Reverse Genetic Techniques Research Articles

Immune Responses to Adjuvanted H7N9 Split Antigen in Aged Mice.

The avian influenza A H7N9 virus has caused severe infection and high mortality in humans. It can be extremely hazardous to the elderly since age might diminish the immune response, and poor immunogenicity of H7 hemagglutinin could diminish the vaccine efficacy in this population. To overcome this issue, adjuvants are used to induce a stronger immune response. In this study, we generated a recombinant H7N9 influenza virus using reverse genetic techniques, consisting of hemagglutinin and neuraminidase genes derived from a human H7N9 virus, with the remaining genes from H1N1 A/Puerto Rico/8/34 (PR8). To evaluate whether the adjuvant can improve immune responses in aged animals, the humoral and cellular immune responses of 18-month-old BALB/c mice to different doses of split avian influenza A H7N9 vaccine with and without the adjuvant MF59 were compared. Our data showed that aged mice immunized with MF59 elicited higher levels of hemagglutination inhibition and microneutralization antibodies and interferon-gamma-specific enzyme-linked immunospot assay (ELISPOT) responses when compared with antigens alone. It is suggested that the split avian influenza A H7N9 vaccine combined with MF59 may significantly improve immune responses to influenza vaccination in elderly humans.

Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro

Although the immunization against swine fever (SF) is compulsory in China, it has still emerged in several areas at times. Herein, this study was conducted to develop an antibody vaccine which can clear the classical swine fever virus (CSFV) immediately after the pathogen invasion. Bovine viral diarrhoea virus (BVDV) infectious cDNA clone pASH28 was used to express a single-chain fragment variable (scFv) antibody against CSFV (CSFV/scFv) by reverse genetic technique. CSFV/scFv was inserted at the N-terminus of the C or Erns gene, generating two rBVDVs (rBVDV/C-CSFV/scFv and rBVDV/Erns-CSFV/scFv). Although both the rBVDVs could stably propagate on MDBK cells, different cellular characteristics existed. Obvious green fluorescence against the CSFV/scFv antibody could be visual on the cytomembrane or outside of the cells infected with rBVDV/Erns-CSFV/scFv, while much weaker fluorescence was observed in rBVDV/C-CSFV/scFv - infected cells. The CSFV/scFv antibodies induced by the two rBVDVs could recognize CSFV, but the rBVDV/Erns-CSFV/scFv induced stronger viral neutralization reaction. It was speculated that the neutralization activity might be associated with the expression location of CSFV/scFv antibody. The datas in this study provide evidence that rBVDV/Erns-CSFV/scFv may be engineered as a new antibody vaccine candidate against CSFV in the future.

The Full Region of N-Terminal in Polymerase of IBDV Plays an Important Role in Viral Replication and Pathogenicity: Either Partial Region or Single Amino Acid V4I Substitution Does Not Completely Lead to the Virus Attenuation to Three-Yellow Chickens.

Infectious Bursal Disease Virus (IBDV) has haunted the poultry industry with severe, prolonged immunosuppression of chickens when infected at an early age and can easily lead to other secondary infections. Understanding the pathogenic mechanisms could lead to effective prevention and control of Infectious Bursal Disease (IBD). Evidence suggests that the N-terminal domain of polymerase in segment B plays an important role, but it is not clear which part or residual is crucial for the pathogenicity. Using a reverse genetics technique, a molecular clone (rNN1172) of the parental vvIBDV strain NN1172 was generated, and its pathogenicity was found to be the same as the parental virus. Then, three recombinant chimeric viruses were rescued based on the rNN1172 and substituted with the counterparts in the N-terminal domain of the attenuated vaccine strain B87: the rNN1172-B87VP1a (substituting the full region of the 1–167 aa residuals), the rNN1172-B87VP1a∆4 (substituting the region of the 5–167 aa residuals), and the rNN1172-VP1∆4 (one single aa residual substitution V4I), to better explore the role of the N-terminal domain of polymerase on the viral pathogenicity. Interestingly, all these substitutions played different roles in the viral pathogenicity: the mortality of the rNN1172-B87VP1a-challenged chickens was significantly reduced from 30% to 0%. No obvious lesion was found in the histopathological examination, and the lowest viral genome copy number was also detected in the bursa when compared to the parental and two other recombinant viruses. The mortalities caused by rNN1172-B87VP1a∆4 and rNN1172-B87VP1∆4, respectively, were all reduced to 10% and had a delayed onset of death. Our results also revealed that the pathogenicity of the IBDV was consistent with the viral replication efficiency in vivo (bursae). This study demonstrated that the full region of the N-terminal of polymerase plays an important role in viral replication and pathogenicity, but the substitutions of its partial region or a single residual do not completely lead to the virus attenuation to Three-Yellow chickens, although that significantly reduces its pathogenicity.

Electroporation-mediated nucleic acid delivery during non-embryonic stages for gene-function analysis in Anopheles sinensis

The delivery of exogenous nucleic acids to eggs or non-embryonic individuals by microinjection is a vital reverse genetics technique used to determine gene function in mosquitoes. However, DNA delivery to eggs is complex and time-consuming, and conventional, non-embryonic-injection techniques may result in unobvious phenotypes caused by insufficient absorption of nucleic acid fragments by cells at target body parts or tissues. In this study, we developed a set of electroporation-mediated non-embryonic microinjections for the delivery of exogenous nucleic acids in Anopheles sinensis. Gene silencing using this method led to down-regulation of target gene expression (AsCPR128) by 77% in targeted body parts, compared with only 10% in non-targeted body parts, thus increasing the defect-phenotype rate in the target area by 5.3-fold, compared with non-shock injected controls. Electroporation-mediated somatic transgenesis resulted in stable phenotypic characteristics of the reporter gene at the shocked body parts during the pupal-adult stages in about 69% of individuals. Furthermore, injecting plasmid DNA near the ovaries of female mosquitoes after a blood meal followed by electric shock produced three germline G1 transgenic lines, with a transformation rate of about 11.1% (calculated from ovulatory G0 females). Among the positive G1 lines, 42%, 40%, and 31% of individuals emitted red fluorescence in the larval stage. When the red fluorescent larvae developed into adults, green fluorescence was emitted from the ovaries of the females upon feeding. These results suggest that electroporation-mediated non-embryonic microinjection can be an efficient, rapid, and simple technique for analyzing gene function in non-model mosquitoes or other small insects.

Zebrafish Models of Craniofacial Malformations: Interactions of Environmental Factors.

The zebrafish is an appealing model organism for investigating the genetic (G) and environmental (E) factors, as well as their interactions (GxE), which contribute to craniofacial malformations. Here, we review zebrafish studies on environmental factors involved in the etiology of craniofacial malformations in humans including maternal smoking, alcohol consumption, nutrition and drug use. As an example, we focus on the (cleft) palate, for which the zebrafish ethmoid plate is a good model. This review highlights the importance of investigating ExE interactions and discusses the variable effects of exposure to environmental factors on craniofacial development depending on dosage, exposure time and developmental stage. Zebrafish also promise to be a good tool to study novel craniofacial teratogens and toxin mixtures. Lastly, we discuss the handful of studies on gene–alcohol interactions using mutant sensitivity screens and reverse genetic techniques. We expect that studies addressing complex interactions (ExE and GxE) in craniofacial malformations will increase in the coming years. These are likely to uncover currently unknown mechanisms with implications for the prevention of craniofacial malformations. The zebrafish appears to be an excellent complementary model with high translational value to study these complex interactions.

What Makes a Weed a Weed? How Virus-mediated Reverse Genetics Can Help to Explore the Genetics of Weediness

Reverse genetics investigates what a gene does by testing how the plant responds when the specific gene is changed. These techniques have been in use for decades to assess whether a given gene underpins interesting phenotypes and gain insight into the function of gene networks and families. Weed science has only recently entered the "genomic era" in which genomic and reverse genetics approaches are used to address hypotheses. This review focuses on two reverse genetic techniques used on a variety of plants including agricultural weeds, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), explaining the biology behind them and highlighting how these tools may be used for gene function validation in weed species for which no other transgenic approaches have been developed.

Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China

Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde virus as a surrogate small animal model for human Lassa fever

ABSTRACT Arenaviruses, such as Lassa virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenavirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde virus (PICV), an arenavirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.

Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders.

Animal models are essential tools for addressing fundamental scientific questions about skeletal diseases and for the development of new therapeutic approaches. Traditionally, mice have been the most common model organism in biomedical research, but their use is hampered by several limitations including complex generation, demanding investigation of early developmental stages, regulatory restrictions on breeding, and high maintenance cost. The zebrafish has been used as an efficient alternative vertebrate model for the study of human skeletal diseases, thanks to its easy genetic manipulation, high fecundity, external fertilization, transparency of rapidly developing embryos, and low maintenance cost. Furthermore, zebrafish share similar skeletal cells and ossification types with mammals. In the last decades, the use of both forward and new reverse genetics techniques has resulted in the generation of many mutant lines carrying skeletal phenotypes associated with human diseases. In addition, transgenic lines expressing fluorescent proteins under bone cell- or pathway- specific promoters enable in vivo imaging of differentiation and signaling at the cellular level. Despite the small size of the zebrafish, many traditional techniques for skeletal phenotyping, such as x-ray and microCT imaging and histological approaches, can be applied using the appropriate equipment and custom protocols. The ability of adult zebrafish to remodel skeletal tissues can be exploited as a unique tool to investigate bone formation and repair. Finally, the permeability of embryos to chemicals dissolved in water, together with the availability of large numbers of small-sized animals makes zebrafish a perfect model for high-throughput bone anabolic drug screening. This review aims to discuss the techniques that make zebrafish a powerful model to investigate the molecular and physiological basis of skeletal disorders.

Application of Reverse Genetics in Functional Genomics of Potyvirus.

Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus–host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.

Validating Plant Genes Involved in Pepper Yellow Leaf Curl Indonesia Virus Infection Using VIGS in Model Plant Nicotiana benthamiana

<p>Pepper yellow leaf curl disease caused by Pepper yellow leaf curl Indonesia virus (PepYLCIV) has become a challenge to chili pepper cultivation. Development of resistant variety by utilizing recessive resistance gene is expected to control the disease in the field. This study aimed to validate three plant genes, namely deltaCOP, hsc70, and BAM1, in PepYLCIV infection by applying Virus-induced Gene Silencing (VIGS) in a model plant, wild type Nicotiana benthamiana. PepYLCIV and construct of Tobacco rattle virus (TRV) which induced silencing of each gene were co-inoculated into N. benthamiana plants through agroinfiltration. Gene expression and the relative amount of viral DNA were determined by quantitative reverse transcription PCR (qRT-PCR) and quantitative PCR (qPCR), respectively, at 15 days post inoculation. The results showed a decreased level of deltaCOP, hsc70, and BAM1 expressions to 66.4%, 53.0%, and 47.0%, respectively, compared to that in the control (100%). Silencing of the three genes decreased the accumulation of PepYLCIV to 0.1%, 18.4%, and 63.0%, respectively, compared to that in the control. deltaCOP and hsc70 genes were indicated to be involved in the viral infection and could be good candidate genes for obtaining chili pepper varieties resistant to PepYLCIV. This result affirmed that the reverse genetics technique could be an alternative approach for identifying plant genes involved in viral infection, including PepYLCIV. The use of an infectious clone in this study allows the virus inoculations could be carried out without rearing and maintaining its natural vector, hence reduces the risk of virus transmission to healthy plants.</p>

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Immunization with the H5N1 Recombinant Vaccine Candidate Induces High Protection in Chickens against Vietnamese Highly Pathogenic Avian Influenza Virus Strains.

Vietnam is one of the countries most affected worldwide by the highly pathogenic avian influenza (HPAI) virus, which caused enormous economic loss and posed threats to public health. Over nearly two decades, with the antigenic changes in the diversified H5Ny viruses, the limited protective efficacy of the available vaccines was encountered. Therefore, it is necessary to approach a technology platform for the country to accelerate vaccine production that enables quick response to new influenza subtypes. This study utilized a powerful reverse genetics technique to successfully generate a recombinant H5N1 vaccine strain (designated as IBT-RG02) containing two surface proteins (haemagglutinin (HA) and neuraminidase (NA)) from the HPAI H5N1 (A/duck/Vietnam/HT2/2014(H5N1)) of the dominant clade 2.3.2.1c in Vietnam during 2012–2014. Importantly, the IBT-RG02 vaccine candidate has elicited high antibody titres in chickens (geometric mean titre (GMT) of 6.42 and 6.92, log2 on day 14 and day 28 p.i., respectively). To test the efficacy, immunized chickens were challenged with the circulating virulent strains. As results, there was a high protection rate of 91.6% chickens against the virulent A/DK/VN/Bacninh/NCVD-17A384/2017 of the same clade and a cross-protection of 83.3% against A/duck/TG/NAVET(3)/2013 virus of clade 1.1. Our promising results showed that we can independently master the reverse genetics technology for generation of highly immunogenic vaccine candidates, and henceforth, it is a timely manner to reformulate avian influenza virus vaccines against variable H5 clade HPAI viruses in Vietnam.

Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue

Objective To investigate the feasibility of using recombinant infectious clones of human coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids (pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12.9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12.9 genes with the reporter gene encoding the green fluorescent protein (GFP) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the rescued virus strains were characterized by immunofluorescence assay (IFA) and Western blot (WB) assay after transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12.9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12.9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12.9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was obtained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infected with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors. Key words: Human coronavirus OC43; Infectious clone; Viral rescue; Green fluorescent protein; Viral vector

Protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV

Objective To evaluate the immune responses and protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV. Methods Two recombinant influenza viruses (rFLU/hMPV/B and rFLU/hMPV/CTL+ Th) carrying hMPV multi-epitope gene segments in NS gene were generated by reverse genetic techniques of eight-plasmid system. BALB/c mice were immunized intranasally with rFLU/hMPV/B and rFLU/hMPV/CTL+ Th twice at a two-week interval. Virus-specific antibody titers and splenocyte cytokines were detected two weeks after the boost immunization. Viral loads in lung tissues and turbinates were detected with digital PCR after the immunized mice were challenged with hMPV and influenza virus. Moreover, HE staining was used to observe lung injuries. Results Specific antibodies against both the influenza virus and hMPV were induced in mice immunized intranasally with rFLU/hMPV/B, while the influenza virus-specific antibody response and hMPV-specific cytotoxic lymphocyte response (significant IFN-γ secretion) were detected in mice immunized with rFLU/hMPV/CTL+ Th. Additionally, balanced Th1/Th2 responses were elicited by rFLU/hMPV/B and rFLU/hMPV/CTL+ Th. Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th conveyed effective protection against subsequent influenza virus and hMPV challenges with significantly alleviated histopathological damages and reduced viral loads. Conclusions Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th can induce specific humoral immune response against hMPV and/or the influenza virus. Moreover, rFLU/hMPV/CTL+ Th can also elicit hMPV-specific CTL immune response. These two recombinant strains can also protect BALB/c mice from the challenges with hMPV and influenza virus, suggesting that they are promising vaccine candidates. Key words: Human metapneumovirus; Influenza virus vector; Antigen epitope; Immune protection

Virus-Induced Gene Silencing of Cell Wall Genes in Flax (Linum usitatissimum).

Plants have developed defense mechanisms against viruses by using an RNA silencing-based process, which has many common features with the endogenous RNA silencing pathway used for regulating the level of transcripts derived from developmental genes. In the virus-induced gene silencing (VIGS) method, it is possible to take advantage of this mechanism by inserting a plant nucleic fragment within the viral genome to knock down the corresponding gene. This tool has been used in many species as a fast and easy reverse genetics technique in order to gain information on the role of genes with poorly understood functions. Here we describe in detail two Agrobacterium-mediated infection protocols in flax, based on a whole plant vacuum infiltration and a leaf syringe infiltration that systemically impact the transcript levels in the stem.

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. Here, we successfully constructed a homozygous strain (Pxα6-KO) with a 2-nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non-homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3-TM4 loop region. When compared to the background strain IPP-S, the knockout strain Pxα6-KO exhibited 229- and 1462-fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3-fold) effects on the toxicities of imidacloprid, abamectin, β-cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2-nt deletion mutation of nAChRα6 in the Pxα6-KO strain. In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6-KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry.

A Fungal Arrestin Protein Contributes to Cell Cycle Progression and Pathogenesis.

Arrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins, Ali1, Ali2, Ali3, and Ali4, in the human fungal pathogen Cryptococcus neoformans Using complementary microscopy, proteomic, and reverse genetics techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression.IMPORTANCE To survive under unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that the disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression.

Construction and immunological evaluation of recombinant Newcastle disease virus vaccines expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP3/GP5 proteins in pigs

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) poses a significant threat to the pig industry, for which vaccination is considered to be an effective means of prevention and control. Here, we developed two recombinant Newcastle disease virus (NDV) LaSota-vectored PRRS candidate vaccines, rLaSota-GP5 and rLaSota-GP3-GP5, using reverse genetic techniques. The two recombinant viruses exhibited a high degree of genetic stability after 10 successive generations in chicken embryos. There was no significant difference in pathogenicity compared with the rLaSota parent strain in poultry, mice and pigs. The recombinant viruses could not be detected in the feeding environment of immunized pigs, but could be detected in the organs and tissues of pigs for no more than 10 days after immunization. Importantly, in contrast to rLaSota-GP5, rLaSota-GP3-GP5 elicited both significant humoral and cellular immune responses in pigs. In particular, the neutralizing antibody titer in the rLaSota-GP3-GP5 group was 1.51 times significantly higher than that of the commercial vaccine group at 42 days post-immunization. At the same time, there was significant difference in the level of IFN-γ between the rLaSota-GP3-GP5 group and the commercial vaccine group. Furthermore, the viral load in the organs and tissues of rLaSota-GP3-GP5-immunized pigs was substantially lower than that of unimmunized pigs after being challenged with HP-PRRS virus GD strain. These results suggest that rLaSota-GP3-GP5 is a safe and promising candidate vaccine, and there is potential for further development of a recombinant virus vaccine for PRRS using NDV.

The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles.

Zika virus (ZIKV) is transmitted by Aedes mosquitoes and exhibits genetic variation with African and Asian lineages. ZIKV Natal RGN strain, an Asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. However, ZIKV Natal RGN strain has not been isolated; its biological features are not yet illustrated. This study rescued and characterized recombinant, single-round infectious particles (SRIPs) of the ZIKV Natal RGN strain using reverse genetic and synthetic biology techniques. The DNA-launched replicon of ZIKV Natal RGN was constructed and contains the EGFP reporter, lacks prM-E genes, and replicates under CMV promoter control. The peak in the ZIKV Natal RGN SRIP titer reached 6.25 × 106 TCID50/mL in the supernatant of prM-E-expressing packaging cells 72 h post-transfection with a ZIKV Natal RGN replicon. The infectivity of ZIKV Natal RGN SRIPs has been demonstrated to correlate with the green florescence intensity of the EGFP reporter, the SRIP-induced cytopathic effect, and ZIKV’s non-structural protein expression. Moreover, ZIKV Natal RGN SRIPs effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. Therefore, the recombinant ZIKV Natal RGN strain was rescued as SRIPs that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates.