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A bacilliform virus was isolated from yellow catfish in China. This virus can directly adapt in cultures of EPC cells. The virus particles, which were rod-shaped approximately 120 nm long and 20 nm wide, were visible in the cytoplasm of EPC cells. The full-length genome of this virus is 26,985 nt. The genome contains four open reading frames that encode polyprotein1ab, spike glycoprotein, M protein, and N protein. There was a putative slippery sequence 14861UUUAAAC14867, which could be modeled into an RNA pseudoknot structure. The predicted amino acid sequence of pp1ab, S, M, and N genes shares 8.7%–40.2% homology with those of the two known Bafinivirus strains—WBV and FHMNV. Based on the viral morphology, genome organization, and sequence homology, this newly identified bacilliform virus appears to be Piscanivirus. To the best of our knowledge, this is the first report of Piscanivirus in yellow catfish and Piscanivirus in China.

A strain of Vibrio diabolicus named SX-2018 was isolated from Callista chinensis in the Zhejiang coastal region of China. The isolate was identified as V. diabolicus by sequencing of the 16S rRNA, housekeeping genes and subclade-associated genes. Antibiotic resistance as well as biochemical and growth characteristics were determined. Animal studies were performed to evaluate the in vivo virulence of SX-2018. The results indicated that SX-2018 was highly related to the known deep-sea hydrothermal vent V. diabolicus and V. diabolicus from Homo sapiens. Artificial infection showed that SX-2018 exhibited strong pathogenicity toward Gomphina aequilatera, but it did not express pathogenicity toward Mactra veneriformis Reeve, Callista chinensis, Mactra chinensis Philippi, Ruditapes philippinarum, Cyclina sinensis, Dosinia corrugate Meretrix meretrix, Tegillarca granosa, Sinonovacula constricta or Danio rerio. Mortality in Gomphina aequilatera at 25 °C and 15 °C was 100% and 27%, respectively. Virulence-related gene sequence analysis showed that SX-2018 encoded the tlh gene but lacked tdh and trh genes. Tlh and hemolysin coregulated protein genes of SX-2018 shared 99.36–99.79% homology to the deep-sea V. diabolicus strain, 98.65.36–99.79% homology to the V. diabolicus strain from Homo sapiens, and 97–97.79% and 84–89.79% similarity to Vibrio alginolyticus and Vibrio parahaemolyticus, respectively. This study will aid in understanding the evolutionary relationship between V. diabolicus and related pathogenic Vibrio species.

The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2′-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S -adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2′-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2′-O methyltransferase (MTase) but not G-N-7 MTase. These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2′-O MTase in paramyxovirus replication and pathogenesis and a new avenue for the development of safe and efficacious live attenuated vaccines for hMPV and other human paramyxoviruses.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2′-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S -adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2′-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. IMPORTANCE Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2′- O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2′- O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.

Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ( 329 RGD 331 ). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329 RGD 331 motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCE Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5β1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 10 6 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 10 6 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo , thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

Vibrio alginolyticus, a Gram-negative bacterium, is one of the Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, the OmpK gene of V. alginolyticus was cloned and expressed. The open reading frame contains 846 bp, and the mature protein consists of 261 amino acid residues. The alignment analysis indicated that OmpK was highly conserved, which could serve as a surface antigen for good vaccine candidate. SDS-PAGE indicated the OmpK could over-express in E. coli BL21 (DE3). The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin. Western blot analysis revealed that the OmpK protein had immunoreactivity. The large yellow croakers vaccinated with the OmpK were significantly protected against the infection by V. alginolyticus. This study showed that the conserved OmpK was an effective vaccine candidate against infection by V. alginolyticus.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker (Pseudosciaena crocea), psuA and pvuA, were cloned and expressed as N-terminal His6-tagged proteins in Escherichia coli BL21(DE3). The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log2 3.25 to log2 9.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals. During infection and induction of the host immune response, outer membrane proteins of bacteria play an important role. In this study, an outer membrane protein gene (ompW) was cloned from V. alginolyticus and expressed in Escherichia coli. The 645 bp open reading frame (ORF) encodes a protein of 214 amino acid residues with a predicted molecular weight of 23.3 kDa. The amino acid sequence showed a high identity with that of Photobacterium damselae (96.2%) and Vibrio parahaemolyticus (94.4%). The alignment analysis indicated that OmpW was highly conserved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the gene was over-expressed in E. coli BL21(DE3). Western blot analysis revealed that the expressed protein had immunoreactivity. The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin. Large yellow croaker vaccinated with the purified OmpW showed significantly increased antibody to OmpW, which could resist the infection by V. alginolyticus. A specific antibody was detected by enzyme-linked immunosorbent assay. This study suggested that the conserved OmpW could be an effective vaccine candidate against infection by V. alginolyticus.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.

Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been reported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral replication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96–129 bp located between ORF A1 and ORF A2 of genomic segment A of IBDV strain HZ2 were deleted, and an Nhe I (GcTaGc) site was inserted at 96–102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chimeric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the pathogenesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses.

Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.

Parvoviridae, which are classified into two subfamilies Parvovirinae and Densovirinae, can infect both vertebrate and insects and are related to a wide range of diseases in insects, animals, and humans. In this report, several new parvoviruses were identified in swine sera collected in southeastern China. The sequence analyses showed that the parvoviruses detected in southeastern China formed a distinct sublineage within the subfamily Parvovirinae. Based on these results, we propose a novel parvovirus sublineage, Cnvirus, to describe these parvoviruses.

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals. During infection and induction of the host immune response, outer membrane proteins of bacteria play an important role. In this study, an outer membrane protein gene (ompW) was cloned from V. alginolyticus and expressed in Escherichia coli. The 645 bp open reading frame (ORF) encodes a protein of 214 amino acid residues with a predicted molecular weight of 23.3 kDa. The amino acid sequence showed a high identity with that of Photobacterium damselae (96.2%) and Vibrio parahaemolyticus (94.4%). The alignment analysis indicated that OmpW was highly conserved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the gene was over-expressed in E. coli BL21(DE3). Western blot analysis revealed that the expressed protein had immunoreactivity. The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin. Large yellow croaker vaccinated with the purified OmpW showed significantly increased antibody to OmpW, which could resist the infection by V. alginolyticus. A specific antibody was detected by enzyme-linked immunosorbent assay. This study suggested that the conserved OmpW could be an effective vaccine candidate against infection by V. alginolyticus.

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.

Recently, we cloned the adult alpha-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these alpha-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid alpha- and beta-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked alpha- and beta-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid alpha- and beta-globin genes.

This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2 was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.