Structure

  • Most rhabdoviruses are bullet shaped but some rhabdoviruses that infect plants are bacilloform1
  • Enveloped1
  • Size: 100-430nm x 45-100nm (length of the virus will be determined by the size of its genome)1
  • Spikes protruding from lipid envelope are viral attachment protein G1
  • Spikes are 5-10 nm long1
  • Genome is 1 linear strand of antisense RNA1
  • RNA genome is surrounded by a helical nucleocapsid1

The nucleocapsid consists of 3 types of proteins1:

  1. Nucleoprotein (NP)
  2. Phosphoprotein (P)
  3. Large Protein (L)
  • The most abundant nucleocapsid protein is NP1.
  • The least abundant nucleoprotein is L which has polymerase activity used for gene expression and genome replication1.

 

Taxonomy

Order:            Mononegavirale2

Family:          Rhabdoviridae2

  • The rhabdoviridae family contains 6 genera with over 200 viruses identified3
  • Viruses in this family have a wide range of hosts including plants, insects, fish, and mammals3

Genera in this family include:

  • Vesiculovirus  (e.g. Vesicular Stomatic Virus)1
  • Lyssavirus  (e.g. Rabies Virus)1
  • Cytorhabdovirus  (e.g. Lettuce Necrotic Yellow Virus)1
  • Nucleuorhadovirus   (e.g. Potato Yellow Dwarf Virus)1
  • Novirhabdovirus (e.g. Infectious haematopoetic necrosis virus)3
  • Ephemerovirus  (e.g. Bovine ephemeral fever virus)3

Image from Dr. Johansens lecture slides

Baltimore Classification:

Rhabdoviruses are Class V (-) ssRNA viruses

Multiplication Cycle

Vesicular somatisis virus (VSV) has been well studied.  Most of what we know about the replication of Rhabdoviruses we know from VSV.

Attachment:

The G protein spikes on the surface of VSV attaches to the host cell11.  VSV has a broad tissue tropism and is able to infect most or possibly all human cell types12.  VSV is also able to infect other mammals and insects***.  A specific receptor that interacts with the G protein of VSV has not been identified.

Entry:

VSV enters the host cell via cell-mediated endocytosis11.

Uncoating:

The G protein causes the viral envelope to fuse with the vesicle membrane13.  The G protein undergoes a conformational change cause by the more acidic pH in the vesicle13.  This conformational change exposes a hydrophobic domain of the G-protein which inserts into the vesicle membrane and allows for membrane fusion13.  Membrane fusion causes VSV released into the cytoplasm11.  The (-) ssRNA genome remains associated with the nucleocapsid proteins to protect it from degradation in the cytoplasm1.

Biosynthesis:

Transcription of VSV occurs in the cytoplasm1..  The nucleoproteins are responsible for transcription.  The L protein is an RNA-dependent-RNA-Polymerase (RdRp) that transcribes mRNA from the (-) ssRNA genome1.  The L protein is also thought to be responsible for adding a 5’ cap to the mRNA1.  A poly-U sequence in the genome encodes for the poly-A tail1.

5 monocistronic mRNAs are produced1:

  1. Glycoprotein (G)
  2. Nucleoprotein (NP)
  3. Phosphoprotein (P)
  4. Large Protein (L)
  5. Matrix protein (M)

Transcription can only be initiated from the 3’ end of the genome1.  Once a gene is transcribed the L protein can continue on to transcribe the next protein or start at the 3’ end of the genome again1.  This phenomenon allows for a level of gene expression control in the virus1.

Genes located closer to the 3’ end of the genome are expressed more and are the proteins required in the greatest abundance1.  Once the viral nucleocapsid proteins produce mature mRNAs, they can be translated using the host cell machinary.

The M protein is believed to shut down host cell gene expression in two different ways.  First the M protein inhibits the activity of RNA Polymerases I and II preventing transcription13.  Additionally, the M protein is thought to inhibit the export of mRNAs from the nucleus preventing translation13.

Genome Replication:

Replication of the genome occurs once there are enough nucleocapsid proteins have been produced to protect the newly synthesized genome from degradation in the cytoplasm.  The L protein synthesizes a (+) ssRNA anti-genome from which many copies of the (-) ssRNA genome are produced.  The P protein has been shown to be essential for DNA replication13.  The P protein may serve to bind and displace the N protein from the viral RNA so that it can be replicated13.  The N protein is important for genome replication because it helps the L protein to copy the entire (-) RNA strand to produce the (+) anti-genome template 13.  The N protein is thought to bind to the leader sequence at the 3’ end of the genome and allow the L protein to ignore the transcription stop sequences in the genome13.

Assembly

The matrix protein (M) associates with the plasma membrane2.  The N protein promotes the encapsidation of the genome13.   Then the nucleocapsid containing  the (-) ssRNA genome interacts with M at the plama membrane2.

Release

Budding releases virions from the host cell2.

VSV particles (purple) can be seen budding from the host cell. Image from: http://www.sciencephoto.com/images/download_lo_res.html?id=770500995

Rhabdoviruses of Plants and Animals

Rhabdoviruses are widespread in the environment. Different genera of rhabdoviruses have the ability to infect plants or animals. Diseases caused by these viral infections result in both devastating medical conditions in humans and huge financial losses in agricultural crops. Only a few genera will be discussed here. While any given genera may have multiple species and diseases associated with it, only one disease will be described for the genera mentioned below.

  • Cytorhabdovirus:
    • One disease caused by a cytorhabdovirus species is Lettuce Necrotic Yellow Virus, which infects select members of the families Chenopodiacea, Compositae, Leguminoase, Liliaceae, and Solanaceae8.
    • This virus is predominately found in Australia and has a temperature-dependent latency period within the vector8.
    • Infected plants show chlorosis (lack of chlorophyll), necrosis, stunted growth, and curling of the leaves. Some plants have a mosaic appearance on the leaves similar to the tomato mosaic virus8.
    • There are also at least four other rhabdoviruses known to infect lettuce. It is difficult to determine which species is infecting the plant based on symptoms alone. Often times, electron microscope must be used to distinguish between the different types of infections8.
    • The image below shows how the disease is spreading from one lettuce plant (top left corner) to another (top middle).

  • Nucleorhabdovirus:
    • One disease caused by Nucleorhabdovirus is Potato Yellow Dwarf Virus (PYDV), which infects plants in the Solanaceae family such as potatoes, tobacco, and chili peppers9.
    • PYDV has been detected primarily in Canada and the United States.
    •  The virus has two serotypes, each being spread by specific species of leaf hoppers. The first serotype, constricta yellow dwarf virus, is transmitted by Agallia constricta. The other serotype, sanguinolenta yellow dwarf virus, is transmitted by Aceratagallia sanguinolenta. PYDV can also be mechanically transmitted, as observed in tobacco plants9.
    • Infected plants will have stunted growth, yellow leaves and tubers, and necrosis. The tubers themselves can transmit the disease once they are infected. There is typically a long incubation period (1 week) within the vector prior to transmission to crops. PYDV  has been shown to remain in the vector over the winter9.
    • The image below depicts the yellowing of the leaves in the potato plants. Outbreaks of this disease are costly to farmers.

  • Vesiculovirus
    • One disease caused by Vesiculovirus is Vesicular Somatisis; a disease that is widespread in the western hemisphere. There are many hosts for this virus including cows, horses, pigs, and humans10.
    • The disease is an “arbovirus” meaning it’s transmitted through arthropod bites. In the case of vesiculovirus, the vector is typically a fly10.
    • The symptoms in livestock consist of malaise, swollen glands, and open sores in the mouth or on the feet. Livestock often recover in 2 weeks but have 90% morbidity. This often results in financial losses from morbidity of production livestock4.
    • Humans do not typically exhibit the lesions observed in other animals with this virus. Other symptoms include headache, fever, and severe aches which can be limb specific. Humans typically recover from the disease in approximately 1 week10.
    • It was estimated that in Central America, approximately 40-100% of the population has been exposed to this virus10.

  • Lyssavirus:
    • One of the most well known diseases in Rhabdoviridae is Rabies, which is in the Lyssavirus family. This disease is found all over the world and infects many mammal species5.
    • There is a long incubation period for the virus within the host, sometimes taking several months to develop symptoms. Once symptoms develop, the disease is untreatable and death typically occurs within a few days5.
    •  Earlier symptoms consist of headache, fever, and severe aches. As the virus enters the nervous system, the symptoms are characterized by encephalitis (inflammation of the brain), changing behavior (typically aggressive or excited), and uncontrolled movements. The virus is named from one of the later symptoms, mania, after the greek goddess Lyssa. Mania is shortly followed by a coma5.
    • Rabies is a growing problem in many suburban communities that share the landscape with wild animals such as skunks, bats, raccoons, and foxes. A recent news segment on Colorado Public Radio discussed the growing concern about rabies on April 11, 2011: http://www.cpr.org/#load_article|Rabies_On_The_Rise_In_Colorado

Availability of Antivirals or Vaccines

Viruses within the family Rhabdoviridae induce diseases in humans, fish, animals, and plants. Outbreaks of these diseases are detrimental to the food supplies and health of individuals within communities. The financial burden of lost crops and livestock to certain species of rhabdovirus is significant. Therefore, many efforts have been focused on developing vaccines to ward off these diseases. Many crops are destroyed by several species of Rhabdoviridae; however, insecticides are used to treat these outbreaks more frequently than vaccines. This is because plants are quite difficult to vaccinate. Most vaccines are administered to humans, their livestock, and their pets.

  • Cytorhabdovirus: No information.
  • Nucleorhabdovirus: No information.
  • Vesiculovirus:  Commercial vaccines are available in Central America. These are killed vaccines however attenuated vaccines have been tested in the US and Colombia with unknown efficacy6.
  • Lyssavirus:
    • The initial vaccine made by Louis Pasteur consisted of the virus harvested from infected rabbits, which was then weakened by allowing it to dry for 5 to 10 days. Vaccines made from this serotype are widely used and protect against the genotype I strains of lyssavirus; however, it does not offer protection against other Lyssavirus strains7.
    • In 1967, the human diploid cell rabies vaccine (HDCV) was made using the attenuated Pitman-Moore L503 strain of the virus. As of 2006, his vaccine has been administered to over 1.5 million humans7.
    • Another vaccine was developed in 1984 in which the glycoprotein gene from rabies was inserted into a vaccinia virus. This V-RG vaccine is trademarked by Merial and has been used to prevent outbreaks in the wild in Belgium, France, Germany and the US7. This vaccine is typically delivered on food bait dropped in the wild for animals to consume.

Current Rhabdovirus Research

Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitos in Cote d’lvoire.

Quan, Phenix-Lan, Junglen, Sandra, Tashmukhamedova, Alla, and Conlan, Sean. January 2010. Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitos in Cote d’lvoire. Virus Res. 147(1).

http://cii.columbia.edu/pub/documents/MoussaVirus.pdf

This paper describes a new species of Rhabdovirus discovered in the African rainforests. The discovery of the virus was prompted by a surveillance program of viruses in mosquitoes in West Africa. RNA isolated from the virus was subjected to high-throughput pyrosequencing and blasted against other members of Rhabdoviridae. This new species, called Moussa Virus (MOUV), has sequence homology to other Rhabdoviruses between the genes encoding the Large protein (L), Glycoprotein (G), and Nucleocapsid protein (N). The ORFs of the Matrix protein (M) and Phoshoprotein (P) genes did not show sequence or amino acid homology to other rhabdoviruses. However, the tertiary structure of the proteins including phosphorylation and glyosylation sites appear to be similar.

The rhabdovirus with the M protein most similar to MOUV is the Wongable virus (WONV), which has 20.7% homologous identity to the new viral M protein. The rhabdovirus with the most similar G protein to MOUV is the infectious hematopoietic necrosis virus (IHNV), which has 23.2% homologous identity to the MOUV glycoprotein. In addition to the 5 typical proteins observed in Rhabdoviridae, MOUV contains additional genes, including one denoted as the “C-protein”. This new virus also has the common 3’ leader sequence which is conserved in Rhabdoviruses that infect mammals . There were also different strains of this virus identified which can be subdivided into forest strains and plantation strains by ANOVA. This new viral classification could have implications on epidemiology and is now incorporated into the disease monitoring program in West Africa.

Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways.

Larrous, Florence,  Gholami, Alireza, Mouhamad, Shahul, Estaquier, Jerome, and Bourhy, Herve. lOctober 2010. Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways.Journal of Virology. 84(19).

http://jvi.asm.org/cgi/content/short/84/19/9897

This paper evaluated the genetic sequence and amino acid sequence of the Rhabdovirus Matrix protein to determine pathways by which this virus causes apoptosis in host cells. This research is important in understanding the virulence of different species of Rhabdoviridae because apoptosis is inversely correlated to pathogenicity in lyssaviruses. Different lyssavirus proteins have been implicated in inducing cell death. While glycoprotein, phosphoprotein, and matrix protein have all be shown to cause cell death, the matrix protein was evaluated closely in this study because this is the protein used by other families in Rhabdoviridae to cause apoptosis.

Matrix protein was obtained from two viruses: Mokola virus (MOK) and rabies from a dog in Thailand (THA). The M proteins were truncated into mutants spanning various regions of the amino acid chain. A span of 20 amino acids between residues 67 and 86 was identified to induce apoptosis through two different pathways. One pathway utilizes the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). This occurs when the M-protein activates capsase 8. The other pathway interferes with the respiratory chain when the matrix protein inhibits cytochrome c (cyt-c) oxidase (CcO).

The mutation analysis revealed that the K amino acid at position 77 is required for M-MOK to inhibit CcO. For CcO to be inhibited by M-THA, position 77 needs to be a K or a R residue. While M-MOK induces apoptosis via the TRAIL pathway when position 81 is N, M-THA is a bit more complicated. Some THA-infected cells have a low sensitivity to the influence of TRAIL due to the expression of the p53 tumor suppressor protein. Understanding how different species and strains of Rhabdoviruses induce apoptosis will allow researchers to produce more effective vaccines or treatments for diseases caused by such viruses.

Systemic Vesicular Stomatitis Virus Selectively Destroys Multifocal Glioma and Metastatic Carcinoma in Brain14

Koray Özduman, Guido Wollmann, Joseph M. Piepmeier, and Anthony N. van den Pol. 2008.  Systemic Vesicular Stomatitis Virus Selectively Destroys Multifocal Glioma and Metastatic Carcinoma in Brain.  The Journal of Neuroscience.  28(8):1882–1893

http://www.jneurosci.org/content/28/8/1882.full.pdf+html

In this study, vesicular stomatisis virus is shown to effectively target brain cancer as well as peripheral tumors in vivo.  Vesicular stomatisis virus is naturally oncolytic, preferentially targeting cancer cells over healthy host cells.  Vesicular stomatisis virus is highly sensitive to interferon antiviral defense.  However, most cancer cells do not have a functional interferon pathway.  This causes cancer cells to be much more susceptible to infection by vesicular stomatisis virus than other cells.  The natural oncolytic capacity of vesicular stomatisis virus makes it ideal for cancer therapy.

In this study, vesicular stomatisis virus was engineered to express green fluorescent protein (GFP).  Several human cancer cell lines were engineered to express red fluorescent protein (RFP).  Brain tumors were induced in mice using the human cancer cell lines expressing RFP.  The florescent markers allowed for laser imaging of the brains in the live mice to analyze vesicular stomatisis virus’ oncolytic capacity for brain tumors.   The laser imaging showed that the vesicular stomatisis virus effectively and selectively targeted the brain tumors.

Brain cancer currently has no effective treatments.  There are several characteristics of brain cancer that makes it an especially difficult cancer to treat.  Brain tumors are difficult to remove surgically as they are commonly multifocal.  Tumor causing cells can also easily be left behind after surgery.  Additionally, the brain is impenetrable to many therapeutic agents that are effective for the treatment of other types of cancers.  This study shows that vesicular stomatisis virus has the potential to be an effective treatment for brain cancer.

Dual DNA vaccination of rainbow trout (Oncorhynchus mykiss) against two different rhabdoviruses, VHSV and IHNV, induces specific divalent protection

Katja Einer-Jensen K, DelgadoL, LorenzenE, BovoG, EvensenO, LaPatra  S and N Lorenzen. 2009.  Dual DNA vaccination of rainbow trout (Oncorhynchus mykiss) against two different rhabdoviruses, VHSV and IHNV, induces specific divalent protection.  Vaccine.  27(8): 1248-1253.

Rainbow trout farmed in the United States and Europe are susceptible to infection with the rhabdoviruses viral haemorrhagic septicaemia virus (VHSV) and infectious haemotopoietic necrosis virus (IHNV).  These infections cause significant losses in the fish populations and therefore economic losses for the fish farming industry.  Novel vaccines for both VHSV and IHNV have been developed.  These vaccines consist of DNA encoding for the G protein of the rhabdoviruses.  As the farmed fish are usually exposed to both VHSV and IHNV, a dual vaccine would be ideal.  A previous study tested the ability of a dual vaccine to elicit an immune response in fish.  They were able to show that G protein specific antibodies were produced for each of the rhabdoviruses.  However, the dual vaccine was not tested for efficacy in preventing infection and mortality.  In this study, the ability one injection of the dual vaccine to prevent mortality caused by subsequent infection with both VHSV and IHNV was analyzed.  When unvaccinated fish were infected with both VHSV and IHNV the mortality rate was 88%.  In fish treated with the dual-vaccine prior to infection mortality was decreased to 13%.  This study demonstrates that the dual vaccine effectively reduces mortality due to VHSV and IHNV in fish.  This dual-vaccine could save the rainbow trout farming industry from significant economic losses in the future.

REFERENCES

  1. Dimmock NJ, Easton AJ, Leppard KN. 2007.Introduction to Modern Virology. 6th edition. Malden: Blackwell Publishing.  516 p.
  2. Swiss Institute of Bioinformatics.  “Rhabdoviridae”.  http://expasy.org/viralzone/all_by_species/2.html (accessed: April 12, 2011)
  3. Microbiology Bytes.  “Rhabdoviruses”.  http://www.microbiologybytes.com/virology/Rhabdoviruses.html (accessed April 14, 2011)
  4. Center for Food Security and Public Health. 2007. “Vesicular Somatisis”. www.cfsph.iastate.edu/DiseaseInfo/ppt/VesicularStomatitis.ppt  (Accessed April 12, 2011).
  5. Wikipedia. “Rabies”. http://en.wikipedia.org/wiki/Rabies  (Accessed April 13, 2011)
  6. World Health Organization (OIE). May 2010. “Vesicular Somatisis ”. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.19_VESICULAR_STOMITIS.pdf  (Accessed April 1, 2011).
  7. Wikipedia. “Rabies Vaccine.”  http://en.wikipedia.org/wiki/Rabies_vaccine (Accessed March 28, 2011)
  8.  Descriptions of Plant Viruses Web. “Lettuce Yellow Necrotic Virus”  http://www.dpvweb.net/dpv/showdpv.php?dpvno=343 (Accessed March 29, 2011).
  9. CABI and EPPO. “Quarantine Pest Potato Yellow Dwarf Virus”.  http://www.eppo.org/QUARANTINE/virus/Potato_yellow_dwarf_virus/PYDV00_ds.pdf
  10. World Health Organization (OIE).  January 2010. “Vesicular Somatisis”.   http://www.cfsph.iastate.edu/Factsheets/pdfs/vesicular_stomatitis.pdf.   (Accessed April 12, 2011)
  11. Wikipedia. “Vesicular stomatic virus”.  http://en.wikipedia.org/wiki/Vesicular_stomatitis_virus (accessed April 14, 2011)
  12. Bloora S, Maelfaitb J, Krumbacha R, Beyaertb R, and F Randowa. 2010. Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus. PNAS.
  13.  Oakland University. “Negative Strand RNA Virus Lecture I (VSV, Ebola)” http://expasy.org/viralzone/all_by_species/2.html (accessed April 14, 2011)
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