Rhabdo Virus:

 

 

Rhabdo viruses are a group of viruses that have wide range of hosts, both plant and animals.  Among them, the two are interesting; they are Lyssavirus and Vesiculo virus.  Vesiculo viral types such as Vesiculo Stomatitis Virus (VSV)) are pathogens of foot and mouth disease of deadly types.  The lyssavirus type is Rabies, which causes, if infected through dog bites, called mad dog disease.  Rabies was known since 1808, and Louis Pasteur was the first to demonstrate it and in 1880s he succeeded in isolating attenuated viruses and used it as vaccine against patients bitten by mad dogs.  Today scientists have developed vaccines for the same virus.

Rabies is a fatal Central Nervous System (CNS) disease responsible for approximately 60,000 annual deaths worldwide, making it the tenth most common lethal infectious disease. The causative agent is a neurotropic virus consisting of non-segmented, negative-stranded RNA contained within a bullet-shaped envelope. Rabies virus (RV) is 1 of 7 serotypes belonging to the genus Lyssavirus and the family Rhabdoviridae. The most common site of RV entry in humans is the skin or mucous membrane, where the virus is delivered into the muscle and subcutaneous tissue through biting, licking or scratching by an RV-infected animal. Disease can be manifested in one of two clinical forms. In the majority of rabies cases, the pathologic manifestation in the CNS is acute encephalomyelitis. This form is known as classic or encephalitic (furious) rabies and comprises 80-85% of rabies cases. It is distinguished by neurotropism, neuroinvasiveness and impaired neuronal functions. The symptoms of classic rabies include hydrophobia, pharyngeal spasms, and hyperactivity leading to paralysis, coma and death. Paralytic rabies is a less common clinical form characterized by the development of prominent and flaccid muscle weakness. Death in both clinical and paralytic rabies ultimately results from neuronal dysfunction due to the dramatically inhibited synthesis of proteins required for maintaining neuronal functions, (Dietzschold et al. 2005), (Dietzschold et al. 2005).(Plotkin 2000), Warrell et al. 2004). (Jackson 2002), (Hankins et al. 2004).

Genera                                   Properties                              Members                              

Lyssa virua

Infect vertebrtes and some insects

Rabies, Makola, Lagos bat viruses

Vesiculo virus

VSV humans and cattles

VSV, Cocal virus,Algoas virus

Ephemero virus

Infects vertebrates,

Bovine ephemeral fever

 

Group V: (-)sense RNA Viruses

Order Mononegavirales

Family

Genus

Type Species

Hosts

Rhabdoviridae

Vesiculovirus

Vesicular stomatitis Indiana virus

Vertebrates

Lyssavirus (Greek, 'frenzy')

Rabies virus

Vertebrates

Ephemerovirus

Bovine ephemeral fever virus

Vertebrates

Novirhabdovirus

Infectious haematopoetic necrosis virus

Vertebrates

Cytorhabdovirus

Lettuce necrotic yellows virus

Plants

Nucleorhabdovirus

Potato yellow dwarf virus

Plants

 

 

Rabies is an 'ancient' disease, first shown to be of infectious origin in 1808, shown to be of viral etiology by Pasteur in the 1880's (when Pasteur and Koch were developing the germ theory of disease - prior to the firm modern definition of 'viruses' by Beijerinick (1898)). Over a decade, Pasteur carried out the serial passage of Rabies virus in rabbits, and eventually succeeded in isolating an attenuated preparation which was used to treat patients bitten by mad dogs (not without some risks). at least in terms of phenotypes. All types of Rhabdo viruses show certain diagnostic symptoms.

vsv_cryoem

Vesicular Stomatitis virus; Bullet shaped morphology; http://www.mcb.uct.ac.za/ http://www.asknature.org/

 

Rabies Rhabdovirus; http://bhavanajagat.com/

Affected regions of the body; https://virusbook.pbworks.com

 

 

Classification:

 

General features:

 

 

 

 

 

Morphology:

 

 

 

 

 

http://medchrome.com/

 

Rabies virions are bullet-shaped with 10-nm spike-like glycoprotein peplomers covering the surface. The ribonucleoprotein is composed of RNA encased in nucleoprotein -(), phosphorylated or phosphoprotein -Illistration of virus, and polymerase -virus.; Another view of bullet shaped Rhabdo virus; http://www.cdc.gov/

 

 

The internal ribonucleoprotein (RNP) core of the rabies virion consists of a negative-sense genome RNA encapsidated by nucleoprotein, polymerase cofactor phosphoprotein, and the virion-associated RNA polymerase. The RNP core is covered in matrix protein and surrounded by a lipid-bilayer envelope.

 

 

 

5’I-------I-------------I--------------I---------I---------I---------I--I3’

      59                6380            1672               838          822       1333      47 =11162  ntds                            

        L                G                 M        NS          N

 

Table showing Genes and gene products and functions

      

Gene

Protein(kd)

Function

L

116-190

Replicase

G

65

TM glycoprotein

M

26

Matrix protein

NS

40/46

NS-replication

N (p)

50

RNA Replicase

 

 

 

 

         

 

The genomic (-) RNA diagram showing coding region for various genes and in VSV the intergenic spacer with 3’ ACUUUUUUU C/GU UUGUC-5’ sequence that helps in termination of that specific gene and provides the poly (A) additional sequence.

Mononegavirales: Gene order; Helical nucleoprotein 13-20nm wide. http://rhabdoviruses.wordpress.com/

 

 

 

 

Replication:

 

CYTOPLASMIC

  1. Attachement of the viral G glycoproteins to host receptors mediates Clathrin-mediated endocytosis of the virus into the host cell.
  2. Fusion of virus membrane with the vesicle membrane; ribonucleocapsid is released into the cytoplasm.
  3. Sequential transcription , viral mRNAs are capped and polyadenylated by polymerase stuttering in the cytoplasm.
  4. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-synthetized antigenomes and genomes.
  5. The ribonucleocapsid binds to the matrix protein and buds via the host ESCRT complexesoccurs at the plasma membrane, releasing new virions. http://viralzone.expasy.org/

 

 

 

 

 

 

 

Recently, by immunoaffinity column chromatography purified two RNA polymerase complexes, the transcriptase and replicase, from VSV-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1 a and heat-shock protein 60. In addition, the complex contains a sub molar amount of cellular mRNA cap guanylyltransferase.

The replicase, on the other hand, is a complex containing the viral protein, L, P, and the nucleocapsid (N), but lacking elongation factor-1 a heat shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3' end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex.

The two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA, as shown in Fig. Understanding the structure and function of the transcriptase and replicase complexes are fundamental to gain insight into the replicative pathways of VSV life cycle.

 

Mechanism:

 

·         Initially, the L/NS complex binds to 3’UTR region of the N covered genomic RNA and adds RNA nucleotides one by one in 5’>3’ direction, till it generates about 47 ntds long RNA segment.  It does not require any primer. All known RNA polymerase transcribe templates with out any primers.

 

Individual cistrons are transcribed by transcriptase enzyme complex and polyadenylated and also cap is added at 5’end; these RNAs are like eukaryotic mRNAs, but monocistronic; Negative-stranded RNA linear genome, about 11-15 kb in size. Encodes for 5 to six proteins; G-Glycoprotein, NP-Nucleoprotein, P-Phosphoprotein, L-Large protein and M Matrix protein;

Imagefrom: http://expasy.org/viralzone/all_by_species/2.html; http://viralzone.expasy.org/

 

man

 

The figure shows the composition of Transcription complex and replication complex; Mol.Biol.

 

 

 

·         Then the short segment dissociates and falls off the strand.  Whether, this leader segment synthesis is repeated or not clear, but every (+) RNA segment possesses the same 47-ntd long leader sequence with a cap at its 5’ end. 

·         It is assumed that L/NS complex, (in association with one of the host factors called H) performs not only the addition of the leader and also capping.

 

·         Once the leader sequence dissociates from the enzyme complex, the enzyme continues and transcribes first coding region. 

 

·         As the enzyme reaches the end of the coding segment, it encounters a sequence, which provides what is called poly (A) signal, in Rabies-3’ ACUUUUUUU C(N)n UUGU G/A. In VSV IGR sequence is 3’ ACUUUUUUU C/GU UUGUC-5’.

 

            Then the enzyme continues to transcribe the stretch of 7Us, when it reaches the last U, it some how slips back to the first of the seven Us with out leaving the transcript, and moves forward and adds another seven A’s. This way it can add few more seven nucleotide poly (A) blocks, then moves into the next coding segment leaving the transcript free from the enzyme. Individual transcripts are added with their 5’ UTR 47ntds long segment.

 

·         Using this unique mechanism it transcribes all segments and produces five distinct mRNAs. Each of the transcripts is added with a 47-48 ntds long leader sequence generated from 3’ transcript. The transcripts are added with a cap at 5’ end and a poly-A tail at its 3’ end, like any other eukaryotic mRNAs

 

·         The first transcript coded is longest of all the transcripts and last but one is the second longest but it is an important segment.  Each of these mRNA segments is translated to produce their respective proteins.

 

·         As more and more of N proteins are produced, they bind to the entire length of RNA. When the RNA is fully covered with the nucleo capsid protein, the L/NS complex that initiates the synthesis of leader sequences, now it doesn’t dissociate from the template and the enzyme continues to produce a full length (+) RNA.  Thus many (+) RNA are generated. 

 

·         At the same time the mRNA transcripts produced are translated and the products accumulate in the cell.  Once the protein products are produced in sufficient quantity, the same enzyme complex, perhaps with one of the host factors, may now switch to full length (+) RNA and initiate (–) RNA from its 3’ end of the (+) RNA. 

 

·         Within about 8-10 hrs of infection, thousands of RNA particles are produced and released.

 

·         The mRNA produced for Glycoprotein is translated and transferred into ER and Golgi, where the protein is properly folded and glycosylated at 3 sites containing Asn-X-Serine.  Among the three sites one is not efficiently glycosylated. The Matrix and Glyco-proteins produced are transferred to endoplasmic reticulum via signal sequence mechanism. From golgi complex proteins are transferred to the plasma membrane, It is here the viral particle gets it envelope. 

 

This figure shows systemic passage of virus and affected body parts; incubation period varies from 2weeks to 3 months.  After entering viruses spread centripetaly a a rate of 100-400mm/d via fast axonal transport to pinal chord and brain. Once virus enters bran the spread becomes Fster and the symptoms visible. http://dualibra.com/

 

 

·         As they are processed and transferred to plasma membrane via Vescicular transport mode. 

·         Full length (-) RNAs, using assembly or packaging sequences bind to plasma membrane where viral proteins are located.  It is here the viral particles organize and bud off continuously. 

 

The released viruses enter into blood stream and reach CNS, where they incubate for a long time before they cause full-fledged disease.