Molecular assessment of voltage-gated sodium channel (VGSC) gene mutations in Rhipicephalus microplus from Guangxi, China.

BACKGROUND: Pyrethroid chemicals are one of the main acaricides used against ticks. Resistance to these chemicals has been reported to be associated with mutations in the voltage-gated sodium channel (VGSC) gene of the Rhipicephalus microplus. This study investigates R. microplus resistance to pyrethroids in Guangxi region of China, marking one of the first research efforts in this area. The findings are intended to provide vital baseline for the effective implementation of localized tick control strategies. METHODS: From March to July 2021, 447 R. microplus tick samples were collected from five prefecture-level cities in Guangxi. Allele-specific polymerase chain reaction (AS-PCR) was used to amplify segments C190A and G215T of the domain II S4-5 linker and T2134A of domain III S6 in the VGSC, to detect nucleotide mutations associated with resistance to pyrethroid acaricides. Subsequent analyses were conducted to ascertain the prevalence, types of mutations, and genotypic distributions within the sampled populations. RESULTS: Mutations within VGSC gene were identified across all five studied populations of R. microplus, although the mutation rates remained generally low. Specifically, the most prevalent mutation was C190A, observed in 4.9% of the samples (22/447), followed by G215T at 4.0% (18/447), and T2134A at 1.3% (6/447). The distribution of mutations across three critical sites of the VGSC gene revealed four distinct mutation types: C190A, G215T, C190A + G215T, and T2134A. Notably, the single mutation C190A had the highest mutation frequency, accounting for 4.3%, and the C190A + G215T combination had the lowest, at only 0.7%. The analysis further identified seven genotypic combinations, with the wild-type combination C/C + G/G + T/T predominating at a frequency of 90.4%. Subsequently, the C/A + G/G + T/T combination was observed at a frequency of 4.3%, whereas the C/C + T/T + T/T combination exhibited the lowest frequency (0.2%). Additionally, no instances of simultaneous mutations at all three sites were detected. Geographical differences in mutation types were apparent. Both samples from Hechi to Chongzuo cities exhibited the same three mutation types; however, C190A was the most prevalent in Hechi, while G215T dominated in Chongzuo. In contrast, samples from Beihai to Guilin each exhibited only one mutation type: G215T occurred in 12.5% (4/32) of Beihai samples, and C190A in 7.5% (4/53) of Guilin samples. CONCLUSIONS: These findings underscore the relatively low frequency of VGSC gene mutations in R. microplus associated with pyrethroid resistance in the Guangxi, China. Moreover, the variation in mutation types and genotypic distributions across different locales highlights the need for regionalized strategies in monitoring and managing pyrethroid resistance in tick populations. This molecular surveillance is crucial for informing targeted control measures and mitigating the risk of widespread resistance emergence.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence.

Rabies is a lethal encephalitis caused by the rabies virus (RABV) with a fatality rate near 100% after the onset of clinical symptoms in humans and animals. Microglia are resident immune cells in the central nervous system. Few studies have been conducted on the functional role of microglia in RABV infection. Here, we performed a transcriptomic analysis of mRNA expression profiles in the microglia of mouse brains intracerebrally infected with RABV. We successfully isolated single microglial cells from the mouse brains. The survival rate of dissociated microglial cells was 81.91%-96.7%, and the purity was 88.3%. Transcriptomic analysis revealed 22,079 differentially expressed mRNAs identified in the microglia of mouse brains infected with RABV strains (rRC-HL, GX074, and CVS-24) of varying virulence at 4 and 7 days post-infection (dpi) compared to the control group. The numbers of DEGs versus the control at 4 and 7 dpi in mice infected with rRC-HL, GX074, and CVS-24 were 3622 and 4590, 265 and 4901, and 4079 and 6337. The GO enrichment analysis showed that response to stress, response to external stimulus, regulation of response to stimulus, and immune system process were abundant during RABV infection. The KEGG analysis indicated that the Tlr, Tnf, RIG-I, NOD, NF-κB, MAPK, and Jak-STAT signaling pathways were involved in RABV infection at both 4 and 7 dpi. However, some phagocytosis and cell signal transduction processes, such as endocytosis, p53, phospholipase D, and oxidative phosphorylation signaling pathways, were only expressed at 7 dpi. The involvement of the Tnf and Tlr signaling pathways prompted us to construct a protein-protein interaction (PPI) network of these pathways. The PPI revealed 8 DEGs, including Mmp9, Jun, Pik3r1, and Mapk12. Notably, Il-1b interacted with Tnf and Il-6 with combined scores of 0.973 and 0.981, respectively. RABV causes significant changes in mRNA expression profiles in the microglia in mice. 22,079 differentially expressed mRNAs were identified in the microglia of mice infected with RABV strains of varying virulence at 4 and 7 dpi. The DEGs were evaluated using GO, KEGG, and PPI network analysis. Many immune pathways were up-regulated in RABV-infected groups. The findings will help elucidate the microglial molecular mechanisms of cellular metabolism dysregulated by RABV and may provide important information for investigating RABV pathogenesis and therapeutic methods.

Whole-genome analyses of APEC carrying mcr-1 in some coastal areas of China from 2019 to 2020.

OBJECTIVES: Polymyxin is considered as one of the 'last lines of defense' for the treatment of multidrug resistant bacteria. Increased use of polymyxin during recent years poses a risk to public health. The purpose of this study was to investigate the carrying situation of the mcr-1 drug-resistance gene in waterfowl in some coastal areas of China from 2019 to 2020. METHODS: Fifty-seven isolated avian pathogenic Escherichia coli strains were selected from 493 APEC isolates for whole-genome sequencing. The 24 mcr-1-positive APEC strains were tested for conjugation and genome-wide analysis, including sequence type (ST) analysis, serotype analysis, and drug-resistance gene analysis. Numerous mcr-1-positive E. coli were downloaded from the National Center for Biotechnology Information (NCBI) for comparative genomic analysis. RESULTS: Antimicrobial susceptibility test results showed that 57 APEC isolates were highly resistant to gentamicin, cefotaxime, and ofloxacin, and 24 mcr-1-positive APEC isolates were resistant to polymyxin. Fourteen isolates of mcr-1-positive APEC plasmids were successfully conjugated to EC600. Both ST156 and ST10 were found in high proportions in human and avian sources through genome-wide analysis; it is worth noting that these two isolates of APEC were detected to contain the blaNDM-1 and blaNDM-4 genes, respectively. CONCLUSION: In this study, the epidemiological investigation of the mcr-1 gene was carried out on APEC in some coastal areas of China from 2019 to 2020, and our results have enriched the data on the transmission of APEC isolates carrying the mcr-1 gene in waterfowl.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

Combining pangenome analysis to identify potential cross-protective antigens against avian pathogenic Escherichia coli.

RESEARCH HIGHLIGHTSPan-RV analysis was used for the first time in the discovery of APEC-protective proteins.A total of 53 potential protective proteins were screened out.Four proteins were verified as potential vaccine candidates using western blotting.

The detection of canine circovirus in Guangxi, China.

Since the first description of canine circovirus (CanineCV)-associated infection, there have been several reports on the distribution of the disease in worldwide. To investigate the prevalence and genetic diversity of CanineCV in China, we conducted PCR screening of 1226 dog serum samples collected from different regions in mainland China between 2014 and 2016. CanineCV DNA was found in 81/926 serum samples from Guangxi Province. Furthermore, 25 full-length genomes of CanineCV from positive samples were sequenced and compared with CanineCV sequences in the GenBank database. Pairwise analysis showed that the determined genome sequences shared 84.9%-100% identity among themselves and 81.4%-90.5% with the other 28 sequences. Phylogenetic analysis revealed that the 52 viral genome sequences could be divided into two genotypes (CanineCV-1 and CanineCV-2). Analysis of the amino acid sequences of the capsid protein revealed the existence of 9 major regions of variation. The present work contributes to the understanding of CanineCV molecular epidemiology.

Vaccination demonstration zone successfully controls rabies in Guangxi Province, China.

BACKGROUND: Guangxi is the province most seriously affected by rabies virus (RABV) in China. Those most affected by RABV each year are people in rural areas, where dogs are the main cause of human infection with the virus. METHODS: In this study, we established a rabies vaccination demonstration program that included eradication, core, and peripheral areas. This program was implemented for 9 years and comprised three stages: 12 counties in the first stage (2008-2010), 21 counties in the second stage (2011-2013), and then extending to all counties of Guangxi Province in the third stage (2014-2016). The program included a dog vaccination campaign, surveillance of clinically healthy dogs who may be potential RABV carriers, monitoring anti-RABV antibody titers in vaccinated dogs, and compiling and reporting statistics of human rabies cases. RESULTS: The target effectiveness was achieved in the eradication, core, and peripheral areas in all three stages. The vaccination demonstration program successfully promoted RABV vaccination of domestic dogs throughout Guangxi Province by drawing upon the experience gained at key points. Compared with a vaccination coverage rate of 39.42-46.85% in Guangxi Province overall during 2003-2007, this rate gradually increased to 48.98-52.67% in 2008-2010, 60.24-69.67% in 2011-2013, and 70.09-71.53% in 2014-2016, thereby meeting World Health Organization requirements. The total cases of human rabies in the province decreased from 602 in 2004 to 41 cases in 2017. CONCLUSIONS: The present pilot vaccination program obviously increased the rabies vaccination and seroconversion rates, and effectively reduced the spread of rabies from dogs to humans as well as the number of human rabies cases, thus successfully controlling rabies in Guangxi.

Evolutionary analysis of rabies virus isolates from Guangxi Province of southern China.

BACKGROUND: Rabies is a severe epidemic in Guangxi province, China, with hundreds of deaths occurring each year. In the past six decades, rabies has emerged three times in Guangxi, and the province has reported the largest number of rabies cases in China. The domestic dog is the principal vector for rabies, and 95% of human cases are associated with transmission from dogs. RESULTS: To understand the genetic relationship between street rabies virus (RABV) from Guangxi, genetic diversity analysis was performed using RABV isolates collected between 1999 and 2012. The N gene of 42 RABV isolates, and the P and M genes, as well as fragments of the 3' terminus (L1-680) and the polymerase activity module of the L gene (Lpam) of 36 RABV isolates were sequenced. In addition, whole genome sequencing was performed for 5 RABV isolates. There was evidence of topological discrepancy in the phylogenetic trees based on different genes of the RABV isolates. Amino acid variation of the deduced N protein exhibited different patterns to those obtained from the P and M proteins reported here, and the previously reported G protein (Tang H. et al., PLoS Negl Trop Dis, 8(10): e3114, 2014), and L1-680 and Lpam. These RABV isolates were divided into three main branches against fixed strains. CONCLUSION: RABV is prevalent in Guangxi province and strains collected over the last two decades belong mainly to three groups (I, II, III). These RABV isolates reveal genetic diversity. Individual RABV genes from Guangxi exhibit different evolutionary characteristics. The results will have benefits for continuing comprehensive rabies surveillance, prevention and control in China.

Genome Sequences of a Novel Recombinant Duck Circovirus in China.

In this study, YN26/2013, a novel recombinant duck circovirus (DuCV), was isolated from a Muscovy duck in Yunnan Province, southern China. The whole genome of YN26/2013 consists of 1,987 nucleotides (nt), the same genomic size as that of the DuCV-2 genotype. However, YN26/2013 shares 91.5 to 94.3% nucleotide identity similarity with previously reported type I (DuCV-1) viruses. Importantly, a novel putative recombinant event between DuCV-1 and DuCV-2 was identified as occurring within the 987- to 1111-nt region of the YN26/2013 genome.

Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4.

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4.

[Canine Circovirus Genome Cloning and Sequence Analysis].

Dog circovirus (DogCV) is a newly discovered mammalian circovirus. To investigate the genomic characteristics and genetic diversity of DogCV spreads in China, the first genome sequence of Chinese isolate, designated as JZ98/2014,was obtained by overlap PCR using the DNA extracted from dog serum as template for amplification. The nucleotide content and genome organization were subsequently analyzed. The results showed that the full-length genome of JZ98/2014 is 2063nt,and contains three major open reading frame: ORF V1 (encodes the 303 amino acid Rep protein),ORF C1(encodes the 270 amino acid Cap protein),and ORF C2(encodes 106 amino acids).JZ98/2014 shared 82.1%-89.5% homology with the complete genome sequences of DogCV isolates from America and Europe. The Rep gene and Cap gene of JZ98/2014 shared 82.1%-89.5%and 84.6%-89.1% homology, respectively, with the same genes from other DogCVs. Phylogenetic tree analysis indicated that there were several different genetic clades of DogCV spread in the world, and JZ98/2014 formed a clade by itself.

A recombinant rabies virus expressing a phosphoprotein-eGFP fusion is rescued and applied to the rapid virus neutralization antibody assay.

Rabies remains a worldwide concern, and dogs are a major vector for rabies virus (RABV) transmission. Vaccination is used in China to control the spread of rabies in dogs, a practice which necessitates effective, efficient, and high-throughput methods to confirm vaccination. The current rapid fluorescent focus inhibition test (RFFIT) method to measure virus-neutralizing antibody titers in the serum involves multiple steps, and more efficient methods are needed to match the increasing demand for this type of monitoring. In this study, based on the parental rRC-HL strain, a recombinant RABV rRV-eGFP expressing enhanced green fluorescent protein (eGFP) fused with RABV P protein was generated by a reverse genetic technique. The rRV-eGFP grew stably and successfully expressed P-eGFP fusion in Neuro-2A (NA) host cells. Furthermore, the P protein was shown to co-localize with eGFP in rRV-eGFP-infected NA cells. Since eGFP is easily detected in infected cells under a fluorescence microscope, rRV-eGFP could be used to establish a more rapid virus-neutralizing antibody titers assay based on RFFIT, designated as the RFFIT-eGFP method. From 69 canine serum samples, the RFFIT-eGFP method was shown to be as specific and as sensitive as the RFFIT method, suggesting that it might represent a faster tool than conventional RFFIT for measuring RABV virus-neutralizing antibody titers in canine sera without sacrificing accuracy.

Re-emergence of rabies in the Guangxi province of Southern China.

BACKGROUND: Human rabies cases in the Guangxi province of China decreased from 839 in 1982 to 24 in 1995, but subsequently underwent a sharp increase, and has since maintained a high level. METHODOLOGY/PRINCIPAL FINDINGS: 3,040 brain samples from normal dogs and cats were collected from 14 districts of Guangxi and assessed by RT-PCR. The brain samples showed an average rabies virus (RV) positivity rate of 3.26%, but reached 4.71% for the period Apr 2002 to Dec 2003. A total of 30 isolates were obtained from normal dogs and 28 isolates from rabid animals by the mouse inoculation test (MIT). Six representative group I and II RV isolates showed an LD50 of 10-5.35/ml to 10-6.19/ml. The reactivity of monoclonal antibodies (MAbs) to group I and II RV isolates from the Guangxi major epidemic showed that eight anti-G MAbs showed strong reactivity with isolates of group I and II with titers of ≥10,000; however, the MAbs 9-6, 13-3 and 12-14 showed lower reactivity. Phylogenetic analysis based on the G gene demonstrated that the Guangxi RV isolates have similar topologies with strong bootstrap values and are closely bonded. Alignment of deduced amino acids revealed that the mature G protein has four substitutions A96S, L132F, N436S, and A447I specific to group I, and 13 substitutions T90M, Y168C, S204G, T249I, P253S, S289T, V332I, Q382H, V427I, L474P, R463K Q486H, and T487N specific to group II, coinciding with the phylogenetic analysis of the isolates. CONCLUSIONS: Re-emergence of human rabies has mainly occurred in rural areas of Guangxi since 1996. The human rabies incidence rate increased is related with RV positive rate of normal dogs. The Guangxi isolates tested showed a similar pathogenicity and antigenicity. The results of phylogenetic analysis coincide with that of alignment of deduced amino acids.

Characterization of the biological properties and complete genome sequence analysis of a cattle-derived rabies virus isolate from the Guangxi province of southern China.

In this study, a street rabies virus isolate, GXHXN, was obtained from the brain of one rabid cattle in Guangxi province of southern China. To characterize the biological properties of GXHXN, we first evaluated its pathogenicity using 4-week-old adult mice. GXHXN was highly pathogenic with a short incubation period and course of disease. Its LD50 of 10(-6.86)/mL is significantly higher than the LD50 of 10(-5.19)/mL of GXN119, a dog-derived rabies virus isolate. It also displayed a higher neurotropism index than the rRC-HL strain. However, the relative neurotropism index of GXHXN was slightly lower than that of GXN119. Analyzing antigenicity using anti-N and anti-G monoclonal antibodies (MAbs), all tested anti-N MAbs reacted similarly to GXHXN, CVS, and rRC-HL, but the reaction of anti-N MAbs to GXHXN was slightly different from GXN119. Moreover, 2/11 tested anti-G mAbs showed weaker reactivity to GXHXN than rRC-HL, whereas 4/11 showed stronger reactivity to GXHXN than CVS and GXN119, indicating that the structures of G might differ. In order to understand its genetic variation and evolution, the complete GXHXN genome sequence was determined and compared with the known 12 isolates from other mammals. A total of 42 nucleotide substitutions were found in the full-length genome, including 15 non-synonymous mutations. The G gene accounts for the highest nucleotide substitution rate of 0.70 % in ORF and an amino acid substitution rate of 0.95 %. Phylogenetic trees based on the complete genome sequence as well as the N and G gene sequences from 37 known rabies isolates from various mammals demonstrated that the GXHXN is closely related to the BJ2011E isolate from a horse in Beijing, the WH11 isolate from a donkey in Hubei, and isolates from dogs in the Fujian and Zhejiang provinces. These findings will be helpful in exploring the molecular mechanisms underlying interspecies transmission and the genetic variation of the rabies virus in different mammal species.

Complete genome sequence of a rabies virus isolate from cattle in guangxi, southern china.

A street rabies virus (RV) isolate, GXHXN, was obtained from brain tissue of rabid cattle in the Guangxi Zhuang Autonomous Region of China in 2009. GXHXN is the first isolate from cattle in China with its entire genome sequenced and is closely related to BJ2011E from horse in Beijing, WH11 from donkey in the Hubei Province, and isolates from dogs in the Guangxi and Fujian Provinces, with homologies of 97.6% to 99.6%. It is more distantly related to isolates from domestic cat, pig, Chinese ferret badger, and vaccine strains, with homologies of 83.1% to 88.0%.