Spot Syndrome Virus In Shrimp Research Articles

A recombinase polymerase amplification and Pyrococcus furiosus Argonaute combined method for ultra-sensitive detection of white spot syndrome virus in shrimp.

White spot disease (WSD) in shrimp is an acute infectious disease caused by white spot syndrome virus (WSSV). WSD has seriously threatened the security of shrimp farming, causing huge economic losses worldwide. As there is currently no effective treatment for WSD, developing early detection technologies for WSSV is of great significance for the prevention. In this study, we have established a detection method for WSSV using a combination of recombinase polymerase amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). We have achieved a detection sensitivity of single copy per reaction, which is more sensitive than the previously reported detection methods. Additionally, we have demonstrated high specificity. The entire detection process can be completed within 75 min without the need for precise thermal cyclers, making it suitable for on-site testing. The fluorescence signal generated by the reaction can be quantified using a portable fluorescence detector or observed with the naked eye under a blue light background. This study provides an ultrasensitive on-site detection method for WSSV in shrimp aquaculture and expands the application of PfAgo in the field of aquatic disease diagnosis.

Functional Peroral Infectivity Complex of White Spot Syndrome Virus of Shrimp.

White spot syndrome virus (WSSV) is a major cause of disease in shrimp cultures worldwide. The infection process of this large circular double-stranded DNA virus has been well studied, but its entry mechanism remains controversial. The major virion envelope protein VP28 has been implicated in oral and systemic viral infection in shrimp. However, genetic analysis of viral DNA has shown the presence of a few genes related to proteins of per os infectivity factor (PIF) complex in baculoviruses. This complex is essential for the entry of baculoviruses, large terrestrial circular DNA viruses, into the midgut epithelial cells of insect larvae. In this study, we aimed to determine whether a PIF complex exists in WSSV, the components of this complex, whether it functions as an oral infectivity complex in shrimp, and the biochemical properties that contribute to its function in a marine environment. The results revealed a WSSV PIF complex (~720 kDa) comprising at least eight proteins, four of which were not identified as PIF homologs: WSV134, VP124 (WSV216), WSSV021, and WSV136. WSV134 is suggested to be a PIF4 homolog due to predicted structural similarity and amino acid sequence identity. The WSSV PIF complex is resistant to alkali, proteolysis, and high salt, properties that are important for maintaining infectivity in aquatic environments. Oral infection can be neutralized by PIF-specific antibodies but not by VP28-specific antibodies. These results indicate that the WSSV PIF complex is critical for WSSV entry into shrimp; the complex's evolutionary significance is also discussed. IMPORTANCE White spot disease, caused by the white spot syndrome virus (WSSV), is a major scourge in cultured shrimp production facilities worldwide. This disease is only effectively controlled by sanitation. Intervention strategies are urgently needed but are limited by a lack of appropriate targets. Our identification of a per os infectivity factor (PIF) complex, which is pivotal for the entry of WSSV into shrimp, could provide new targets for antibody- or dsRNA-based intervention strategies. In addition, the presence of a PIF complex with at least eight components in WSSV, which is ancestrally related to the PIF complex of invertebrate baculoviruses, suggests that this complex is structurally and functionally conserved in disparate virus taxa.

RNA Nanovaccine Protects against White Spot Syndrome Virus in Shrimp.

In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Visual LAMP Method for Detection of White Spot Syndrome Virus in Shrimp and Crayfish

White spot syndrome virus (WSSV) has caused large economic losses to the aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. Therefore, rapid detection of white spot syndrome virus (WSSV) in shrimp is crucial. This study was conducted to establish a rapid visual detection method for WSSV in shrimp and on the basis of loop-mediated isothermal amplification (LAMP). The color change of the amplification products was observed using a fluorescence amplification curve (with the SYTO-9 fluorescent dye) and LAMP visualization (with calcein), and the method`s specificity, sensitivity, and reproducibility were analyzed. It showed that this method can specifically detect WSSV, with a detection sensitivity of 1 fg/μL and has good reproducibility and reliability. This method was used to analyze cultured shrimp and crayfish samples; among 329 suspected clinical samples, the positive diagnostic rate of WSSV was 27.96%. The infection activity of WSSV was the highest at temperatures of 20–26°C. The LAMP-based method for rapid visual detection of WSSV does not need complex experimental instruments, and an equipment with a stable heat source is sufficient for the reaction. It is easy to use and can be used at shrimp breeding sites.

Design and Evaluation of a PCR Method for Detecting White Spot Syndrome Virus in Shrimp

Design and Evaluation of a PCR Method for Detecting White Spot Syndrome Virus in Shrimp

Field-usable aptamer‑gold nanoparticles-based colorimetric sensor for rapid detection of white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is one of the most threatening viral pathogens of shrimp worldwide. VP28, the major capsid protein of WSSV, is reported to play an essential role in the interaction with the host cells. Usually, the diagnostic test of WSSV is performed by one-step PCR, which is neither field-usable nor rapid enough. Therefore, the development of early diagnostic tests is imperative for the management of disease and to prevent huge economic losses. In this work, a rapid colorimetric aptasensor with high selectivity and sensitivity was introduced. First, a new ssDNA aptamer for the detection of WSSV was designed from a random pool of aptamer sequences based on the docking score and bioconjugate free energy, and then, evaluated experimentally. The designed aptamer was used to prepare an aptasensor conjugate gold nanoparticles (AuNPs) as a recognition element that increased the resistance of AuNPs to salt-induced aggregation. Therefore, the AuNPs with spherical morphology and average particle size of about 20 nm were synthesized for this reason. The results showed that the aptamer didn't attach to the other pathogens (i.e., IHHN and Vibrio harveyi) and healthy shrimp cells, and remained stable. Interestingly, the gray-blue color of aptasensor was seen only by the presence of WSSV, indicating the aptasensor assay showed good specificity to WSSV. The limit of detection (LOD) of the aptasensor was 104 copies of WSSV that could be detected only utilizing naked eyes. It was concluded that the designed aptamer had excellent potential for the detection of WSSV as a rapid visual colorimetric assay in aquaculture shrimp farmers.

Oral administration of Saccharomyces cerevisiae displaying VP28-VP24 confers protection against white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is the major pathogen that leads to severe mortalities in cultured shrimp worldwide. The envelope proteins VP28 and VP24 of WSSV are considered potential vaccine candidate antigens. In this study, we utilized a Saccharomyces cerevisiae (S. cerevisiae) surface display system to demonstrate the feasibility of this platform for developing a vaccine candidate against WSSV. EBY100/pYD1-VP28-VP24 was generated, and the fusion protein VP28-VP24 was present on the surface of S. cerevisiae. Penaeus vannamei (P. vannamei) was used as an animal model. Oral administration of EBY100/pYD1-VP28-VP24 could induce significant activities of immune-related enzymes such as superoxide dismutase (SOD) and phenoloxidase (PO). Importantly, WSSV challenge indicated that oral administration of EBY100/pYD1-VP28-VP24 could confer 100% protection with a corresponding decrease in the viral load. The collective results strongly highlight the potential of a S. cerevisiae-based oral vaccine as an efficient control strategy for combating WSSV infection in shrimp aquaculture.

A multi-target dsRNA for simultaneous inhibition of yellow head virus and white spot syndrome virus in shrimp

Outbreaks of diseases caused by yellow head virus (YHV) and white spot syndrome virus (WSSV) infection in shrimp have resulted in economic losses worldwide. DsRNA-mediated RNAi has been used to control these viruses, and the best target genes for efficient inhibition of YHV and WSSV are the protease and ribonuleotide reductase small subunit (rr2), respectively. However, one dsRNA can suppress only one virus, and therefore the production of multi-target dsRNA to effectively inhibit both YHV and WSSV is needed. In this study, plasmids pETpro-rr2_one stem and pETpro-rr2_two stems were constructed to produce two different forms of multi-target dsRNA in E. coli, which were designed specifically to both YHV protease and WSSV rr2 genes. The potency of each dsRNA in inhibiting YHV and WSSV and reducing shrimp death were investigated in L. vannamei. Shrimp were injected with the dsRNAs into the hemolymph before challenge with YHV or WSSV. The results showed that both dsRNAs could inhibit the viruses, however the one stem construct was more effective than the two stems construct when shrimp were infected with WSSV. This study establishes a potential strategy for dual inhibition of YHV and WSSV for further application in shrimp aquaculture.

A novel and convenient bacterial platform for production of encapsidated double‐stranded RNA targeting white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is a highly infectious virus that can cause 100% mortality in farmed shrimp in the matter of days, leading to significant economic losses to shrimp aquaculture worldwide. To mitigate the adverse impact of WSSV, effective antiviral strategies are necessary. Currently, double‐stranded RNA (dsRNA), which can silence viral gene expression through an RNA interference (RNAi) pathway, is among the most promising therapeutic tools for WSSV, but its farm‐scale utilization is held back by high costs and the lack of delivery approach. Recently, virus‐like particles (VLPs) have been applied as nanocarriers to deliver dsRNA into shrimp tissues, resulting in prolonged half‐life of the encapsidated dsRNA and stimulation of shrimp innate immunity. However, recombinant VLPs and dsRNA are traditionally expressed in two different E. coli strains: a protease‐deficient BL21(DE3) strain and an RNase III‐deficient HT115(DE3) strain, respectively. The ability to produce both VLPs and dsRNA simultaneously in a cell will reduce the costs and steps associated with the production, but attempts at VLP‐dsRNA coexpression in each of these strains resulted in degradation of either VLP or dsRNA due to the presence of proteases or RNaseIII, respectively. Here, to streamline VLP‐dsRNA production and allow simultaneous expression of VLP and dsRNA in the same cell, P1 transduction was employed to generate a novel E. coli strain which is both protease‐ and RNase III‐deficient. The newly engineered E. coli strain is able to express both MrNV‐VLP and VP28 dsRNA efficiently. Therefore, this new expression platform will pave the way for development of effective and low‐cost production of VLP‐encapsulated dsRNA for protection against viral infection in farmed shrimp.Support or Funding InformationThe authors would like to thank the following institutions for supporting research and scholarship to graduate student: Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA) and Mahidol University.

Determining the Dosage and Time of Injection for WSSV VP28 Double Stranded RNA to Penaeus indicus in Providing Effective Protection Against WSSV

Bala Amarnath, C.; Saravanan, P., and Subhendu Kumar Otta, 2019. Determining the dosage and time of injection for WSSV VP28 double stranded RNA to Penaeus indicus in providing effective protection against WSSV. In: Jithendran, K.P.; Saraswathy, R.; Balasubramanian, C.P.; Kumaraguru Vasagam, K.P.; Jayasankar, V.; Raghavan, R.; Alavandi, S.V., and Vijayan, K.K. (eds.), BRAQCON 2019: World Brackishwater Aquaculture Conference. Journal of Coastal Research, Special Issue No. 86, pp. 102–106. Coconut Creek (Florida), ISSN 0749-0208.White spot syndrome virus of shrimp is best known for its wide distribution and high virulence in bringing mass mortality and huge economic loss to farmers. Though the virus has been causing wide spread mortality to all the cultured penaeid shrimps for quite a long time, no effective treatment strategies have been identified so far. RNAi has been recognized as a powerful tool and found to be functional in shrimp. An attempt was made to verify the possibility of using this technology in laboratory-based experiment through double stranded RNA (dsRNA) construction and injection to Indian white leg shrimp, Penaeus indicus. Amongst the different dosages tried, 1 µg/gm of shrimp administered 24-48 hours before WSSV infection was found to be effective in providing full protection. While trying to further refine the time interval by injecting the shrimp with 1 µg of VP28 dsRNA/gm of shrimp at 3, 6, 12 and 18 hours before WSSV infection, no protection was observed in any of these time intervals indicating that administration of dsRNA 24 hours before infection to be the most suitable. Real time PCR analysis further indicated that only the shrimps injected with dsRNA 24 hours prior to infection were virus free, and the others were with high copy numbers of WSSV. This experiment indicates that dsRNA can be used as a prophylaxis to further prevent the infection by WSSV.

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals.

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population-level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease-specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer-reviewed research. A systematic review identified only 12 peer-reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence-based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer-reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.

Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp

White spot disease (WSD), caused by white spot syndrome virus (WSSV), is among the most severe diseases of cultivated shrimp. Here, the CRISPR-Cas12a system coupled with nucleic acid amplification was optimized for the detection of WSSV. The CRISPR-Cas12a system was used to specifically cleave the WSSV amplicons, simultaneously releasing a quenched reporter molecule resulting in fluorescence that could be detected with a simple UV transilluminator or a microplate reader. This specific cleavage accompanied by fluorescence simultaneously revealed the presence of the amplicon and confirmed its identity, preventing false positive test results from non-specific amplicons. When coupled with PCR or recombinase polymerase amplification (RPA), the Cas12a platform was capable of detecting as few as 200 copies WSSV per reaction and displayed no cross-reactivity with other shrimp DNA viruses. The method was also un-interfered by the presence of large amounts of unrelated background DNA. Moreover, the RPA-Cas12a protocol from start to finish could be performed at a constant temperature near 37 °C and required <1 h, without the need for complex equipment. Overall, our results demonstrated that the CRISPR-Cas12a method is robust, specific, confirmatory, user-friendly and potentially adaptable for in-field diagnosis of shrimp diseases.

Identification of Functional Gene Modules Associated With STAT-Mediated Antiviral Responses to White Spot Syndrome Virus in Shrimp.

White spot syndrome virus (WSSV) is one of the major threats to shrimp aquaculture. It has been found that the signal transducer and activator of transcription (STAT) protein plays an important role in the antiviral immunity of shrimp with a WSSV infection. However, the mechanism that underlies the STAT-mediated antiviral responses in shrimp, against WSSV infection, remains unclear. In this work, based on the gene expression profiles of shrimp with an injection of WSSV and STAT double strand RNA (dsRNA), we constructed a gene co-expression network for shrimp and identified the gene modules that are possibly responsible for STAT-mediated antiviral responses. These gene modules are found enriched in the regulation of the viral process, JAK-STAT cascade and the regulation of immune effector process pathways. The gene modules identified here provide insights into the molecular mechanism that underlies the STAT-mediated antiviral response of shrimp, against WSSV.

Quantitative analysis of the dose-response of white spot syndrome virus in shrimp.

White spot syndrome virus (WSSV) is an important cause of mortality and economic losses in shrimp farming. Although WSSV-induced mortality is virus dose dependent and WSSV infection does not necessarily lead to mortality, the relationships between virus-particle dose, infection and mortality have not been analysed quantitatively. Here, we explored WSSV dose-response by a combination of experiments, modelling and meta-analysis. We performed dose-response experiments in Penaeus vannamei postlarvae, recorded host mortality and detected WSSV infection. When we fitted infection models to these data, two models-differing in whether they incorporated heterogeneous host susceptibility to the virus or not-were supported for two independent experiments. To determine the generality of these results, we reanalysed published data sets and then performed a meta-analysis. We found that WSSV dose-response kinetics is indeed variable over experiments. We could not clearly identify which specific infection model has the most support by meta-analysis, but we argue that these results also are most concordant with a model incorporating varying levels of heterogeneous host susceptibility to WSSV. We have identified suitable models for analysing WSSV dose-response, which can elucidate the most basic virus-host interactions and help to avoid underestimating WSSV infection at low virus doses.

Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28.

Correction for Xu et al., "Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp".

Correction for Xu et al., "Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp".

Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp.

Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, ΔMjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.

Oral administration of Anabaena-expressed VP28 for both drug and food against white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is one of the most prevalent and lethal viruses for shrimp, and oral administration of VP28 is a promising approach to protect shrimp against WSSV. Although seven heterologous expression systems have been successful since 2004, no one has been commercialized in shrimp industry. This paper presented the idea of “both drug and feed from the same feedstock” to develop oral vaccine against WSSV. Filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120, was used to express major envelope protein, VP28, for feeding post larvae of shrimp. The expression of vp28 gene in transgenic mutant was analyzed by Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). For vaccination, Litopenaeus vannamei was orally administrated by feeding the wild type and mutants of Anabaena cells for 10 days, and was challenged with WSSV during 10 days post vaccination. The post larvae were fed with the mutant harboring vp28 (10 μg shrimp−1). The post larvae were divided five groups and their survivals were as follows: (1) negative control (neither vaccination nor challenge), 100 %; (2) positive control (challenge and no vaccination), 0 %; (3) feeding the wild type, 15.5 %; (4) feeding blank, 20.6 %; and (5) feeding the mutant harboring vp28, 68.0 %. Moreover, the post larvae fed the mutant harboring vp28 grew better than no feed. Also, the photosynthetic features indicated the transgenic mutant was easier to cultivate. These data show that transgenic mutant harboring vp28 was favorable not only to use both for drug and feed but also to cultivate in scale.

An enhanced immuno-dot blot assay for the detection of white spot syndrome virus in shrimp using antibody conjugated gold nanoparticles probe

White Spot Syndrome (WSS) is a worldwide ailment of penaeid shrimp, a lethal and contagious disease in shrimp caused by the White Spot Syndrome Virus (WSSV). It is essential to control; handle early findings and routine screenings under field conditions. Though PCR affirmed sensing of the causative viral nucleic acid has many advantages, for its routine use custom necessitate an advanced equipment and technical expertise. In comparison, protein-based immuno detecting methods are easier to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this study, we focused on a sensitive immuno detection method which developed by gold nanoparticles coupled with alkaline phosphatase conjugated secondary antibody in turn recognized primary anti-serum raised against WSSV. We successfully enhanced the sensitivity of immuno-dot blot assay by 80 fold over the conventional method and visually it can be detected up to 1 ng/mL of purified WSSV. The approach described in this study is a prototype for the development of simple and inexpensive diagnostic tool will provide the routine screening of WSS in shrimp farms.

Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation

The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.