Resistance to permethrin alters the gut microbiota of Aedes aegypti.

Insecticide resistance has emerged as a persistent threat to the fight against vector-borne diseases. We compared the gut microbiota of permethrin-selected (PS) strain of Aedes aegypti relative to the parent (KW) strain from Key West, Florida. Bacterial richness but not diversity was significantly higher in PS strain compared to KW strain. The two mosquito strains also differed in their gut microbial composition. Cutibacterium spp., Corynebacterium spp., Citricoccus spp., Leucobacter spp., Acinetobacter spp., Dietzia spp., and Anaerococcus spp. were more abundant in PS strain than in KW strain. In contrast, Sphingomonas spp., Aquabacterium spp., Methylobacterium spp., Flavobacterium spp., Lactobacillus spp., unclassified Burkholderiaceae and unclassified Nostocaceae were more abundant in KW strain compared to PS strain. PS strain was enriched with propionate metabolizers, selenate reducers, and xylan, chitin, and chlorophenol degraders while KW strain was enriched with sulfur oxidizers, sulfur metabolizers, sulfate reducers and naphthalene and aromatic hydrocarbons degraders. These findings demonstrate an association between the gut microbiota and insecticide resistance in an important vector species and sets the foundation for future studies to investigate the contribution of gut microbiota to evolution of insecticide resistance in disease vectors.

Permethrin Resistance in Aedes aegypti Affects Aspects of Vectorial Capacity.

Aedes aegypti, as one of the vectors transmitting several arboviruses, is the main target in mosquito control programs. Permethrin is used to control mosquitoes and Aedes aegypti get exposed due to its overuse and are now resistant. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world and the potential influence on vectorial capacity needs to be studied. Here we selected a permethrin resistant (p-s) Aedes aegypti population from a wild Florida population and confirmed the resistance ratio to its parental population. We used allele-specific PCR genotyping of the V1016I and F1534C sites in the sodium channel gene to map mutations responsible for the resistance. Two important factors, survival rate and vector competence, that impact vectorial capacity were checked. Results indicated the p-s population had 20 times more resistance to permethrin based on LD50 compared to the parental population. In the genotyping study, the p-s population had more homozygous mutations in both mutant sites of the sodium channel gene. The p-s adults survived longer and had a higher dissemination rate for dengue virus than the parental population. These results suggest that highly permethrin resistant Aedes aegypti populations might affect the vectorial capacity, moreover, resistance increased the survival time and vector competence, which should be of concern in areas where permethrin is applied.

Profiling Transcripts of Vector Competence between Two Different Aedes aegypti Populations in Florida.

A Chikungunya virus (CHIKV) outbreak in Italy in 2007 spread to include the islands of the Caribbean and most of the Americas and still circulates in Europe and Africa. Florida being close in distance to the Caribbean islands experienced a CHIKV outbreak in 2014 and continues to have a few travel-related cases each year. It is known that different environmental conditions in different regions can result in genetic variation that favor changes in competence to arbovirus. We evaluated the vector competence of Florida Aedes aegypti for CHIKV and determined if there is a geographic component that influences genes involved in CHIKV competence. We utilized a genomic approach to identify the candidate genes using RNA sequencing. The infection and dissemination results showed that field populations were more competent vectors for CHIKV than a lab population. The differentially expressed genes in the two field-collected CHIKV-infected populations, compared to the Rockefeller strain, were related to the Wnt/Notch signaling pathway, with similarity to genes scattered throughout the signaling pathway. This result suggested the possibility of identifying genes involved in the determination of vector competence in different gene pools of Ae. aegypti.

Effect of Oral Infection of Mayaro Virus on Fitness Correlates and Expression of Immune Related Genes in Aedes aegypti.

Mayaro virus is a mosquito-borne Alphavirus endemic to forests of tropical South America with a sylvatic cycle involving non-human primates and Haemagogus mosquitoes. Human infection with Mayaro virus causes a febrile illness and long-lasting arthralgia and cases are often associated with exposure to tropical forest habitats. Human movement between tropical forest habitats and urban settings may allow for imported cases and subsequent local transmission by domestic mosquito Aedes aegypti. The relative importance of Ae. aegypti as a vector of Mayaro virus may depend on the pathogenic effects of the virus on fitness correlates, especially those entomological parameters that relate to vectorial capacity. We performed mosquito infection studies and compared adult survival and fecundity of females from Brazilian and Floridian populations of Ae. aegypti following oral ingestion of uninfectious (control) and Mayaro virus infectious blood. Mayaro virus infected and refractory mosquitoes had similar or 30-50% lower fecundity than control (unexposed) mosquitoes, suggesting a reproductive cost to mounting an immune response or phenotypic expression of refractoriness. Survival of adult female mosquitoes and targeted gene expression in the Toll and IMD pathways were not altered by Mayaro virus infection. Adult lifespan and fecundity estimates were independent of measured viral titer in the bodies of mosquitoes. The lack of adverse effects of infection status on female survival suggests that Mayaro virus will not alter vectorial capacity mediated by changes in this parameter.

Size of Openings in Water-Holding Containers: Impact on Oviposition by Culex (Culex) Mosquitoes.

To assess how a grate covering a catch basin impacts oviposition by Culex mosquitoes, a field study was conducted in south Florida using containers with two types of covers, with openings of equal area, but different configurations. One opening mimicked a catch basin grate with 16 small openings, while the other cover had just one large opening. The number and presence of egg rafts in six of each container and cover combination was recorded over 18 nights at two field sites, consisting of a wastewater management facility area and residential subdivision. Three mosquito species, all belonging to the subgenus Culex accounted for more than 99% of total egg rafts collected: Culex nigripalpus (n = 1766), Culex quinquefasciatus (n = 754) and Culex coronator (n = 526). Approximately 90% of Cx. nigripalpus and Cx. coronator egg rafts were deposited in the containers with the large opening cover; whereas more Cx. quinquefasciatus egg rafts were laid in the containers with small opening covers than those of Culex nigripalpus and Culex coronator combined. Similar patterns of egg laying activity were noted at each sampling stations. These results suggest that for locating oviposition sites Culex quinquefasciatus may rely more on olfactory clues, while other Culex species depend more on visual clues.

Studies on the Anti-Angiogenic Activities of Wild and Cultivated Orostachys japonicus Extracts in Human Umbilical Vein Endothelial Cells.

Orostachys japonicus has traditionally been used as a food product and a fork medicine in Asia to treat various diseases. Angiogenesis is a critical process that contributes to various chronic diseases via excessive delivery of oxygen and nutrients. Common anti-angiogenic drugs have serious problems related to high costs and side effects; thus, natural products with low costs and no cytotoxicity have garnered increasing interest. In this study, we evaluated and compared the anti-angiogenic effects and phenolic compound contents between wild (WOEs) and cultivated O. japonicus extracts (COEs) prepared under various extract conditions. WOEs and COEs suppressed cell proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited vascular endothelial growth factor-induced chemotactic migration, invasion, and capillary-like tube formation in HUVECs. Among COEs, that prepared by 70% EtOH (70% CE) showed the most effective anti-angiogenic activity in HUVECs. When compared to WOEs, total polyphenol and total flavonoid contents were 1.28 to 4.38 times higher in COEs, and 70% CE contained the greatest flavonoid contents (28.28 ± 0.93 mg%), as well as the highest levels of major phenolic compounds including gallic acid (21.84 µg/mL), epicatechin-gallate (6.58 µg/mL), kaempferol (6.32 µg/mL), and quercetin (8.55 µg/mL). Although further studies are required to identify the molecular mechanisms behind these anti-angiogenic effects, 70% CE could be used as an herbal medicine, functional food ingredient, and potent angiogenesis inhibitor. PRACTICAL APPLICATION: Environmental factors such as altitude, nutrients, exposure to sunlight, and temperature can influence the type and quantity of bioactive components in plants. The advantage of cultivated plants is that the above-mentioned factors can be artificially adjusted compared to wild plants. Based on economic efficiency, productivity, and consistent quality, anti-angiogenesis activity of cultivated O. japonicus is of greater commercial value as a functional food than wild O. japonicus.

Transcriptional Profile of Aedes aegypti Leucine-Rich Repeat Proteins in Response to Zika and Chikungunya Viruses.

Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever, and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, accessed on: August 22, 2018, GEO DataSets). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins related genes, AaeLRIM1 and AaeAPL1, and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into the transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.

Homologs of Human Dengue-Resistance Genes, FKBP1B and ATCAY, Confer Antiviral Resistance in Aedes aegypti Mosquitoes.

Dengue virus (DENV) is transmitted by mosquitoes and is a major public health concern. The study of innate mosquito defense mechanisms against DENV have revealed crucial roles for the Toll, Imd, JAK-STAT, and RNAi pathways in mediating DENV in the mosquito. Often overlooked in such studies is the role of intrinsic cellular defense mechanisms that we hypothesize to work in concert with the classical immune pathways to affect organismal defense. Our understanding of the molecular interaction of DENV with mosquito host cells is limited, and we propose to expand upon the recent results from a genome-scale, small interfering RNA (siRNA)-based study that identified mammalian host proteins associated with resistance to dengue/West Nile virus (DENV/WNV) infection. The study identified 22 human DENV/WNV resistance genes (DVR), and we hypothesized that a subset would be functionally conserved in Aedes aegypti mosquitoes, imparting cellular defense against flaviviruses in this species. We identified 12 homologs of 22 human DVR genes in the Ae. aegypti genome. To evaluate their possible role in cellular resistance/antiviral defense against DENV, we used siRNA silencing targeted against each of the 12 homologs in an Ae. aegypti cell line (Aag2) infected with DENV2 and identified that silencing of the two candidates, AeFKBP1 and AeATCAY, homologs of human FKBP1B and ATCAY, were associated with a viral increase. We then used dsRNA to silence each of the two genes in adult mosquitoes to validate the observed antiviral functions in vivo. Depletion of AeFKBP1 or AeATCAY increased viral dissemination through the mosquito at 14 days post-infection. Our results demonstrated that AeFKBP1 and AeATCAY mediate resistance to DENV akin to what has been described for their homologs in humans. AeFKBP1 and AeATCAY provide a rare opportunity to elucidate a DENV-resistance mechanism that may be evolutionarily conserved between humans and Ae. aegypti.

Genome-Wide Transcriptome Profiling Reveals Genes Associated with Meiotic Drive System of Aedes aegypti.

Aedes aegypti is an important mosquito vector of several arboviruses, including dengue, yellow fever, Zika, and Chikungunya, which cause significant human morbidity and mortality globally. In certain populations of this mosquito, a native meiotic drive system causes abnormal spermatogenesis that results in highly male-biased progenies from some matings. Although the basic genetics and cytogenetics of the drive mechanism were elucidated, very little is known on a transcriptome level about how the meiotic drive phenotype is expressed in individual males. To address this question, we conducted a whole-genome microarray expression study of testes from a meiotic-drive-carrying strain (T37) in comparison with testes from a non-drive-carrying strain (RED). Based on bioinformatics analyses of the microarray data, we identified 209 genes associated with the meiotic drive phenotype that were significantly differentially expressed between the two strains. K-means cluster analysis revealed nine clusters, in which genes upregulated in T37 testes were assigned to five clusters and genes downregulated in T37 testes were assigned to four clusters. Our data further revealed that genes related to protein translation, phosphorylation, and binding, as well as to G-protein-coupled receptor (GPCR) and peptidase activities, are differentially upregulated in testes from males with the meiotic drive genotype. Based on pathway analysis of these differentially expressed genes, it was observed that the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway may play a role in the meiotic drive system. Overall, this investigation enhances our understanding of whole-genome gene expression associated with the meiotic drive system in Ae. aegypti.

Optimization of double-stranded RNAi intrathoracic injection method in Aedes aegypti.

RNA interference is widely used to analyze gene functions via phenotypic knockdown of target transcripts in mosquitoes, which transmit numerous mosquito-borne diseases. Functional analysis of mosquito genes is indispensable to understand and reduce transmission of mosquito-borne diseases in mosquitoes. Intrathoracic injection of double-stranded RNA (dsRNA) remains the simplest and most customizable method in mosquitoes for functional analysis of the genes of interest. However, achieving consistent and effective knockdown by dsRNAi is often elusive and may require extensive optimization. We tested the effectiveness of gene silencing by intrathoracic injection of four different quantities of dsRNA targeting two Ae. aegypti genes, cysteine desulfurylase (Nfs1) and short-chain dehydrogenase (SDH). We found that Nfs1 gene has a lower expression level upon silencing than SDH gene. In the case of the gene that is easier to silence, Nfs1 gene expression was significantly silenced by all four tested quantities of dsRNA up to 21 d.p.i., but silencing of SDH, the gene that is difficult to silence, was less effective, with knockdown lasting up to 9 d.p.i. only when 1,000 ng of dsRNA was used. Based on our observation, intrathoracic injection of 500 ng of dsRNAs per mosquito is recommended to achieve effective knockdown for well-silenced transcripts such as Nfs1 for up to 3 weeks. This includes most in vivo bioassays involving arboviral infections in Ae. aegypti. The estimated quantities of dsRNA described in this study should be applicable to most Ae. aegypti dsRNAi studies and thus provide a guideline to develop efficient dsRNAi in other experimental investigations.

The Effect of Permethrin Resistance on Aedes aegypti Transcriptome Following Ingestion of Zika Virus Infected Blood.

Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into seven categories of biological processes. The 863 transcripts were expressed at significantly different levels between the two mosquito strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in insecticide resistant Ae. aegypti with implications for mosquito control strategies.

Culex quinquefasciatus (Diptera: Culicidae) From Florida Transmitted Zika Virus.

We report a laboratory colony of Culex quinquefasciatus mosquitoes were experimentally able to salivate Zika virus (ZIKV, Flaviviridae; Flavivirus) at 16 days post infection (dpi). ZIKV RNA was detected in bodies and in saliva deposited on filter paper cards with subsequent studies demonstrating the presence of live ZIKV in saliva.

Transcription Profiling for Defensins of Aedes aegypti (Diptera: Culicidae) During Development and in Response to Infection With Chikungunya and Zika Viruses.

Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These viruses encounter a variety of induced defense responses from the innate immune system of the mosquito. We cloned defensin A from Ae. aegypti using laboratory populations originating from Key West and Orlando, Florida. To characterize inducible immune defensin peptides, we examined the defensin A (DefA) and defensin C (DefC) expression through time course studies using quantitative real-time PCR. We observed that ingestion of chikungunya virus (CHIKV) and Zika virus (ZIKV) infected blood triggered early upregulated expression of DefA and DefC at 3 h after blood feeding. At 10-d post infection, there was significant downregulation of DefA and DefC in CHIKV-infected females and significant upregulation of DefA and DefC in ZIKV-infected females compared with control mosquitoes fed uninfected blood. Our studies demonstrate that the relative activity of DefA and DefC changed depending on whether Ae. aegypti was infected with CHIKV or ZIKV, suggesting differences in antiviral defense responses. In addition, we also examined DefA and DefC gene expression during the different developmental stages. Significant qualitative and quantitative differences were found in DefA and DefC transcripts between Key West and Orlando strains. We found that adult males consistently had higher expression than adult females of different ages. Together, these data show that members of the Ae. aegypti defensin gene family play a role in both Zika and chikungunya antiviral response.

Dengue serotype-specific immune response in Aedes aegypti and Aedes albopictus.

BACKGROUND: Dengue viruses (DENV) are considered one of the most important emerging pathogens and dengue disease is a global health threat. The geographic expansion of dengue viruses has led to co-circulation of all four dengue serotypes making it imperative that new DENV control strategies be devised. OBJECTIVES: Here we characterize dengue serotype-specific innate immune responses in Aedes aegypti and Aedes albopictus using DENV from Puerto Rico (PR). METHODS: Ae. aegypti and Ae. albopictus were infected with dengue serotype 1 and 2 isolated from Puerto Rico. DENV infected mosquito samples were collected and temporal change in expression of selected innate immune response pathway genes analyzed by quantitative real time PCR. FINDINGS: The Toll pathway is involved in anti-dengue response in Ae. aegypti, and Ae. albopictus. Infections with PR DENV- 1 elicited a stronger response from genes of the Toll immune pathway than PR DENV-2 in Ae. aegypti but in infected Ae. albopictus expression of Toll pathway genes tended to be similar between the serotypes. Two genes (a ribosomal S5 protein gene and a nimrod-like gene) from Ae. albopictus were expressed in response to DENV. MAIN CONCLUSIONS: These studies revealed a role for antiviral genes in DENV serotype-specific interactions with DENV vectors, demonstrated that infections with DENV-2 can modulate the Toll immune response pathway in Ae. aegypti and elucidated candidate molecules that might be used to interfere with serotype specific vector-virus interactions.

Dengue serotype-specific immune response in Aedes aegypti and Aedes albopictus

BACKGROUND Dengue viruses (DENV) are considered one of the most important emerging pathogens and dengue disease is a global health threat. The geographic expansion of dengue viruses has led to co-circulation of all four dengue serotypes making it imperative that new DENV control strategies be devised. OBJECTIVES Here we characterize dengue serotype-specific innate immune responses in Aedes aegypti and Aedes albopictus using DENV from Puerto Rico (PR). METHODS Ae. aegypti and Ae. albopictus were infected with dengue serotype 1 and 2 isolated from Puerto Rico. DENV infected mosquito samples were collected and temporal change in expression of selected innate immune response pathway genes analyzed by quantitative real time PCR. FINDINGS The Toll pathway is involved in anti-dengue response in Ae. aegypti, and Ae. albopictus. Infections with PR DENV- 1 elicited a stronger response from genes of the Toll immune pathway than PR DENV-2 in Ae. aegypti but in infected Ae. albopictus expression of Toll pathway genes tended to be similar between the serotypes. Two genes (a ribosomal S5 protein gene and a nimrod-like gene) from Ae. albopictus were expressed in response to DENV. MAIN CONCLUSIONS These studies revealed a role for antiviral genes in DENV serotype-specific interactions with DENV vectors, demonstrated that infections with DENV-2 can modulate the Toll immune response pathway in Ae. aegypti and elucidated candidate molecules that might be used to interfere with serotype specific vector-virus interactions.

Evidence of Zika Virus RNA Fragments in Aedes albopictus (Diptera: Culicidae) Field-Collected Eggs From Camaçari, Bahia, Brazil.

A major mosquito-borne viral disease outbreak caused by Zika virus (ZIKV) occurred in Bahia, Brazil, in 2015, largely due to transmission by the mosquito, Aedes aegypti (L.). Detecting ZIKV in field samples of Ae. aegypti has proven problematic in some locations, suggesting other mosquito species might be contributing to the spread of ZIKV. In this study, several (five) adult Aedes albopictus (Skuse) mosquitoes that emerged from a 2015 field collection of eggs from Camaçari, Bahia, Brazil, were positive for ZIKV RNA; however, attempts to isolate live virus were not successful. Results from this study suggest that field-collected Ae. albopictus eggs may contain ZIKV RNA that require further tests for infectious ZIKV. There is a need to investigate the role of Ae. albopictus in the ZIKV infection process in Brazil and to study the potential presence of vertical and sexual transmission of ZIKV in this species.

The Effect of West Nile Virus Infection on the Midgut Gene Expression of Culex pipiens quinquefasciatus Say (Diptera: Culicidae).

The interaction of the mosquito and the invading virus is complex and can result in physiological and gene expression alterations in the insect. The association of West Nile virus (WNV) and Culex pipiens quinquefasciatus mosquitoes results in measurable changes in gene expression; 22 gene products were shown previously to have altered expression. Sequence analysis of one product, CQ G1A1, revealed 100% amino acid identity to gram negative bacteria binding proteins (CPQGBP) in Cx. p. quinquefasciatus, Aedes aegypti (70%) and Anopheles gambiae (63%) that function in pathogen recognition. CQ G1A1 also was differentially expressed following WNV infection in two populations of Cx. p. quinquefasciatus colonized from Florida with known differences in vector competence for WNV and showed spatial and temporal gene expression differences in midgut, thorax, and carcass tissues. These data suggest gene expression of CQ G1A1 is influenced by WNV infection and the WNV infection-controlled expression differs between populations and tissues.

Assessment of esterase gene expression as a risk marker for insecticide resistance in Florida Culex nigripalpus (Diptera: Culicidae).

Esterases are enzymatic proteins known to play a role in insecticide resistance formation. To further our understanding of the development of insecticide resistance, we tested the gene expression level of a gene implicated in insecticide resistance (Temsha est-1) from Culex nigripalpus Theobald (Diptera: Culicidae) in field mosquitoes. We found that the level of expression of TE-1 differed depending on the frequency of exposure to organophosphate insecticide through expression studies. Temsha est-1 cDNA is 1,808 base pairs and fully sequenced with up to 96% nucleotide sequence identity to esterase B genes of other mosquito species. The genes from five different species, including TE-1, were closely related by genetic distance and phylogenetic analysis. Differential expression of this gene that is correlated to differences in susceptibility towards organophosphate would provide the ability to use Temsha est-1 as an indicator of the formation of tolerance/resistance. This would greatly enhance mosquito control efforts by allowing targeted application of insecticides to mosquito populations that are most susceptible. Also, it would provide resistance information so that a rational design could be used for insecticide rotation schedules.

An evaluation of the effectiveness of hyaluronidase in the selective nerve root block of radiculopathy: a double blind, controlled clinical trial.

STUDY DESIGN: Prospective, double-blind, randomized controlled trial. PURPOSE: To determine the ability of hyaluronidase to provide longer lasting pain relief and functional improvement in patients with lumbar radiculopathy. OVERVIEW OF LITERATURE: Selective nerve root block (SNRB) is a good treatment option in lumbar radiculopathy. We studied the effectiveness of hyaluronidase when added to the traditional SNRB regimen. METHODS: A sample size of 126 patients per group was necessary. A sample of 252 patients who underwent an injection procedure with or without hyaluronidase due to radiculopathy was included in this study. The patients were randomly divided into two groups: the control (C) group and the hyaluronidase (H) group. After SNRB due to radiculopathy, the visual analog scale (VAS) was compared at 2, 4, 6, 8, and 12 weeks between the two groups, and the Oswestry disability index (ODI) was compared at 12 weeks between the two groups. RESULTS: Both groups seemed to have general improvement in VAS, but in C group, the VAS was higher than the H group 2 and 4 weeks after the surgery, and the difference in time-group change between 2 groups was statistically significant (p <0.05). ODI improved in both groups, and the difference in time-group change between 2 groups was not statistically significant (p >0.05). CONCLUSIONS: The rebound pain (the re-occurrence of pain within 2-4 weeks after injection) that occurs within 2-4 weeks after the injection of the routine regimen can be reduced when hyaluronidase is added to the routine SNRB regimen.