Literature Reports of VIRAL TYPE DIVERSITY for Rabies virus (with special reference to raccoon rabies variant):

Viral Type Diversity (Subtype / Subspecies)

• Rabies strains are highly related to one another (90 to 98% amino acid identity between strains). There are a variety of recognised wild strains and also "fixed" laboratory strains. The most variable protein is glycoprotein G, the major viral antigen. Variations develop because the viral polymerase lacks a proof-reading mechanism and fails to produce exact copies of the virus's genetic material.

In vitro differences

Strains of rabies virus may be distinguished by indirect fluorescent antibody testing using a panel of monoclonal antibodies (antigenic typing), or by nucleotide sequence analysis following extraction of RNA and amplification by RT-PCR. In antigenic typing, a panel of monoclonal antibodies against the nucleocapsid (N) protein has been used most commonly, although antibodies against the phosphoprotein or glycoprotein antigens have been used by some researchers.  

Raccoon rabies variant

• Using a panel of monoclonal antibodies to nucleocapsid (N) proteins, raccoon rabies virus variant can be distinguished from other terrestrial variants (and bat rabies variants) in the USA. It has been shown that raccoon rabies virus variant from the mid-Atlantic raccoon rabies outbreak is the same as that of the southeastern raccoon rabies endemic area, and that rabies isolates from other terrestrial hosts within the raccoon rabies area are raccoon rabies virus variant. Differentiation between raccoon rabies virus variant and other rabies virus variants has also been demonstrated using a multiplex PCR protocol. Phylogenetic analysis has shown that raccoon rabies virus variant is well separated from other rabies virus variants. 

In vivo differences

Different strains of rabies virus are characteristic of different host species, vary in their infectivity and pathogenicity in a given host species, and may result in different clinical features in a given host species. A virus strain may be maintained within a host species due to adaptation of that strain to the particular host, as well as because contact between hosts occurs more commonly within the host species than between species. "Spillover" cases of rabies from the reservoir host into other species do not generally result in sustained transmission in the other species. The genetic changes in the rabies virus associated with adaptation to different hosts are not known. 

Raccoon rabies variant

• The raccoon rabies variant is adapted to raccoons (Procyon lotor - Common Raccoon), but spillover occurs into a wide variety of other species. In vivo differences have been documented for this rabies strain compared to other rabies strains in various species including raccoons (higher susceptibility of raccoons to raccoon rabies than to skunk rabies), skunks (shorter duration of clinical signs for skunks with raccoon rabies than with an Ontario skunk rabies strain) and squirrels (lower infective dose with the raccoon rabies strain than with one bat rabies strain).
  • For further information on the species in which raccoon rabies has been reported see: Rabies virus - Definitive Mammal Host Species (Viral Reports)

• There are a variety of recognised wild strains and also "fixed" laboratory strains produced by passage in either avian embryos or cell culture to produce consistent infections or for attenuated strains for production of vaccines. (B336.76.w76)
• Rabies strains are highly related to one another with 90-98% amino acid identity between strains. (B352.2.w2)
• The most variable protein is glycoprotein G, the major viral antigen. (B352.2.w2)
• Because the viral polymerase of RNA viruses such as lyssaviruses lacks a proof-reading mechanism, a diverse viral population can be produced, with the ability to escape host defence mechanisms and to inhabit new conditions. (B414.3.w3, J64.23.w1)
  • Normally, "mutations are distributed about a relatively constant consensus sequence." However, when transmission is constrained, only a subset of the virus population (a founder lineage) is transmitted. If this founder lineage has beneficial (to the virus) mutations, it could quickly spread through the susceptible host population. (B414.3.w3)
  • Molecular typing methods can be used to distinguish a founder lineage from other, pre-existing, rabies virus lineages. (B414.3.w3)

In vitro differences

Virus strain types may be distinguished by indirect fluorescent antibody testing using a panel of monoclonal antibodies (antigenic typing), or by extraction of viral RNA, use of RT-PCR for amplification, then nucleotide sequence analysis for a 205-base-pair portion of the nucleoprotein gene (equivalent to nucleotides 1,112 - 1,316 of the laboratory standard CVS rabies virus). (B414.3.w3, J4.218.w4)

• Monoclonal antibodies with specific reactivity have been used to characterise and classify rabies virus strains and rabies-related viruses. (J64.23.w1)
• Monoclonal antibodies against the N (nucleocapsid) protein have been used most widely for distinguishing rabies virus strains; antibodies against P (phosphoprotein) or G (glycoprotein) have also been used by some researchers. (B414.3.w3, J100.166.w1)
• Strain typing, to distinguish between different isolates, may be performed using characteristic reaction patterns to a panel of MAbs, usually directed against the N gene (nucleocapsid proteins), used with immunofluorescent staining. (J15.23.w4, J93.24.w1, J101.126.w2)
• A panel of anti-glycoprotein monoclonal antibodies has been developed which can distinguish between different rabies virus strains in formalin-fixed tissues. (J217.77.w1)
• A panel of monoclonal antibodies against the relatively nonconserved rabies virus phosphoprotein was shown to distinguish some different rabies strains, for example distinguishing between arctic fox rabies strain and other strains co-circulating in the same geographical area of North America. (J93.38.w8)
• Nucleotide sequence analysis of a 200 base pair region of the N gene has been shown to distinguish between groups of samples of rabies virus from different geographical areas. This method was able to distinguish between rabies virus strains from different areas even though they were antigenically similar to one another. (J100.166.w1)
• An in situ hybridisation method using discriminatory probes has been developed for strain typing of rabies virus in formalin-fixed tissues. (J93.41.w6)

Raccoon rabies variant

Antigenic typing

• Different strains of rabies virus can be distinguished from one another using a panel of monoclonal antibodies (mAbs), with different strains reacting with some mAbs but not others. Raccoon rabies variant is distinguishable in this manner from other variants of rabies virus found in North America. The mAb reaction patterns of rabies virus isolates from raccoons in the south eastern and in the mid-Atlantic raccoon rabies areas were the same as one another, while isolates from raccoons in other areas matched the pattern on the local rabies virus isolate. (J100.149.w1)
• Antigenic typing showed that the rabies involved in the Mid-Atlantic raccoon rabies outbreak and that in rabid raccoons in the south-eastern USA were identical. (D239)
• Using monoclonal antibodies to nucleocapsid proteins, rabies virus isolates from raccoons and other mammals collected in the southeastern USA and in the mid-Atlantic areas, in raccoon rabies areas, over a period of eight years, showed a distinctive, and shared reaction pattern, which was not obtained with isolates from any other geographical areas. Isolates from rabid raccoons from other geographical areas gave reaction patterns identical to those seen for the dominant vertebrate host for the same area, except that in Texas, a cluster of cases including one case in a raccoon (as well as cases in gray foxes, dogs and a cow) were found to be identical to dog rabies isolates from Mexico, rather than the prevailing variant from striped skunks in the region. (J93.24.w1)
• Characterisation of North American rabies isolates using a panel of monoclonal antibodies showed that raccoons in the southeastern and mid-Atlantic states were infected with a single virus subtype. (B360.3.w3)
• Using a panel of antinucleocapsid monoclonal antibodies, identical results were obtained for rabies virus isolates from raccoons in Florida in 1984, from raccoons and skunks in raccoon epizootic areas of Pennsylvania collected 1982 - 1985, and from four Marmota monax - Woodchuck, three Vulpes vulpes - Red fox, one Odocoileus virginianus - White-tailed deer, six Bos taurus - Domestic cattle, two equines, two Felis catus - Domestic cat, one Canis familiaris - Domestic dog and one domestic Oryctolagus cuniculus - European rabbit from Pennsylvania. (B360.3.w3)
• Rabies virus isolates from the southeastern USA raccoon rabies area (100 samples) and from the mid-Atlantic raccoon rabies area (78 samples) have been shown to give a distinctive pattern with a panel of monoclonal antibodies to different epitopes of the rabies N protein; patterns from isolates from the two areas are identical. (J237.36.w1)  
• Using monoclonal antibodies to nucleocapsid proteins, 84 rabies virus isolates from raccoons and other terrestrial mammals collected in Florida during 1988 were tested and 83 of these were identified as being the raccoon rabies variant (the exception reacted with all monoclonal antibodies in the panel, a pattern typical of laboratory or vaccine strains and it was not possible to rule out laboratory contamination as the cause of this). A variety of different reaction patterns were obtained from various bats. (J1.26.w6)
• Strain typing using monoclonal antibodies to epitopes on the rabies virus N protein has shown that rabies virus isolates from other species within the raccoon rabies areas are identical with raccoon rabies variant. (J93.24.w1, J100.149.w1, J237.36.w1)
• A multiplex PCR protocol has been developed, using strain-specific primers targeting the N (nucleoprotein) gene, to distinguish the raccoon rabies variant from other rabies strains (arctic fox strains and bat strains) present in Ontario, Canada. (J217.57.w1)
• A panel of anti-glycoprotein monoclonal antibodies has been developed which distinguished between different rabies virus strains, including raccoon rabies strain, in formalin-fixed tissues. (J217.77.w1)
• Both restriction fragment length polymorphism of the products of PCR, and strain-specific RT-PCR, may be used to distinguish raccoon rabies virus strain from other virus strains (such as arctic fox rabies virus strain and bat rabies strains). (J217.75.w1)
• N.B. Monoclonal antibodies recognise epitopes created by protein folding, and may identify on or two amino acids critical for the correct conformational folding of the protein, allowing antibody recognition, but do not identify the complete amino acid sequence. Occasionally an isolate will not respond as expected to a given monoclonal antibody, due to a single amino acid change. For raccoon rabies, examples of this include two Virginia isolates and one Alabama isolate, each of which failed to react to MAb CR54, due to a change from lysine at position 112. The unusual isolates from the two areas were not closely related to one another but rather the Virginia isolates were closely related to other raccoon rabies isolates from Virginia. Another antigenic variant was detected in raccoons in Florida, relating to leucine at position 36. (B414.3.w3)

Genetic typing

• The raccoon rabies variant in the USA has been shown by phylogenetic analysis to be well separated from other rabies variants. Isolates from rabid raccoons north and south along the east coast of the USA from Florida to Maine share about 99% sequence homology. The next most closely related variant is that found in skunks in the south central US states. (J308.6.w1)
• "The genetic stability of the raccoon rabies variant in the context of 15% to 20% divergence from the rabies variants maintained in other reservoir species, implies long-standing, isolated and intraspecific transmission within the raccoon population. (J308.5.w1) 
• Tests run on more than 100 raccoon rabies virus isolates, from all along eastern North America (Florida, USA to New Brunswick, Canada), using a 516 base segment from the non-coding region between the G and L genes, amplified by a hemi-nested RT-PCR and analysed phylogenetically using the neighbour-joining method, showed three lineages. The first, apparently ancestral linage, was found in Florida, Alabama and Georgia, the second was found only in Florida and was represented by only a few isolates. The third was more broadly distributed, through the remainder of the raccoon rabies range, with an origin traced back to Virginia, and appeared to be evolving independently from the ancestral viral clade. The study also found that, for the Canada isolates, while isolates from Ontario were closely related to New York isolates, there was a separate cluster formed by isolates from New Brunswick, consistent with the New Brunswick isolates originating from a different source to the Ontario isolates. (P102.16.w1)

In vivo differences

• Different strains of rabies are characteristic of different host species. (J15.23.w3)
• Different strains of rabies virus vary in their infectivity. (J15.23.w3)
• A given host may show different susceptibilities towards different rabies virus strains. (J1.8.w2)
• Different strains of rabies vary in their pathogenicity in a given host species and may result in different clinical features (e.g. average morbidity periods) in that host. (J214.187.w1)
• A given virus isolate may show different pathogenicity in different host species. (J237.36.w1)
• Different rabies strains may result in, for a given host species, differences in susceptibility, incubation period, excretion of virus in saliva, duration of clinical signs and frequency with which aggressive behaviour occurs. (J270.10S4.w3)
• The genetic changes in the rabies virus associated with adaptation to different hosts are not known; changes may involve just one or two amino acid changes in the genome. (B414.3.w3)
  • A single amino acid change, at position 333 of the G (glycoprotein) distinguishes pathogenic from nonpathogenic ERA and CVS laboratory rabies strains. (B414.3.w3)
• It is probable that a given rabies variant is maintained within a given host in a particular area due to virus adaptation, and also probably because contact leading to virus transmission occurs more common within than between species. (B414.3.w3)
• "Spillover" cases of rabies from the reservoir host into other species do not generally result in sustained transmission of that variant in the other species; if such sustained transmission did occur, with sustained independent transmission in the new host species, a new virus variant should emerge in that species. (B414.3.w3)
• Four different rodent species inoculated with a fox rabies virus showed different susceptibility as indicated by the LD₅₀ of the virus, and also some differences in incubation period. (J1.8.w2)
• Raccoons (Procyon lotor - Common Raccoon) vary in their susceptibility to different rabies virus strains: 
  • Raccoons (Procyon lotor - Common Raccoon) survived inoculation with up to 10^4.8 MICLD₅₀ of a skunk rabies virus, not developing disease during a 92-day period following inoculation into the masseter muscle, nor excreting virus in saliva, but succumbed to a canine rabies strain (New York City/Georgia) given at 10^3.2 MICLD₅₀ (a dose of 10^1.8 MICLD₅₀ had been determined to be lethal to raccoons). (J1.28.w8)
• Different species show different susceptibilities to the same virus strain:
  • Two raccoons (Procyon lotor - Common Raccoon) survived at least 273 days after inoculation (intramuscular, into the masseter muscle) with 10^5.9 MICLD₅₀ of a skunk rabies virus (isolated from a naturally infected Mephitis mephitis - Striped skunk in Iowa), and did not excrete rabies virus in their saliva (tested 25 to 273 days post inoculation) while five Mephitis mephitis - Striped skunk inoculated with 10^0.7 or 10^2.1 MICLD₅₀ of the same isolate died within 35 days of inoculation. (J1.29.w10)

Raccoon rabies variant

• The raccoon rabies variant is adapted to raccoons. The origin of this variant is not known. The genetic stability of the raccoon rabies variant, and its divergence from variants in other reservoir species, suggests long-standing isolated transmission of this variant in raccoons. It is therefore probable that enzootic raccoon rabies existed in the Florida raccoons long before it was first reported but that the disease was only noticed in the 1950s with changes in human demographics leading to greater contact between humans and raccoons. (J308.5.w1)
• Spillover of raccoon rabies occurs into other species, including a wide variety of domestic and wild species. (J1.22.w6, J1.33.w11)
  • For further information on the species in which raccoon rabies has been reported see: Rabies virus - Definitive Mammal Host Species (Viral Reports)
• Reports of rabies in skunks in raccoon rabies areas have increased greatly, but there is no evidence as yet that the virus is establishing an independent maintenance cycle in skunks. (J84.9.w24)
• Experimentally, preliminary studies indicated a shorter duration of clinical signs for skunks inoculated with raccoon rabies strain than for skunks inoculated with an Ontario skunk variant. (J270.10S4.w3)
• Experimentally, it has been shown that raccoon variant rabies can be transmitted to Sciurus carolinensis - Eastern grey squirrel and that rabid squirrels had rabies virus in their salivary glands. The lethal dose for raccoon rabies virus in the squirrels was much lower than the lethal dose of a bat rabies virus (from a naturally infected Florida yellow bat Dasypterus floridarus), and squirrels developing rabies were more likely to have rabies virus detectable in the salivary glands if infected with the raccoon rabies strain than if infected with the bat rabies strain. (J1.8.w2)