Patent Application
20250127883
This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Nov. 29, 2024, is name “15290-035US1.xml” and is 70,289 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.
The invention relates to the general field of animal health. In particular, the invention relates to a vaccine composed of a nonvirulent wild-isolate virus which is useful against a virulent virus in a beetle larvae system.
The superworm (Zophobas morio) and its close relative, the mealworm (Tenebrio molitor), are species of darkling beetles whose protein-rich larvae are dietary staples for captive reptiles, birds, and amphibians worldwide. Darkling beetles are omnivorous, and eat plants, animals, and in some cases Styrofoam™. Superworms are also under investigation as an alternative animal protein source. Commercial production of Zophobas morio larvae (superworm) is a growing industry, with applications in animal feed. Research using this species has also made recent popular press headlines for the species' potential as a next-generation approach to plastic waste management and degradation.
Yet, industrial production of the superworm is complicated by mass die-offs due to infectious disease. Reports from commercial and hobbyist growers of Z. morio that farm larval populations indicate that some growers were experiencing up to 100% mortality. The larvae are typically housed in enclosures containing many individuals because dense packing inhibits pupation. Darkling beetles are known to eat deceased or moribund members of their own species, which exacerbates the spread of infectious disease.
One disease that affects these beetles is caused by viruses of the family Parvoviridae (common name: parvoviruses) and subfamily Densovirinae (common name: densoviruses). Colonies that become infected with densoviruses can experience mortality rates of up to 100%. Insects experience symptoms such as blackening, liquefaction of the organs, and uncoordinated movement. A composition for controlling densovirus infection in live insects has not been described. Therefore, there is a need in the art for methods to prevent and treat infection by Parvoviridae.
Parvoviruses (26 PV) are small icosahedral viruses with a non-enveloped virion and linear, ssDNA genome. The family Parvoviridae possesses a remarkably diverse host spectrum of both protostome and deuterostome invertebrates as well as vertebrates. Although two out of the three subfamilies within the Parvoviridae—Densovirinae and Hamaparvovirinae—include viruses of invertebrate hosts, traditionally all invertebrate-infecting PVs are referred to as densoviruses (DVs). DVs in general display high virulence and pathogenicity, and can affect insects in their larval stage, crustaceans, mollusks, and echinoderms. Mass-reared arthropods are especially in danger of DV infection.
Members of the family Parvoviridae are united by their genome size and organization and presence of certain conserved protein domains, as well as their capsid protein structure. All parvoviruses thus far harbor a small, single-stranded DNA genome of 3.7 to 6.3 kb in length, flanked by partially double-stranded, hairpin-like DNA secondary structures, essential for parvovirus replication. The coding region of the genome contains two major expression cassettes: rep, which expresses the non-structural (NS) proteins, and cap, which expresses between one and four isoforms of the structural protein (VP). These are flanked by structured genomic termini named “inverted terminal repeats” (ITRs), which are hairpin-like and partially double-stranded. The cassette closer to the 5′ end, named rep, is capable of expressing a varied number of non-structural (NS) proteins. The cassette closer to the 3′ end, named cap, encodes for one to four structural proteins (VP). The VPs share an overlapping C-terminal region and differ from each other in N-terminal extensions.
The conserved genome organization, presence of certain domains, and capsid protein structure are characteristic of the family Parvoviridae. PV infect diverse hosts from protostome and deuterostome invertebrates to humans. Most members of the vertebrate infecting Parvovirinae and all members of the Densovirinae harbor a phospholipase A2 (PLA2) enzymatic domain in the unique N-terminal extension (VP1u) of their largest minor capsid proteins. The PLA2 domain is essential for endolysosomal egress during PV and DV intracellular trafficking.
Current approaches for avoiding this disease in commercial growing are largely dependent on rearing practices by the producer. At present, culling the entire population is the only known response to an outbreak at a facility. The disease is poorly understood, and there has been insufficient time since discovery of the causal organism for effective treatments to arise. Superworms are mainly reared for sale as pet food. Mortality of the reared insects causes loss of salable product. Avoiding this cause of mortality could therefore increase revenues for superworm sellers.
Thus, there exists a need in the art for a method to treat and prevent Parvovirus infection in superworms and mealworms, to improve husbandry for these species.
The invention described here relates to a vaccine method useful in prevention and treatment of “Zophobas morio black wasting disease (Zmbwd)” using the NJ2-molitor virus to infect larvae.
In particular embodiments, the present invention relates to a method of inhibiting Zophobas morio black wasting disease morbidity and mortality in a darkling beetle colony in need thereof, comprising: (a) isolating a strain of densovirus from Tenebrio molitor; and (b) administering the strain of densovirus to the darkling beetle colony, wherein the strain of densovirus is non-pathogenic to the recipient beetle colony. In preferred embodiments, the strain of densovirus is SEQ ID NO:5. In some embodiments, administering is by injection, dripping, spraying or ingestion.
In certain embodiments, the invention comprises a prophylactic vaccine composition, comprising the corpses of Z. morio larvae infected with a non-pathogenic strain of densovirus selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any combination thereof.
In certain embodiments, the invention comprises a prophylactic vaccine composition, comprising the corpses of darkling beetle larvae infected with a strain of ZmBWV that is isolated from Tenebrio molitor and does not cause mortality in the recipient species at a dose of 109 genomes. Preferably, the strain is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any combination thereof. In preferred embodiments, the darkling beetle is of the species Zophobas morio.
In certain embodiments, the invention relates to a prophylactic vaccine composition comprising purified virions of a non-pathogenic strain of densovirus and a pharmaceutically acceptable medium, wherein the sequence identity between the non-pathogenic strain of densovirus and strain NJ2-molitor (SEQ ID NO: 5) is about 96% or greater.
In certain embodiments, the invention relates to a prophylactic vaccine composition comprising: a pharmaceutically acceptable medium and a purified virions of a non-pathogenic strain of densovirus selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any combination thereof.
In certain embodiments, the invention relates to a prophylactic vaccine composition, comprising one or more nonpathogenic densovirus strain, wherein the NS3 canonical ATG start codon is mutated, truncating the NS3 protein to fewer than 200 amino acids, instead of the length of 221 residues in the pathogenic, highly virulent strain UT-morio (SEQ ID NO: 1). Preferably, the DNA sequence identity from NJ2-molitor (SEQ ID NO: 5) is about 96% or greater. In addition, preferably the pharmaceutically acceptable medium is phosphate-buffered saline. The vaccine preferably is formulated for injection, spraying, or dripping.
Certain embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.
A small, commercial insect-rearing facility experienced repeated Z. morio colony collapse in 2022. At approximately 8 weeks of age and 25 mm in length, Z. morio larvae showed signs of distressed locomotion, uncoordinated wiggling, and rigor followed by death. Moribund larvae quickly blackened as their inner organs lost structure, essentially becoming liquefied. See
The work disclosed here has successfully identified Zophobas morio black wasting virus (ZmBWV), characterized its structure, sequence, and pathogenesis as well as identified a prophylactic mechanism that can be used to diminish it. The present invention relates to products for and methods of preventing disease in a darkling beetle population by contacting the insects with non-pathogenic densovirus. In a preferred embodiment, the invention comprises a prophylactic product that is orally available as a feed for Z. morio larvae. In a second preferred embodiment, the invention comprises a prophylactic product that is applied topically to Z. morio larvae.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.
In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element or step modified by the article.
As used herein, the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125±0.025, and “about 1.0” means 1.0±0.2. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of “less than 10” can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.
As used herein, the term “subject” refers to insect larvae of Z. morio or T. molitar, including individual larvae and populations of larvae. Preferably the larvae are captive.
As used herein, the term “subject in need” refers to any Z. morio or T. molitar larva or population of larvae that are susceptible to exposure to ZmBWV, including symptomatic and asymptomatic individuals and populations and diagnosed or undiagnosed individuals and populations.
As used herein, the term “prevent,” “prevention,” “prophylaxis” and their cognates refer to (a) complete prevention of the condition, disease, or symptom(s) thereof from occurring in a subject; (b) decreasing the likelihood of a subject contracting or developing the disease or condition; (c) inhibiting the condition, or disease or symptom thereof, such as, arresting or delaying its development in a subject; (d) causing the disease or condition to occur less frequently in a subject; (e) reducing the occurrence of the condition, disease, or symptom thereof in a population; (f) shortening the duration or reducing the severity of a disease or condition in a subject; and/or (g) relieving, alleviating or ameliorating the condition, disease, or symptom thereof, such as, for example, causing regression of the condition or disease or symptom thereof.
As used herein, the term “vaccine” refers to a pharmaceutical compound or composition that prevents a disease, condition, or a symptom thereof in a subject or a population of subjects.
As used herein, the term “densovirus” refers to any virus listed in the 9th Report of the International Committee on the Taxonomy of Viruses as belonging to the subfamily Densovirinae, and additionally to any non-listed virus which has a higher sequence similarity to a virus listed as belonging to the subfamily Densovirinae than to a virus listed as belonging to another subfamily. These viruses contain a single-stranded DNA genome, an isometric capsid of 20-30 nm in diameter, and a genome length of 3,000 to 8,000 nucleotides.
As used herein, the term “ZmBWV” refers to Zophobas morio black wasting virus, a densovirus with a single-stranded DNA genome and an isometric capsid between 20 and 30 nm in diameter that infects beetles of the family Tenebrionidae (common name: darkling beetles). This term also can refer to any DNA virus with at least 90% sequence identity to SEQ ID NO:1.
The causative agent of Zophobas morio black wasting disease was identified by electron microscopy and named Zophobas morio black wasting virus (ZmBWV). The etiological role of ZmBWV was confirmed by injecting ZmBWV into healthy beetles, which then exhibited the characteristic symptoms of ZmBWD. ZmBWV was detected in beetles from: Utah, Minnesota, Georgia, Maryland, New Jersey, New York, Mississippi, Ohio, Arkansas, Indiana, Pennsylvania, Oregon, Louisiana, and Florida.
Using larval corpse homogenate as a diagnostic specimen, the structure of two variants of the agent at 2.7 Å and 2 Å resolution, respectively, were identified and named Zophobas morio Black Wasting Virus (ZmBWV). This resolution was sufficient to identify the agent as a virus of subfamily Densovirinae, family Parvoviridae. Mass-reared arthropods are known to be at high risk for densovirus (DV) infection.
Several pieces of information indicate that this agent (ZmBWV) is the etiologic agent behind ZmBWV. First, it is uncommon for a virus to grow to such high abundance in a diseased animal unless it is the cause of that animal's disease. In this study, ZmBWV was far more abundant than all other viruses, and much more abundant than the native bacteriophages. Second, there is ample precedent for a densovirus to cause symptoms and death in farmed insects. Third, our isolation of ZmBWV from this mortality event echoes the molecular detection of ZmDV from similar outbreaks in Europe. Related DVs infecting Z. morio are believed to have a worldwide distribution. Fourth, we detected ZmBWV by quantitative polymerase chain reaction in beetles with symptoms of Zophobas morio black wasting disease from multiple states. Finally, we successfully infected unexposed larvae with purified ZmBWV virions, resulting in all individuals exhibiting the symptoms of Zophobas morio black wasting disease, which was followed by a mortality rate of 100% (See
Parvoviruses are notably resistant to alcohol-based sanitizers (unlike, for example, SARS-CoV-2). Therefore, ethanol is not sufficient for cleaning enclosures and other items that have come into contact with infected beetles. Bleach should be used in these cases. Similarly, by analogy to other parvoviruses (and in contrast to SARS-CoV-2), we expect the virus to last for a long time on surfaces but not to spread particularly effectively through the air. It is likely that transmission can occur through tools and clothing as well as co-housing. There is some precedent for asymptomatic carriage of DVs. Therefore, when bringing new beetles into a breeding colony, avoiding overtly symptomatic individuals might not be sufficient. As a best practice, when introgressing exogenous stock into a colony, housing part of the colony separately for a couple generations is advisable to avoid loss of the whole colony in case the new beetle(s) carry this or other pathogens. It is unknown whether immunity to ZmBWV is heritable.
Results presented here suggest that ZmBWV has reached a nation-wide epidemic status, which appears to have started four years ago. Acheta domesticus densovirus (AdDV) of the same subfamily regularly disseminates cricket rearing facilities, causing mass mortality in its wake in Europe since 1977 as well as in North America since 2009. AdDV has completely transformed the cricket rearing culture worldwide, requiring the rearing of orthopteran species that exhibit less susceptibility to the virus than the common house cricket (Acheta domesticus). Although this is the first ZmBWV outbreak to reach an epidemic scale, it is important to note that this virus also exhibits a worldwide distribution, similarly to AdDV. The broad host spectrum of ZmBWV, as well as its subclinical presence and multiple genotypes indicate that the epidemic may remain active for a long a time to come.
There are several different ZmBWV strains circulating currently in the United States, affecting both the T. molitor and the Z. morio species. Despite this, we did not observe a similar scale epidemic in T. molitor. The lack of virulence shown here by the NJ2-molitor strain in the Z. morio host suggests that the current epidemic has a distinct origin. This is also supported by the phylogenetic calculations, which suggest a single introduction event. The studies presented here, however, indicate that there is cross-protectivity between these two strains. This phenomenon can be employed to provide protection against virulent strains of ZmBWV.
As part of these studies, it became apparent that ZmBWV spreads within a colony by the oral-fecal route. The midgut has been shown to play an important role in DV infection. Lepidopteran protoambidensoviruses cross the midgut wall by transcytosis, in order to reach the true site of replication, which may be the fat bodies or the wall of the visceral trachea and hemocytes, while lepidopteran iteradensoviruses and bidnaviruses replicate exclusively in the columnar midgut cells. In both cases, however, the infection runs its course fast and larvae die within 7-10 d.p.i.
The pathogenesis of the ZmBWV virulent strain UT-morio required almost three times longer to result in mortality. Moreover, at the affected farms the newly hatched larvae were already infected, yet symptoms manifested only at the age of eight weeks. Once the larvae display the initial signs of ZmBWD, death is expected to set in less than five days.
Here, we suggest a model of pathogenesis of the virus replicating in the midgut columnar cells following uptake by contaminated feed, which eventually leads to the invasion of the fat body once the midgut wall is too damaged to fulfill its barrier function. The invasion may happen directly before the onset of symptoms. Although DV-infected caterpillars fail to pupate, ZmBWV-infected Z. morio larvae completed their entire life cycle, if they managed to pupate before the onset of symptoms. The ability of ZmBWD to cause a chronic infection in reproducing beetles may be the major reason to why the epidemic has been persistent.
ZmBWV is a member of the Blattambidensovirus genus, yet its biology differs from that of its most studied member i.e., Blattella germanica densovirus (BgDV). The minor VPs of BgDV run significantly heavier when subjected to SDS-PAGE than suggested by their predicted molecular mass due to ubiquitination. Moreover, BgDV exhibits two spliced transcripts when expressing its minor capsid proteins, which results in the protein sequence of cap1 to comprise the N-terminus of both VP1 and VP2. The ZmBWV VPs display an SDS-PAGE running profile that corresponds with their predicted molecular weights, implying that they are not subjected to the same post translational modifications. Moreover, only the protein sequence of VP1 corresponded with cap1, implying that ZmBWV only expresses one spliced VP transcript. However, we could identify a glycan at the ZmBWV surface, probably due to the N-glocalization. Although this type of post-translational modification has not been described in DVs, members of the Parvovirinae, such as adeno-associated virus (AAV), have been shown to be subjected to N-glocalization. In the case of AAV8, N-glocalization was only detectable in secreted capsids as opposed to the ones acquired by cell lysis. It is plausible that ZmBWV also acquires this modification during the process of cellular egress, which implies that it does not rely exclusively on cell lysis, leading to intact midgut cells to possibly secrete virions. This finding is in concordance with the slow initial phase of the ZmBWV pathogenesis.
This invention provides the first method and product for inhibition of ZmBWV disease in Z. morio colonies. The method involves exposure of the larvae in a colony to a nonpathogenic virus as a vaccine which reduces morbidity and mortality in the colony produced by Z. morio black wasting virus.
Specimens of Z. morio (16 pools) and of T. molitor (8 pools) were collected from breeders who had experienced mass mortality events clinically consistent with ZmBWD (9 breeders; 11 farms), or from mail-order services (3), or from local stores (2). Using the whole-genome sequence, a diagnostic PCR targeting the NS1 gene was developed. 100% of Z. morio pools obtained from breeders with symptomatic larvae tested positive by PCR. At local stores, staff or customers reported observing the typical pathology and pools were positive from both. Mail-order Z. morio from two vendors did not exhibit symptoms; these alone were PCR-negative. These findings satisfy Koch's First Postulate. ZmBWV was detected in 11 states representing all regions of the lower 48 of the United States, and ZmBWV should therefore be considered endemic nationwide.
We did not obtain or attempt to obtain samples from other countries. The NCBI GenBank, however, already included nine metagenomic sequences of Chinese, Malaysian and European origin, derived from bird, bat and pangolin metagenomes, harboring 94-97% identity to the reference ZmBWV strain. This implies a worldwide distribution.
Breeders reported that mealworms would occasionally exhibit black wasting but economically-significant mortality was never observed. We detected a ZmBWV-like virus from all 3 mail-order T. molitor batches tested, although no signs or symptoms were observed. Likewise, 3 of 4 T. molitor pools from breeders of Z. morio were PCR-positive; two of these colonies had a few overtly symptomatic individuals and one had none. We concluded that the ZmBWV-like virus of T. molitor is of mild pathogenicity in its native host. Genomic sequences were obtained for 8 Z. morio and 5 T. molitor samples.
Full genome phylogenetic calculations (
A densovirus associated with mass mortality in Z. morio larvae at a small-sized insect rearing facility in the western United States was investigated. Z. morio larvae approximately two months of age and about 25 mm in length were observed to show signs of distressed locomotion, uncoordinated wiggling, and rigor followed by death. The deceased larvae quickly blackened as their inner organs lost structure, essentially becoming liquefied (see
Interestingly, an outbreak of similar pathology occurred in the Z. morio larvae stock of the Moscow Zoo in 2015, with PCR detection revealing a DV as the causative agent. The partial genome of this DV has been deposited to the GenBank under the name Zophobas morio densovirus (ZmDV). ZmDV was first described in Hungary in 2014, with similar symptoms. The DV sequence, revealed in both studies, disclosed 97% nucleotide sequence identity with Blattella germanica densovirus-like virus (BgDVLV), a member of the Densovirinae genus Blattambidensovirus. BgDVLV has a genome of over 5.1 kb in length (the length of the 77 yet-unsequenced genome termini are unknown), and it utilizes an ambisense gene expression strategy.
The primer pair 5′-GACAGCGGATACTATGTGTCA-3′ (SEQ ID NO: 11) and 5′-AATTTCAAGAGGAAGTCTTTG-3′ (SEQ ID NO:12) was designed to target an approximately 300 nucleotide long, highly conserved region of the NS2 gene. The primers were designed to be capable of amplifying the respective genome region of all members within the Blattambidensovirus incertum1 species, hence this PCR system could be used for diagnostic purposes, i.e., detecting the presence of ZmBWV DNA in the sample. Amplification was executed in a 25 μL final reaction volume, including 2 μL of purified DNA target, 0.5 μL of both primers in 50 pmol concentration, 0.5 μL dNTP mix with 8 pmol of each nucleotide, and 0.1 μL of DreamTaq™ DNA polymerase enzyme (Thermo Fisher™) PCR reactions were executed under a program of 5 minutes denaturation at 95° C. followed by 35 cycles of 30 seconds denaturation at 95° C., 30 seconds annealing at 50° C., and 1 minute of elongation at 72° C. The final elongation step was 5 minutes long at 72° C.
Quantitation of the viral DNA was carried out by real-time PCR amplification (qPCR), using an Applied Biosystems QuantStudio 5 instrument. This quantitation process was executed to estimate viral titers in the samples and in vaccine formulations. A 300-bp-long target sequence was amplified by the primers mentioned above. For dsDNA quantitation the SYBR™ Green PCR Master Mix (Applied Biosystems™) was used, with an amplification program of 5-minute denaturation at 95° C. followed by 45 cycles of 30 second denaturation at 95° C., 15 seconds annealing at 55° C., and 30 seconds of elongation at 72° C. Results were analyzed by the QuantStudio™ Pro software (Applied Biosystems™). This was used exclusively for diagnostic purposes. Material from PCR-positive larvae was subjected to whole-genome sequencing, either without further purification or after purification of virions by sucrose gradient centrifugation.
The vaccines according to embodiments of this invention include the following strains of ZmBWV, deemed non-virulent,
Vaccine compositions according to the invention can take several forms. According to certain embodiments, the composition is produced for administration to beetle larvae per os. In such cases, the composition comprises beetle larvae that have been infected by any of injection, spraying, dipping, or administration as a feed, and then killed. Killing preferably is accomplished by freezing, suffocation by carbon dioxide exposure, and the like, as is convenient. The killed larvae may be administered as is, without any further processing, or may be crushed, sliced, freeze-dried, air-dried, or the like for suitable packaging for sale. Vaccine compositions for oral administration are added to an enclosure containing larvae to be treated with the vaccine. The larvae eat the infected material and are thereby infected with the vaccine densovirus. See
In alternative embodiments, vaccine compositions include solutions of densovirus material in a pharmaceutically acceptable carrier such as phosphate-buffered saline. The carrier also can contain optional additives such as antibacterial agents, pH modifiers, antifungals, non-ionic surfactants, stabilizing agents, and the like. Such solutions preferably are formulated as a spray or dip solution, or alternatively for injection. See
The dose of the vaccine for an individual larva is about 107 genome copies (gc) to about 1011 gc; preferably about 108 gc to about 1010 gc; and most preferably about 109 gc for administration by injection. The dose of the vaccine for administration by feeding for an individual larva is about 1010 gc to about 1015 gc; preferably about 1011 gc to about 1013 gc; and most preferably about 1012 gc. For administration to an individual by spraying or dipping, the larvae is subjected to the virus suspension in PBS by direct application on the cuticle.
For a population of individual larvae (such as 1000 individuals) the dosage of the vaccine is about 1011 gc to about 1013 gc; preferably about 1×10012 gc to about 5×1012; and most preferably about 1×10012 gc to about 2×1012 per 10 ml of injectable suspension, dosing each larva by ˜10 μl. For spraying, about 10 mL to about 20 mL per thousand individuals of a solution of 1014 gc in buffer, e.g. phosphate buffered saline, at a concentration of about 1013 gc/ml to about 1014 gc/ml; preferable about 1013 gc/ml to about 5×1013 gc/ml; and most preferably about 1013 gc/ml is administered to the population.
This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety; nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Specimens of Z. morio were shipped directly to our laboratory from 11 farms, located in the states of Arkansas, Florida, Georgia, Maryland, Mississippi, Ohio, New Jersey, New York, and Utah, with two farms located in the state of New York. T. molitor samples were obtained from the farms in Ohio, New Jersey and one New York farm. Z. morio larvae also were purchased from two facilities, located in the states of Oregon and Pennsylvania. T. molitor samples were obtained the same way from both facilities, as well as from Louisiana. Samples of both T. molitor and Z. morio, bred in the states of Indiana and Minnesota, were purchased at local pet stores. Dubia roaches and buffalo beetles originated from one of the farms located in New York.
Z. morio larvae were housed in plastic insect breeder boxes at room temperature and were given a piece of fresh carrot every second day. Oatmeal bran was used as bedding. Experimentally inoculated larvae were housed in single use plastic dessert cups, also on oat bran bedding. Animals were euthanized prior to processing in either dry ice or by freezing at −80° C. Strain NJ2-molitor was isolated from Tenebrio molitor. It is not pathogenic in Zophobas morio.
Deceased Z. morio larvae were subjected to tissue homogenization in 1× phosphate-buffered saline (PBS), followed by three cycles of freeze-thaws. Following this, the homogenate was combined with an equal volume of 1×TN™ pH8 (50 mM Tris pH8, 100 mM NaCl, 0.2% Triton X-100, 2 mM MgCl2) and the debris were removed by centrifugation at 3700×g at 4° C. for 15 minute intervals until the supernatant was sufficiently cleared. The supernatant was mixed with 1×TNET pH8 (50 mM Tris pH8, 100 mM NaCl, 0.2% Triton X-100, 1 mM EDTA) in a 1:3 ratio and concentrated on a cushion of 20% sucrose in TNET using a type 45 Ti rotor for 3 h at 4° C. at 42,300 rpm on a Beckman Coulter S class ultracentrifuge. The pellet was resuspended in 1 mL of 1×TN™ pH8 and, following overnight incubation, purified on a 5 to 60% sucrose step gradient for 3 hours at 4° C. at 35,000 rpm, using the same instrument with a SW 41 Ti swinging bucket preparative ultracentrifuge rotor. Both visible bands were aspired by a single needle puncture and a 10-mL volume syringe. The purified fractions were dialyzed against 1×PBS in order to remove the sucrose.
Quantifoil™ R1.2/1.3 300 mesh grids were glow discharged and coated with a 2.62-nm-thick carbon film. The film was fabricated by electron-beam deposition on cleaved mica using a Leica™ EM ACE600 instrument and floated onto a surface of ultrapure water through which the discharged grids were lifted. In case of the ZmBWV NJ2-molitor strain UltrAfoil™ R1.2/1.3 300 grids were used. Samples were plunged-frozen into liquid ethane using a Vitrobot Mark IV (FEI) at 100% humidity and ambient temperature. The grids were clipped into autoloader grids and imaged using a Talos Arctica™ transmission electron microscope (TEM) (Thermo Fisher™), equipped with a Gatan K2 direct electron detector, operated in low dose mode.
D. Identification of a Parvovirus by cryoEM
Z. morio carcasses received from the Utah facility were homogenized and subjected to sucrose gradient fractionation. Two bands, at the interfaces between the 20% and 25% sucrose steps and between the 35 and 40% sucrose steps, were obtained. Both contained isometric viral particles of approximately 26 nm in diameter; the more-buoyant particles were hollow but the less-buoyant particles contained dense intracapsid material corresponding to the viral nucleic acid. This virus is referred to here as “Zophobas morio black wasting virus” (ZmBWV).
3D maps of the empty capsids and full virions at 2.7 and 2.9 Å resolution, respectively, were obtained. Both were T=1 icosahedral capsids with a single capsid protein per asymmetric unit with a jelly roll core. The Cu backbone was manually traced by building polyalanine chains of 423 and 425 residues in length per asymmetric unit, respectively. Qualitatively, we assessed that the backbone model exhibited a prototypical parvovirus fold. Quantitatively, we queried all extant protein structures from the Protein Data Bank (PDB) using DALI (Holm 2019) to detect homologs, and obtained three hits with a z-score over 20, namely: Galleria mellonella densovirus capsid (GmDV) (1DNV, z-score of 29.9), Acheta domesticus densovirus (AdDV) capsid (4MGU, z-score of 29.4) and Bombyx mori densovirus (BmDV) capsid (3POS, z-score of 23). All of these are members of the Densovirinae subfamily of the Parvoviridae. Automated, background-knowledge-agnostic tools were used to reproduce this determination. We automatically traced, and assigned sequence to, the cryoEM density using ModelAngelo (Jamali25b. DALI search of the longest detected chain revealed the same result pattern, albeit with lower z-scores: GmDV, AdDV, and BmDV followed by PVs of other subfamilies. Meanwhile, a HHblits search of the sequence profile from ModelAngelo against UniProt detected similarity to Blatella germanica densovirus (BgDV) with a p-value of 10−77. Sequencing later confirmed that these results were completely correct: ZmBWV is a densovirus whose closest known relative is BgDV. We conclude that cryo-EM can be used for sequencing-free discovery of novel viral species from clinical or environmental samples.
Selected cryoEM grids were subjected to high resolution data collection, using electron microscopy (operated at 200 kV, with a 10-s-long exposure and a total dose of 43.16 e−/A297, using a frame length 0.2 seconds). Movie frames were recorded in counting mode using the Serial EM suite (Mastronarde 2018) at a sampling of 1.038 Å/pixel (ZmBWV Utah) and 0.658 Å/pixel (ZmBWV Nj-molitor). The collected movies were aligned by the MotionCorr2 application with dose-weighting. The cisTEM software was used for single-particle image reconstruction to obtain an initial model. High resolution single particle reconstruction was carried out by Relion 4.0 (Zivanov) and CryoSparc (Punjani). Micrograph quality was assessed by CTF estimation using a box size of 512. The subset of micrographs with the best CTF fit values were included in further processing.
Particles were automatically boxed by the particle selection subroutine of CisTEM, at a threshold value of 2.0 or by the blob picking subroutine of CryoSparc live, during on-the-fly processing. Boxed particles were subjected to 2D classification, imposing icosahedral symmetry at 35 classes. Particles of classes, which failed to display a clear 2D-class average of the icosahedral particle, were eliminated from the reconstruction, resulting in the incorporation of 42,219 and 2265 particles in the full and empty capsid reconstructions, respectively. Ab initio model generation was carried out in iterations, imposing icosahedral symmetry. The obtained startup volume was subjected to automatic refinement under icosahedral constraints and underwent iterations until reaching a stabile resolution. To improve the resolution, corrections for higher-order aberrations, beam tilt, trefoil and anisotropic magnification were implemented, as well as astigmatism was fitted for each micrograph and CTF parameters were fitted per particle. In case of using the Relion suite, particles were subjected to Bayesian polishing, obtaining the training parameters based on 10 000 particles. The automatic high-resolution refinement was repeated in the presence of a mask with a soft edge. The maps were subjected to sharpening or to the post processing subroutine to obtain the final reconstructions. The final maps were achieved by sharpening at a post cutoff B-factor of 20. The resolution of each reconstructed map was calculated based on a Fourier shell correlation (FSC) of 0.143. The obtained cryoEM maps were visualized in Coot to model the backbone of one subunit. Visualization was carried out by UCSF Chimera. Data collection parameters and refinement statistics are shown in Table 1, below.
The cryoEM maps were visualized in Coot to model the backbone of one subunit. Visualization was carried out by UCSF Chimera. The density was modeled by Coot and ISOLDE (Croll) and the obtained models were refined in PHENIX.
The cryo-EM structure provided important details about the viral genome, which allowed determination of the most economical sequencing option to obtain its complete sequence. A PCR-based diagnostic tool was developed and used to investigate the extent and pathogenesis of the ZmBWV epidemic. This diagnostic pipeline, however, has no limitations to invertebrate animals only; it can be implemented in the identification and monitoring of human-infecting viral pathogens with similar efficiency.
Z. morio larvae were fixed in 4% paraformaldehyde. To preserve the healthy larvae in a straightened-out position, they were first placed to 4° C. for 15 minutes and only in this immobile state were they placed to the paraformaldehyde solution. Specimens were washed in 1×PBS three times then stained in aqueous 1% iodine and 2.5% potassium iodide to enhance internal features. Staining was carried out for 24 hours. Following staining, the sample was washed in a 0.9% sodium chloride solution. The specimens were scanned in a Skyscan 1272 instrument, at the voltage of 60 kV with a 166 μm source current. Images were collected at the pixel size of 4.5 μm, as the frame average of three per a 4° rotation step. The completed 2D scan images were reconstructed by the Skyscan Nrecon software and rendered in the Amira software (Thermo Fisher™)
ZmBWV (strain “Utah,” from the index case) was administered to healthy, 4-week-old, PCR-negative Z. morio larvae. Three forms of administration were contrasted: injection of purified virus into the fat body, dripping purified virus suspension onto larval cuticles, or (exploiting the naturally cannibalistic tendency of Z. morio) feeding blackened carcasses of infected insects (ZmBWV titer in carcasses at about 1016 gc/mL). See
Time to symptoms and death varied with route of administration and with titer. Direct injection was the most lethal; the LD50 by injection is below 105 gc but is between 109 and 1013 gc by dripping. 50% mortality was achieved by 8 days post-infection (d.p.i.) on injection of 1013 gc, 11 d.p.i. at 109 gc, and 12 d.p.i. at 105 gc. Administration by cannibalism led to a slower course of infection but full mortality was observed. No symptomatic individual recovered in any experiment. At 14 d.p.i., among larvae exposed to ZmBWV by feeding, viral load (by qPCR of NS1 gene) was 6.6×1012 gc/mL in presymptomatic larvae and higher still at 2.2×1013 gc/mL and 1.7×1013 gc/mL in symptomatic and deceased larvae, respectively. Meanwhile, dead larvae infected by injection at 1015 or 1013 gc/mL had viral loads of 7.9×1012 and 3.9×1012 gc/mL, respectively. No sequence differences were identified in virus recovered from larvae experimentally inoculated with the strain UT-morio, confirming Koch's Fourth Postulate.
To assess the time course of natural infection, we obtained Z. morio individuals in various stages of life from an affected farm in New York, viz., 8-week-old symptomatic or asymptomatic larvae, surviving pupae and beetles, as well as one-week-old larvae that were the offspring of surviving beetles. Virus yield (by NS1 qPCR) varied extensively by life stage, with titers from ˜1×109 gc/mL in newly hatched larvae to ˜2×1016 gc/mL for blackened carcasses (see
Symptomatic larvae exhibited a dark area of miscoloration along the midgut prior to gross blackening. This, and the high viral load in larvae infected by feeding, suggested that the midgut plays a crucial role in the ZmBWV infection. We fixed and iodine-stained healthy and freshly-deceased larvae for microscale X-ray computed tomography (microCT). While healthy larvae had an intact midgut wall, the midgut wall of dead larvae was extremely thin and was disrupted by frequent fenestrations. The preserved ring-shaped structures suggest that the longitudinal muscles of the outer midgut wall remained intact, while the inner layer (which is composed mainly of columnar cells) was destroyed by ZmBWV infection. Not wishing to be bound by theory, it is possible that when infected per os, the virus somehow breaches the midgut wall. Lepidopteran densoviruses, for example, use transcytosis and then disseminate through the hemocoel and devastate fat bodies. However, ZmBWV likely directly infects the midgut epithelium as do many flaviviruses and alphaviruses as well as the distantly related bidnaviruses.
Next, the difference in virulence between the T. molitor and Z. morio-derived ZmBWV strains was investigated using the same inoculation experiments, using Z. morio as the host. Along with the reference strain UT-morio, we used purified virus of the T. molitor-derived “NJ2-molitor” strain. Fat body injections by the pathogenic strain UT-morio mirrored the results of the previous inoculation experiment i.e., all larvae died 10 to 21 d.p.i., in a dosage-dependent manner. Larvae inoculated by 1013 gc of the NJ2-molitor strain displayed ˜65% mortality by 3 d.p.i., albeit ˜30% of them survived beyond 30 d.p.i., the timepoint at which the experiment was terminated. Lower inoculation titers of 109 gc and 105 gc, resulted in 70% and 90% survival 30 d.p.i., respectively, suggesting that the large scale die-off of the highest inoculation titer may be due to acute viral toxicity.
The feeding experiment was repeated by offering the healthy larvae either blackened Z. morio larvae, infected by the strain UT-morio, or blackened T. molitor larvae, exhibiting titers of the strain NJ2-molitor of about 1016 gc/mL. Although the survival curve of the strain UT-morio infected group echoed the previous results of 100% mortality to set in at 25 d.p.i., we could not observe any symptomatic larvae in the NJ2-molitor-fed group, nor could we collect any carcasses, resulting in 100% survival at 30 d.p.i. These larvae were kept alive for another four months. There was no significant difference in the virus yield of the two earlier timepoints at 2.68×109 gc/mL and 1.15×1010 gc/mL, respectively. The virus was still detectable at four months post inoculation, albeit at a low titer of 1.78×107 gc/mL.
To investigate whether nonpathogenic strains of ZmBWV can be used to confer protection against disease and death, we inoculated the healthy larvae with the NJ2-molitor strain at a dose of 109 gc by injection. The larvae were challenged at 21 d.p.i by injection with either the strain UT-morio at 107 gc or with an equivalent volume of saline. Larvae that were inoculated exclusively by the strain UT-morio, reached 100% mortality 21 d.p.i. In case of the double-inoculated larvae, a 30% survival could be observed at the termination of the experiment (32 d.p.i.). This group also showed a seven-day-long delay in the onset of the first symptoms, compared to the single, strain UT-morio-inoculated treatment group. See
Infected Z. morio larvae carcasses were subjected to sucrose cushion and sucrose step gradient purification. In the step gradient, which included fractions of 5-60% sucrose at 5% step intervals, two well-defined protein bands could be observed at the 20-25% and the 35-40% interfaces, respectively. See
Upon subjecting the purified particles to SDS-PAGE, five bands corresponding to the approximate sizes of 85 kDa, 74 kDa, 65 kDa, 50 kDa and 48 kDa, could be observed. See
By means of electron microscopy, we identified a DV of genus Blattambidensovirus in connection with an outbreak of mass-mortality in captive Z. morio larvae, designated Zophobas morio black wasting virus (ZmBWV). The ZmBWV capsid has a densovirus-like structure with a unique surface morphology. The plunge-frozen grids with the viral particles were subjected to high-throughput cryoEM data collection to determine the atomic structure of the ZmBWV capsid by single-particle reconstruction. We resolved the ZmBWV capsid structure for both the genome packaging (full), particles and for the empty, high buoyancy particles, at the nominal resolutions of 2.9 A and 3.3 A, respectively. See
Because cryoEM had revealed that ZmBWV is a parvovirus, we knew the genome must be comprised of linear ssDNA, which is refractory to ligase-based next-generation sequencing (NGS) preparation but amenable to transposase-based NGS preparation. Parvoviral genomes are short and therefore need few total reads to achieve good coverage. We obtained a complete genome of the index case of ZmBWV by transposase-based NGS. The genome is 5,452 nt long, with I-shaped inverted terminal repeats (ITRs) of 180 nt at both termini. Its coding region harbored five major open reading frames (ORFs) over the (+) and (−)-sense frames, suggesting an ambisense replication strategy. Three of these, located on the right strand, were homologues of the NS1, NS2 and NS3 proteins of DVs classified to genus Blattambidensovirus of the Densovirinae subfamily (protein sequence identity of 45-98%, according to homology searches by BLASTP).
Blattambidensoviruses, such as the type species, Blattella germanica densovirus (BgDV), express three capsid proteins encoded by two ORFs: cap1 and cap2. While cap2 gives rise to major capsid protein VP3, cap1 provides the N-terminal extensions to these in order to express minor VP2 and VP1 via alternative splicing. ZmBWV, contains homologues of cap1 and cap2 (amino acid identity of 41% and 49.5%, respectively, with their BgDV counterparts). Analyzing either empty capsids or full virions by SDS-PAGE, four protein bands were observed at sizes of 85 kDa, 74 kDa, 50 kDa and 48 kDa, with the 50 kDa fraction being the most abundant. Protein sequencing by tandem mass spectrometry (MS/MS) revealed that the three smaller-sized fractions (designated as VP4, VP3 and VP2) all were products of cap2 exclusively, while only the largest fraction encompassed the almost complete cap1. Consequently, we designated this protein VP1. See sequences above.
Viral DNA was extracted either from purified virus particles or directly from ZmBWV-infected insects. In the first case 0.25 mg of full ZmBWV particles were incubated for three hours in 1× TE puffer, pH 8.7 (10 mM tris-HCl, 1 mM EDTA) supplemented with 10 μl 10% sarcosyl and 4 μl proteinase K in 10 mg/ml concentration. The DNA was extracted by the DNeasy™ kit from Qiagen™. To isolate viral DNA directly from insects, we pooled five individuals in a 5-ml conical tube and homogenized them in 1 mL 1× TE buffer, using a handheld homogenizer with a sterile pestle. DNA was isolated from 100 μL homogenized suspension, which was digested overnight with the same components as the virions. The isolation step was carried out by the Monarch PCR & DNA Cleanup kit from New England Biolabs™, utilizing the single-stranded DNA specific protocol. The acquired DNA preps were shipped to a commercial Oxford nanodrop sequencing service, provided by Plasmidsaurus™ (Eugene, OR, USA) where they were processed as linear amplicons.
The obtained complete genomes were aligned by T-Coffee (Notredame) and converted to nexus format using Unipro Ugene. Model selection was carried out by the nucleic acid model selection subroutine of IQ-Tree (Nguyen™). Phylogenetic inference was calculated by BEAST.
The engineered vaccine strain which forms an embodiment of this invention is derived from a non-pathogenic virus derived from a related species of beetle (Tenebrio molitor) and is referred to herein as “NJ2-molitor.” differs from the natural strain in that one or more mutations are introduced that reduce the length or expression level of the NS3 protein. In one embodiment, the start codon of the NS3 gene is mutated to a codon other than the canonical ATG start codon. In another embodiment, the NS3 gene was truncated at the amino-terminus by use of a second start codon.
For direct fat body injections, a ZmBWV virus stock with a known titer, diluted to the desired concentration, was used. Approx. 10 μl of these was injected into each larvae, using a 1 ml insulin syringe with a delicate needle. The fat bodies of the first five abdominal segments were targeted by the needle. Mock-infected individuals were injected the same way with 1×PBS. To inoculate the larvae with contaminated food, we used deceased blackened individuals. In case of Z. morio larval carcasses, one carcass was provided for every 10 individuals, while one carcass was provided per every three individuals in case the deceased T. molitor larvae. In order to infect the Z. morio larvae by dripping virus suspension on their cuticules, the ZmBWV virus stocks were diluted to the desired concentration with 1×PBS and a 100 μl of these were dripped dropwise on the healthy larvae for every 10 individuals.
Frozen corpses of larvae (T. molitor) are prepared as follows: We aim to test two methods of preparation for this product. In one case, healthy two-week old Z. morio larvae is exposed to the vaccine strain by feeding. Briefly, a virus suspension in the concentration of 1013 to 1015 gc/mL of the ZmBWV vaccine strain is dripped on carrot slices, which the animals eagerly consume. The exposed larval population ts monitored on a weekly basis for viral concentration and the larvae are placed on dry ice when the viral yield reaches 1014 to 1015 gc/mL, established by using a pool of five randomly selected larvae. In another method, the same virus suspension in 1×PBS is dripped on the cuticle of the larvae, followed by the same screening and freezing procedure as detailed above. In another method, a gelling agent with excipients and flavorants, such as gelatin with glucose syrup, is mixed with heated water. The virus suspension is added before the mixture sets into a solid gel. Pieces of the gel are fed to Z. morio larval colonies.
For vaccination, this product is fed to Z. morio larval colonies or otherwise administered, for example by spraying or dipping. To obtain these suspensions, approximately 4-week old Z. morio larvae are individually injected by 107 gc of the ZmBWV vaccine strain. The larval colony is regularly subjected to virus titer quantification, to establish that the highest yield of the viral vaccine strain was achieved. When the viral yield reaches 1014 to 1015 gc/mL, the vaccine strain is purified directly from these larvae, hoping to yield a suspension of 1014 to 1015 gc/mL concentration. Approximately 50 individuals yield 2 mL suspension at the desired concentration. As this concentration is acutely toxic to utilize directly as a vaccine, the obtained suspension is diluted with 1×PBS by a 1000- to 10,000-fold.
The optimal vaccination timepoint and of the necessity of boosters can be determined by the person of skill, however vaccination can start as soon as possible as the virulent ZmBWV strains are capable of successfully infecting larvae of only a couple of days of age. Preliminary data indicates that the vaccine strain is maintained by the larvae for at least four months post inoculation, which suggests that frequent optional boosters may not be required. As for quantities, studies show that acceptable vaccine strain titers can be achieved by feeding one larva per every 30 individuals to be vaccinated at the age of two weeks. This quantity may be doubled every two weeks, e.g. for 4-week old larvae 15 individuals may be vaccinated per larva.
To produce a vaccine product in solution, the larvae can be vaccinated in batches, as they need to be devoid of substrate. The vaccine suspension is directly sprayed on their cuticle, followed by a thorough manual mixing of the batch. The vaccinated batch of larvae are left without adding fresh substrate for 24 hours, after which they are housed in their regular substrate of wheat bran or oatmeals. If administered by injection, each larva is held firmly in one hand, while injected into the abdominal fat bodies with a delicate insulin needle. One should be cautious to avoid puncturing the posterior section of the midgut. Each animal should be administered approximately 10 μL of vaccine suspension. If administered by feeding dead larvae, the vaccine strain containing frozen larvae is thawed at room temperature and warmed to at least 20° C. Following this, these larvae are mixed evenly among the individuals to be vaccinated. Feeding success may be increased by intermittent stirring of the bin, to ensure that each individual gets access to the vaccine carrying larvae. If administered by feeding a gel, the gel is placed in the bin with larvae and the bin may be intermittently stirred to ensure that each individual gets access to the gel.
Groups of larvae were reared and injected using syringes with either saline (control) or the protective vaccine virus “NJ2-molitor” (SEQ ID NO:5) at a dose of 109 genome copies (gc) into the fat body. After three weeks (21 days post-inoculation), the larvae were injected with either saline (control) or the pathogenic virus that causes Zophobas morio black wasting disease (ZmBWV strain UT-morio at 107 gc). The strain UT-morio previously was shown to be infectious by dripping/spraying, and by adding dead infected larvae to be eaten by the live larvae.
Control larvae that were inoculated exclusively by the strain UT-morio with no prior inoculation with vaccine, reached 100% mortality 21 d.p.i. The double-inoculated larvae (larvae injected with the NJ2-molitor strain prior to ZmBWV exposure), exhibited a 30% survival at the termination of the experiment (32 d.p.i.). This group also showed a seven-day-long delay in the onset of the first symptoms, compared to the single, strain UT-morio-inoculated treatment group. These data show that the vaccine is effective in reducing larval death and morbidity in captive colonies of the superworm (Zophobas morio). See
All references listed below and throughout the specification are hereby incorporated by reference in their entirety.
This application claims the benefit of U.S. provisional application Ser. No. 63/591,484, filed 19 Oct. 2023. The entire contents of the aforementioned provisional application is hereby incorporated by reference as if fully set forth herein.
| Number | Date | Country | |
|---|---|---|---|
| 63591484 | Oct 2023 | US |
