epitope sequences from the Immune Epitope Database (IEDB) based on the reference strain of each genotype. ResultsResults showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in Borrelia garinii (B.g) and Borrelia afzelii (B.a) strains. B.g strains were divided into three subclusters and two scattered strains JC1-7 and JC2-2 according to the amino acid sequences of P66. The P66 sequences of 15 Xinjiang strains represented by XI91-12 in the B.g subcluster 1, changed from CAA to TAA codon at 508aa position, resulting in early termination. Bases A and C were inserted at sequence position 1 523 bp of strains FP1, LB20, LB21, and SZ21 in the B.a genotype, which resulted to early termination at position 511 aa. G base was inserted at 438 bp of LIP94-11 strain, which led to early termination at position 172 aa. ConclusionIn P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of B.g strains had a certain regional character. One of the characteristics of Xinjiang B.g isolates might be the variation at the 508aa location in 15 Xinjiang B.g strains, which may be related to the strains’ pathogenicity in this area.

Profiling of Brevetoxin Metabolites Produced by Karenia brevis 165 Based on Liquid Chromatography-Mass Spectrometry.

In this study, Karenia brevis 165 (K. brevis 165), a Chinese strain, was used to research brevetoxin (BTX) metabolites. The sample pretreatment method for the enrichment of BTX metabolites in an algal culture medium was improved here. The method for screening and identifying intracellular and extracellular BTX metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The results show that the recovery rates for BTX toxins enriched by a hydrophilic–lipophilic balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTX metabolites at different growth stages of K. brevis 165. This is the first time a Chinese strain of K. brevis has been reported that can produce toxic BTX metabolites. Five and eight kinds of BTX toxin metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites. The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages show that the total toxic BTX metabolite contents in single cells ranged between 6.78 and 21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27 and 449.11 μg/L. There were significant differences in the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

Differential expression of TgMIC1 in isolates of Chinese 1 Toxoplasma with different virulence

BackgroundThe predominant genotype of Toxoplasma in China is the Chinese 1 (ToxoDB#9) lineage. TgCtwh3 and TgCtwh6 are two representative strains of Chinese 1, exhibiting high and low virulence to mice, respectively. Little is known regarding the virulence mechanism of this non-classical genotype. Our previous RNA sequencing data revealed differential mRNA levels of TgMIC1 in TgCtwh3 and TgCtwh6. We aim to further confirm the differential expression of TgMIC1 and its significance in this atypical genotype.MethodsQuantitative real-time PCR was used to verify the RNA sequencing data; then, polyclonal antibodies against TgMIC1 were prepared and identified. Moreover, the invasion and proliferation of the parasite in HFF cells were observed after treatment with TgMIC1 polyclonal antibody or not.ResultsThe data showed that the protein level of TgMIC1 was significantly higher in high-virulence strain TgCtwh3 than in low-virulence strain TgCtwh6 and that the invasion and proliferation of TgCtwh3 were inhibited by TgMIC1 polyclonal antibody.ConclusionDifferential expression of TgMIC1 in TgCtwh3 and TgCtwh6 may explain, at least partly, the virulence mechanism of this atypical genotype.Graphical

Molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014 to 2019.

The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses.

Potential of waste reduction of agro-biomasses through Reishi medicinal mushroom (Ganoderma lucidum) production using different substrates and techniques

One of the most important priorities in the medicinal mushrooms cultivation is the selection and preparation of the substrate, which depends on factors such as price and easy access to different lignocellulosic compounds. In order to investigate the effect of mixed and non-mixed agro-waste substrates on some vegetative, reproductive, nutritional and medicinal properties of two Iranian and Chinese strains of Ganoderma lucidum, the factorial experiment was conducted as a completely randomized design with three replications. The experimental substrates of different poplar wood chip (PWC), wheat bran (WB), soybean meal (SM), date palm leaf (DPL) and olive oil extraction waste (OOEW) were used. They were enriched with chemical supplements (ammonium nitrate, manganese sulfate and Nano manganese oxide) using two cultivation techniques (open bag door and Polika-Cotton) for fruiting body production.The results showed that various substrates significantly affect dry weight, moisture content quantity, vegetative and fruit yield, total ganoderic acids content, total polysaccharide value and mineral elements amount (nitrogen, potassium and calcium) in fruiting body. The highest amount of N content in the fruiting body of both Iranian and Chinese strains (5.88 and 6.25 mg/100 g dry matter) was dedicated to the combined PWC + SM (70:30) substrate enriched with Nano manganese oxide supplement and Polika-Cotton method. The highest total fresh of Iranian and Chinese strains weight (109.60 and 127.66 g, respectively) obtained in the combined PWC + WB (70:30) substrate enriched with manganese sulfate. The highest amount of total ganoderic acids in fruiting organs in both strains (0.52 and 0.42 μg/mg dry matter) is achieved using a combined PWC + SM (70:30) enriched with manganese sulfate.

Analysis of the evolution and transmission dynamics of the field MDV in China during the years 1995-2020, indicating the emergence of a unique cluster with the molecular characteristics of vv+MDV that has become endemic in southern China.

Marek's disease (MD) continues to threaten the sustainability of the world poultry industry. In this study, the sequences of the meq gene of 220 MDV strains isolated during the years 1964-2020 were analysed, including 50 from our group plus 170 isolates from the GenBank. Analyses, using phylogenetic trees, amino acid (aa)-mutation screening, evolutionary studies and transmission dynamics were all performed. All strains were divided into two clusters (Clusters 1 and 2), and Cluster 1 includes the mild strains, the vaccine strains and the foreign virulent strains, while Cluster 2 was dominated by the Chinese field strains. Our study identified that the Chinese field strains in Cluster 2 during the years 1995-2020 likely originated in the 1980s from abroad, and the estimated genetic diversity of these strains experienced two growth phases in the years 2005-2007.5 and 2015-2017. Viral phylogeography identified 3 major geographic provincial regions for the Chinese field strains of Cluster 2: the Northeastern Region (Jilin, Liaoning and Heilongjiang), the East-central Region (Henan, Shandong and Jiangsu) and the Southern Region (Guangxi, Guangdong and Yunnan). The spread of Northeastern strains to East-central chicken flocks and the further spread from Guangxi to Guangdong are strongly indicated. The emergence of the mutations A88T and Q93R together in the Southern strains during the years 2017-2020 with molecular characteristics of vv+MDV were also found later than those in the Northern strains. Overall, the Chinese field strains in Cluster 2 in southern China in recent years have been rapidly evolving. Guangxi Province has become an epicentre for these viruses and the chicken flocks in the Southern region have been facing the adverse effects of the emerging vv+MDV.

Isolation and Identification of a Recombinant Porcine Epidemic Diarrhea Virus With a Novel Insertion in S1 Domain.

Porcine epidemic diarrhea virus (PEDV) is the major pathogen that causes diarrhea and high mortality in newborn piglets with devastating impact to the pig industry. Recombination and mutation are the main driving forces of viral evolution and genetic diversity of PEDV. In 2016, an outbreak of diarrhea in piglets occurred in an intensive pig farm in Central China. A novel PEDV isolate (called HNAY) was successfully isolated from clinical samples. Sequence analysis and alignment showed that HNAY possessed 21-nucleotide (nt) insertion in its S1 gene, which has never been reported in other PEDV isolates. Moreover, the sequence of the insertion was identical with the sequence fragment in PEDV N gene. Notably, the HNAY strain exhibited two unique mutations (T500A and L521Y) in the neutralizing epitopes of the S1 protein that were different from those of other PEDV variant strains and CV777-based vaccine strains. Additionally, PEDV HNAY might be derived from a natural recombination between two Chinese variant PEDV strains. Animal experiments demonstrated that HNAY displayed higher pathogenicity compared with two other clinical isolates. This study lays the foundation for better understanding of the genetic evolution and molecular pathogenesis of PEDV.

Detection of Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Slovenia.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains—three detected in non-vaccinated flocks and one in a flock vaccinated against ILT—were identical or very similar to the chicken embryo–origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

Canine Parvovirus Infections in Taiwanese Pangolins (Manis pentadactyla pentadactyla).

Canine parvovirus type 2 (CPV-2) is among the most important and highly contagious pathogens that cause enteric or systemic infections in domestic and nondomestic carnivores. However, the spillover of CPV-2 to noncarnivores is rarely mentioned. Taiwanese pangolins (Manis pentadactyla pentadactyla) are threatened due to habitat fragmentation and prevalent animal trafficking. Interactions between Taiwanese pangolins, humans, and domestic animals have become more frequent in recent years. However, information about the susceptibility of pangolins to common infectious agents of domestic animals has been lacking. From October 2017 to June 2019, 4 pangolins that were rescued and treated in wildlife rescue centers in central and northern Taiwan presented with gastrointestinal signs. Gross and histopathological examination revealed the main pathologic changes to be necrotic enteritis with involvement of the crypts in all intestinal segments in 2 pangolins. By immunohistochemistry for CPV-2, there was positive labeling of cryptal epithelium throughout the intestine, and immunolabeling was also present in epidermal cells adjacent to a surgical amputation site, and in mononuclear cells in lymphoid tissue. The other 2 pangolins had mild enteritis without crypt involvement, and no immunolabeling was detected. The nucleic acid sequences of polymerase chain reaction (PCR) amplicons from these 4 pangolins were identical to a Chinese CPV-2c strain from domestic dogs. Quantitative PCR revealed a higher ratio of CPV-2 nucleic acid to internal control gene in the 2 pangolins with severe intestinal lesions and positive immunoreactivity. Herein, we present evidence of CPV-2 infections in pangolins.

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus.

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

Whole genome comparison of Pakistani Corona virus with Chinese and US Strains along with its predictive severity of COVID-19

Initially submitted 784 SARS-nCoV2 whole genome sequences on NCBI Virus database were selected for phylogenetic analysis to look into their similarities with two of Pakistani sequenced coronavirus strains having accessions of MT240479 and MT262993. The MT240479 named (Gilgit1-Pak) was found in close proximity to MT184913 named (CruiseA-USA), while MT262993 named (Manga-Pak) was in neighboring to MT039887 named (WI-USA) strain, which were further chosen for variant calling analysis along with reference genome NC_045512 as out-group to construct concluding cladogram and looked for evolutionary distance with PAUP software in this article. Aforementioned Pakistani strains each of having 29,836 bases were compared with MT263429 (WI-USA) of 29,889 bases and MT259229 (Wuhan-P.R. China) of 29,864 bases. Whole genome variant calling pipeline revealed 31 variants in both Pakistani strains collectively (Manga-Pak vs USA having 2del & 7SNPs, while different from Chinese strain with 2del & 2SNPs, similarly Gilgit1-Pak vs USA having 10SNPs, while different from Chinese strains having 8SNPs). These variants harbour ORF1ab, ORF1a and N genes having their role is viral replication/translation, host innate immunity and viral capsid formation respectively. These novel variants may be one of the reasons for low mortality rate in Pakistan with 385 deaths as compared to USA with 63,871 and P.R. China with 4633 by May 01, 2020. However functional characterization of these variants and their integrations with other viral proteins including variability of human receptors (ACE2 & NRP1) may be the other reasons for unlikely COVID-19 statistics in Pakistan which need further confirmatory studies. Moreover, mutated N and ORF1a proteins in Pakistani strains were also analyzed by 3D structure modeling, which give another dimension of comparing these alterations at amino acid level. In a nutshell, these novel variants are correlated with reduced mortality of COVID-19 severity in Pakistan while more robust results can be obtained by wet lab experimentation. This also gives insight of genomic landscape of these indigenous strains to develop diagnostics kits, vaccines and therapeutic interventions.

A New Strain of Virus Discovered in China Specific to the Parasitic Mite Varroa destructor Poses a Potential Threat to Honey Bees.

The ectoparasitic mite, Varroa destructor, feeds directly on honey bees and serves as a vector for transmitting viruses among them. The Varroa mite causes relatively little damage to its natural host, the Eastern honey bee (Apis cerana) but it is the most devastating pest for the Western honey bee (Apis mellifera). Using Illumina HiSeq sequencing technology, we conducted a metatranscriptome analysis of the microbial community associated with Varroa mites. This study led to the identification of a new Chinese strain of Varroa destructor virus-2 (VDV-2), which is a member of the Iflaviridae family and was previously reported to be specific to Varroa mites. A subsequent epidemiological investigation of Chinese strain of VDV-2 (VDV-2-China) showed that the virus was highly prevalent among Varroa populations and was not identified in any of the adult workers from both A. mellifera and A. cerana colonies distributed in six provinces in China, clearly indicating that VDV-2-China is predominantly a Varroa-adapted virus. While A. mellifera worker pupae exposed to less than two Varroa mites tested negative for VDV-2-China, VDV-2-China was detected in 12.5% of the A. mellifera worker pupae that were parasitized by more than 10 Varroa mites, bringing into play the possibility of a new scenario where VDV-2 could be transmitted to the honey bees during heavy Varroa infestations. Bioassay for the VDV-2-China infectivity showed that A. cerana was not a permissive host for VDV-2-China, yet A. mellifera could be a biological host that supports VDV-2-China’s replication. The different replication dynamics of the virus between the two host species reflect their variation in terms of susceptibility to the virus infection, posing a potential threat to the health of the Western honey bee. The information gained from this study contributes to the knowledge concerning genetic variabilities and evolutionary dynamics of Varroa-borne viruses, thereby enhancing our understanding of underlying molecular mechanisms governing honey bee Varroosis.

A recombinant Chinese measles virus vaccine strain rMV-Hu191 inhibits human colorectal cancer growth through inducing autophagy and apoptosis regulating by PI3K/AKT pathway

The potential therapeutic effects of oncolytic measles virotherapy have been verified against plenty of malignancies. However, the oncolytic effects and underlying mechanisms of the recombinant Chinese measles virus vaccine strain Hu191 (rMV-Hu191) against human colorectal cancer (CRC) remain elusive. In this study, the antitumor effects of rMV-Hu191 were evaluated in CRC both in vitro and in vivo. From our data, rMV-Hu191 induced remarkably caspase-dependent apoptosis and complete autophagy in vitro. In mice bearing CRC xenografts, tumor volume was remarkably suppressed and median survival was prolonged significantly with intratumoral treatment of rMV-Hu191. To gain further insight into the relationship of rMV-Hu191-induced apoptosis and autophagy, we utilized Rapa and shATG7 to regulate autophagy. Our data suggested that autophagy was served as a protective role in rMV-Hu191-induced apoptosis in CRC. PI3K/AKT signaling pathway as one of the common upstream pathways of apoptosis and autophagy was activated in CRC after treatment with rMV-Hu191. And inhibition of PI3K/AKT pathway using LY294002 was accompanied by enhanced apoptosis and decreased autophagy which suggested that PI3K/AKT pathway promoted rMV-Hu191-induced autophagy and inhibited rMV-Hu191-induced apoptosis. This is the first study to demonstrate that rMV-Hu191 could be used as a potentially effective therapeutic agent in CRC treatment. As part of the underlying cellular mechanisms, apoptosis and autophagy were involved in the oncolytic effects generated by rMV-Hu191. And the cross-talk between these two processes and the PI3K/AKT signaling pathway was well identified.

Morpho-molecular and mating-type locus diversity of Ustilaginoidea virens: an incitant of false smut of rice from Southern parts of India.

To characterize the geo-distinct isolates of Ustilaginoidea virens for morpho-molecular and mating-type locus diversity. Sixty-one isolates of U.virens collected from Southern India exhibited significant diversity in mycelial width (3·45-5·50µm), colony colour (yellow, pale yellow, and white), and growth pattern (thick leather mat, raised-fluffy, flat-fluffy, and raised). Field-borne chlamydospores of each isolate were significantly smaller in size (3·34-5·26µm2 ) compared to those formed on culture media (18·6-100·89µm2 ). The phylogenetic study based on internal transcribed sequences revealed two clusters; however, most isolates (n=54) were grouped in cluster-I, indicating common ancestral origin. We also identified 42 haplotypes; among them, Hap_3 has the highest number of isolates (n=19). Mating-type locus (MAT1) analysis revealed all sixty-one isolates as heterothallic, wherein 37 and 24 isolates belonging to MAT1-1-1 and MAT1-2-1 heterothallic mating types, respectively. The microsynteny analysis of MAT1 loci of one of the Indian strain (Uv-Gvt) along with Uv-8b (China) strain revealed synteny conservation at MAT1 locus, which is flanked by conserved genes SLA2 and a hypothetical protein in the upstream and APN2, COX12 and APC5 in the downstream of the locus. Morpho-molecular study revealed the significant diversity among geo-distinct isolates, and MAT1 loci analysis indicated the distribution of heterothallic mating types in south Indian paddy fields. And also, complete synteny conservation between Indian and Chinese strain was observed at the MAT1 locus. This is the first report describing the sexuality of Indian strains of the U.virens, which would help better understand the genetic diversity of the U.virens prevailing in Southern India and aid in developing resistant rice cultivars against this pathogen population.

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018-2019.

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.

Identification and genomic characterization of emerging goose astrovirus in central China, 2020.

Astroviruses are a non-enveloped virus with large host range breadth. AstV-associated gastroenteritis in human and animal, nephritis in chicken, gout in gosling and hepatitis in duckling pose great threats to public health and poultry industry. Since early 2020, continuous emergence of fatal goose astrovirus (GAstV) infections characterized by articular and visceral gout was reported in China. Here, we described two outbreaks of emerging gout disease in two different goose farms of central China. Two virulent GAstV strains, designated as HNKF-1/China/2020 and HNSQ-6/China/2020, were isolated, and the fifth passage of the isolates could cause urate crystals accumulated in the allantoic fluid and even deposited around great vessels and embryo bodies. Meanwhile, the source of these GAstV outbreaks was tracked to goose hatcheries. The prevalence of GAstV in the goose embryos with hatch failure was confirmed, and embryo-origin HNXX-6/China/2020 was further isolated. The complete genome of these three newly isolates was then sequenced and analysed. The results showed that Chinese GAstVs have formed two distinct groups, and the three GAstV isolates, as well as most of the Chinese GAstVs, belong to the G-I group. There are several amino acid mutations in the three newly identified GAstVs, such as A520T, S535R, V555I and A782T in ORF1a and Q229P in ORF2, suggesting the field stains, HNKF-1/China/2020 and HNSQ-6/China/2020, might derive from the weak goose embryo via vertical transmission. Moreover, the phylogenetic analysis of the complete viral genome and individual viral proteins revealed that Chinese GAstV strains have been constantly evolving towards more complicated and various directions. Our study reported the recently emerging GAstV outbreaks in central China, and further analysed the genetic characteristics of three virulent G-I GAstV isolates from commercial goose farms and goose hatchery, indicating the diverse transmission of the virus and providing a basis for developing effective preventive measures and control strategies.

Isolation and evolutionary analysis of Senecavirus A isolates from Guangdong province, China.

Senecavirus A (SVA), an emerging swine pathogen, has been reported in many provinces of China since the first outbreak in 2015 in Guangdong province. In this study, 10 lymph nodes positive for SVA, collected between 2018 and 2019 from slaughterhouses in Guangdong province, were subjected to virus isolation. Rapid and evident cytopathic effects (CPEs) were observed in SVA-infected PK-15 cells, including shrinking, rounding and detaching, with peak titers being reached at 24h post infection (hpi). Electron microscopy showed that SVA particles are spherical and approximately 30nm in diameter, and exist as crystalline lattices in cytoplasm revealed by ultra-thin sectioning. Phylogenetic analysis based on the whole genome sequences of all available isolates showed that SVA globally can be divided into two groups with each being further divided into two subgroups (Ia-b and IIa-b), and with the Guangdong isolates obtained here and other Chinese strains belonging to subgroups IIa and IIb. Evolutionary analysis showed that the mean substitution rate of SVA was 2.696×10-3 per site per year based on whole genomic sequences, with subgroup IIb isolates having evolved faster than those of subgroup IIa. Analysis of efficient population size showed that the outbreak point of SVA worldwide occurred at the end of 2013 with that of subgroup IIb, the current dominant group, in mid 2014.

Molecular Evolution of Infectious Pancreatic Necrosis Virus in China.

Passive virus surveillance was performed in twenty-nine salmon and trout farms from seven provinces and districts in China during the period 2017–2020. A total of 25 infectious pancreatic necrosis virus (IPNV) isolates were obtained, mainly from rainbow trout (Oncorhynchus mykiss). The molecular evolution of these Chinese IPNV isolates and the previously reported Chinese IPNV strains ChRtm213 and WZ2016 was analyzed, based on their VP2 gene coding region sequences (CDS). All 27 Chinese IPNV isolates clustered within genogroups I and V, with 24 of the IPNV isolates belonging to genogroup I (including ChRtm213 and WZ2016), and only three isolates clustering in genogroup V. The Chinese genogroup I IPNV isolates lacked diversity, composing six haplotypes with 41 polymorphic sites, and the identity of nucleotide and amino acid sequences among the entire VP2 gene CDS from these isolates was 97.44%–100% and 98.19%–100%, respectively. Divergence time analyses revealed that the Chinese genogroup I IPNV isolates likely diverged from Japanese IPNV isolates in 1985 (95% highest posterior density (HPD), 1965–1997), and diverged again in 2006 (95% HPD, 1996–2013) in China. Each of the three Chinese genogroup V IPNV isolates has a unique VP2 gene CDS, with a total of 21 polymorphic sites; the identity of nucleotide and amino acid sequences among all VP2 gene CDS from these isolates was 98.5%–99.5% and 98.6%–99.0%, respectively. The data demonstrate that genogroups I and V are more likely the currently prevalent Chinese IPNV genotypes.

Isolation, characterization, culturing, and formulation of a new Beauveria bassiana fungus against Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is a worldwide destructive citrus pest. It aids in the transmission of a group of phloem-limited bacteria (Candidatus Liberibacter spp.) that leads to Huanglongbing (HLB) disease. In this study, a new Chinese native fungal strain (F-HY006) was isolated from the cadavers of D. citri collected from Ganzhou, Jiangxi in China (114°94′ E, 25°87′ N). The isolate was virulent towards adult female D. citri with corrected mortality of 76% and LT50 of 4.09 d after application of a suspension containing 107 conidia/ml (400 µl/citrus seedling). Morphologic and phylogenetic analyses identified this isolate as Beauveria bassiana. Based on its virulence against D. citri and growth characteristics (2.89 ± 0.28 × 105 conidia/cm2) on the PDA plate, biphasic (liquid–solid) cultivation for conidia production of B. bassiana F-HY006 was optimized using several nutritional and environmental factors. Soybean meal + cottonseed powder (1:1) and continuous fluorescent light resulted in the highest conidial production (5.7 × 109/g dry substrate). Moreover, the oil-based conidial formulation was optimized using sesame oil as an effective carrier, which resulted in a significant enhancement of the conidial shelf life, tolerance to thermal stress, and virulence against D. citri compared to unformulated conidia. Therefore, we have prepared a potential mycopesticide for the biological control of D. citri based on the optimized biphasic culture growth and formulation of an isolated virulent fungus, B. bassiana F-HY006.