Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus.

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.

Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala).

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus).

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

MicroRNA miR-214 inhibits snakehead vesiculovirus replication by targeting the coding regions of viral N and P.

Snakeheadvesiculovirus (SHVV), a new member of the family Rhabdoviridae, has caused enormous economic losses in snakehead fish culture during the past years in China; however, little is known about the molecular mechanisms of its pathogenicity. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in virus infection. In this study, we identified that SHVV infection downregulated miR-214 in striped snakehead (SSN-1) cells in a time- and dose-dependent manner. Notably, transfecting SSN-1 cells with miR-214 mimic significantly inhibitedSHVV replication, whereas miR-214 inhibitor promoted it, suggesting that miR-214 acted as a negative regulator of SHVV replication. Our study further demonstrated that N and P of SHVV were the target genes of miR-214. Over-expression of P, but not N, inhibited IFN-α production in SHVV-infected cells, which could be restored by over-expression of miR-214. Taken together, these results suggest that miR-214 is downregulated during SHVV infection, and the downregulated miR-214 in turn increased N and P expression and decreased IFN-α production, thus facilitating SHVV replication. This study provides a better understanding of the molecular mechanisms on the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Transcriptomic profiles of striped snakehead fish cells (SSN-1) infected with red-spotted grouper nervous necrosis virus (RGNNV) with an emphasis on apoptosis pathway.

Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. However, the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood. In this study, the transcriptomic profiles of striped snakehead fish (Channa striatus) cells (SSN-1) infected with red-spotted grouper NNV (RGNNV) were investigated using deep RNA sequencing technique. From 254,955,234 raw reads, a total of 253,338,544 clean reads were obtained and they were assembled into 93,372 unigenes. Differentially expressed genes (DEGs) were identified from RGNNV-infected or mock-infected SSN-1 cells, including 1184 up-regulated and 1456 down-regulated genes at 3 h (h) post of infection (poi), and 1138 up-regulated and 2073 down-regulated genes at 24 h poi, respectively. These DEGs were involved in many pathways related to viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, apoptosis pathway, oxidative phosphorylation, PI3K-Akt signaling pathway, and MAPK signaling pathway. Subsequent analysis focusing on the apoptosis pathway showed that the expression of Endonuclease G (EndoG) was up-regulated upon RGNNV infection at both 3 and 24 h poi. Therefore, EndoG gene was cloned and its function was further characterized. The results showed that over-expression of EndoG could also induce cellular apoptosis in SSN-1 cells, indicating that RGNNV infection might induce apoptosis of SSN-1 cells via EndoG-associated mitochondrial pathway. These results will shed a new light on the pathogenesis of NNV.

Glutamine is required for red-spotted grouper nervous necrosis virus replication via replenishing the tricarboxylic acid cycle.

Glutamine, one of the most important nutrients, plays a vital role in carbon metabolic pathway and has been reported to be required for the replication of several human DNA viruses. However, whether glutamine is required for RNA virus replication and the related mechanism remains elusive. Nervous necrosis virus (NNV), a positive-stranded RNA virus, can infect a number of important aquatic species and has caused great economic losses in aquaculture industry worldwide. In this study, the effects of glutamine on red-spotted grouper nervous necrosis virus (RGNNV) replication were investigated. The results showed that lack of glutamine did not affect the cell viability, but dramatically inhibited RGNNV replication, indicating that glutamine was required for RGNNV replication. Glutamine can be converted to α-ketoglutarate (α-KG) by glutaminase (GLS) to join in the tricarboxylic acid (TCA) cycle. Inhibiting the activity of GLS by a GLS inhibitor: bis-2-5-phenylacetamido-1,3,4-thiadiazol-2-ethyl sulfide (BPTES) significantly inhibited RGNNV replication, while adding the TCA cycle intermediates: α-KG, oxaloacetic acid (OAA), or pyruvate significantly restored RGNNV replication in glutamine-free medium, indicating that the requirement of glutamine for RGNNV replication was due to replenishing the TCA cycle. Taken together, these data revealed that glutamine could regulate RGNNV replication via TCA cycle, which will pave a new way for the prevention of the RGNNV infection in the future.

Glutamine is required for snakehead fish vesiculovirus propagation via replenishing the tricarboxylic acid cycle.

Snakehead fish vesiculovirus (SHVV), a member of the family Rhabdoviridae, has caused mass mortality in snakehead fish culture in China. Previous transcriptomic sequencing of SHVV-infected and non-infected striped snakehead fish cells (SSN-1) showed that glutaminase (GLS), the critical enzyme of glutamine metabolism, was upregulated upon SHVV infection. It therefore drew our attention to investigating the role of glutamine in SHVV propagation. Glutamine deprivation significantly reduced the expression of the mRNAs and proteins of SHVV, and the production of virus particles, indicating that glutamine was required for SHVV propagation. Glutamine can be converted to glutamate by GLS, and then be converted to α-ketoglutarate, to join in the tricarboxylic acid (TCA) cycle. Addition of the TCA cycle intermediate α-ketoglutarate, oxaloacetic acid or pyruvate significantly restored SHVV propagation, indicating that the requirement of glutamine for SHVV propagation was due to its replenishment of the TCA cycle. Inhibiting the activity of GLS in SSN-1 cells by an inhibitor, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, decreased SHVV propagation, while overexpression of GLS increased SHVV propagation. Taken together, our data have revealed the relationship between glutamine metabolism and SHVV propagation.

Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates.

Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates.