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NEW RABIES VIRUS IN BATS IN SOUTH-WEST SIBERIA. Rassadkin Y., Axionov V., Ternovoi V., Tomilenko A., Scharf B., Shestopalov A. OBJECTIVES. Rhabdovirus Reminder Details about Expeditions and Results. Rabies Virus. Family – Rhabdoviridae Order – Mononegavirales Genus – Lyssavirus
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NEW RABIES VIRUS IN BATS IN SOUTH-WEST SIBERIA Rassadkin Y., Axionov V., Ternovoi V., Tomilenko A., Scharf B., Shestopalov A.
OBJECTIVES Rhabdovirus Reminder Details about Expeditions and Results
Rabies Virus • Family – Rhabdoviridae • Order – Mononegavirales • Genus – Lyssavirus Viruses of the family Rhabdoviridae known to infect humans are classified into 2 genera: 1. Vesiculovirus 2. Rabies Rhaboviruses that cause rabies and some diseases of fish appear to have life cycles confined to vertebrates.
All other rhabdoviruses are thought to be transmitted by arthropods which may be the original hosts from which all rhabdoviruses evolved (Shope & Tesh) Genome Strand ______________________________ N - Nucleocapsid (single stranded RNA) L & P – Minor polymerase proteins G – integrated glycosylated protein M – Peripheral Matrix Protein
Only 1 Serogroup has been established within the Lyssavirus genus. Placement within the genus is determined by serological cross-reaction of viral antigens (e.g. Complement fixation, immunoflourescence test, particularly antigenic sites on the N protein. Placement of a virus as rabies or rabies – related is determined by anti-genic sites on the glycoprotein (as recognized in virus neutralization tests) along with phylogenetic analysis of limited N gene sequences.
Rabies Serogroup Rabies (RAB) genotype I Duvenage (DUV) A IV Bats V European bat type I (EB – 1) VI European bat type II (EB - 2) Kotonkan (KOT) A – midges & mosquitos Lagos Bat (LB) A II – midges & mosquitos Rodents – Mokola (MOK) A III – midges & mosquitos Obodhiang (OBD) A – midges & mosquitos Rochambeau (RBU) VII Australian bat Lyssavirusa New strain of Duvenage virus, two red fox variants, Irkut virus, WCBV (Western Caucaus Bat Virus), Khujand, and Aravan viruses arguably have brought the total genotype to 13. All rabies virus isolates from hematophagous, insectivorous and frugivorous bats from the Americas belong to the rabies virus species.
The two Lyssavirus species from European insectivorous bats (EBL type I & II) are classified as rabies - related viruses because of the antigenic differences from rabies virus. Although rabies-related viruses are pathogenic for mammals (including humans) and infection with these viruses can result in a rabies like encephalitis the importance of these agents as disease entities is still a matter of controversy.
Rabies virus G protein is the only antigen capable of inducing and reacting with virus neutralizing antibodies. G Protein has been cloned and sequenced. All sequences contain a single open – Reading frame encoding a 524 amino acid protein. There is approximately a 90% homology at the amino acid level of G proteins of rabies virus strains sequenced so far. Mokola virus is 2 a.a. shorter than rabies virus G protein. Only 54.3% homology w/rabies.
Nucleoprotein (N) 450 Residues long and there is a high degree of homology (98% - 99.6%) amongst fixed rabies strains. Sequencing of rabies related virus strains (Lagos bat, Duvenage, EBL I & II) showed 77% homology at the N protein. M protein contains 202AA L protein is the longest and contains 2,142 a.a in rabies virus.
Why Look Into Rabies Virus in Bats? All insectivorous 10 Siberian species Biological and genetic drift? Reservoir Species
Map of Russia Highlighting the Novosibirsk and Altay Regions
Late May 2002 Expedition Highlighting the Maslanino Territory where the Barsookovskya and Yegorievskya caves are located
March 2003 Expedition Highlighting the Altay Region where four caves were searched for bats.
Our Team • Andrei Tomilenko, bat specialist • Bruce Scharf, US collaborator • Alexander Shestopalov, Laboratory Head • Vasily Axionov, Novosibirsk Diagnostic Laboratory • Vasily’s Son, our driver
Bruce with Shestopalov Laboratory personnel, Sergei and Alexandra.
Andrei Tomilenko, our bat specialist, is completing his doctorate in bat biology and ecology at Novosibirsk State University.
Preparing To Enter the Caves • On the May 2002 expedition we collected ten bats from the Barsookovskya and eight bats from the Yegorievskya caves. Two out of eight were positive for antigen from the Yegorievskya cave and four out of ten bats were positive for viral antigen from the Barsookovskya cave.
Entering the Yegorievskya Cave • This cave was extremely wet, cold and difficult to move within due to the necessity of ropes to go into the cave’s shaft.
Myotis Daubentoni • During the May 2002 expedition all eighteen bats collected from both caves were this species of bat. All of the bats appeared healthy. It is very interesting that bats seem to have active rabies virus and not get sick. They are thought to be a reservoir for rabies virus.
Myotis Daubentoni • These bats tend to huddle together in cave crevices. When we collected our May 2002 samples the bats were in torpor since it was mid-day and temperatures are at their hottest. They were hanging upside down with their wings folded in at the time of collection. These bats were easy to put into a bag with adequate space for oxygenation for the journey back to Vector.
Myotis Daubentoni • This species of bat is primarily insectivorous. Many insects are inside the caves. Despite -30C temperatures. Mosquitoes were observed inside the Altay caves close to where the bats roost.
Murina Leucogaster • In the March 2003 expedition a total of nine bats were collected. Three species of bats were observed in the caves, one of which is this species.
Myotis Brandti • All of the species of bats observed are primarily insectivorous. Bats were again collected in torpor during the March 2003 expedition and placed in breathable bags. They were separated, packaged and placed into liquid nitrogen in Biisk for transportation back to Vector where tissue processing occurred.
One of our subjects, May 2002 Bats heads were painlessly cut off with scissors, blood was drained, lungs, salivary glands and heads were then individually wrapped and placed in liquid nitrogen. Bruce should be wearing a mask and gloves, but he likes to look good for his photos. Bruce is vaccinated against rabies. Bear in mind that the vaccine only protects against genotypes 1 and 6 out of a total of 7 found in the world. CVS strain is in genotype 1.
Dr. Rassadkin and laboratory personnel processed the tissues • The cerebellum and posterior spinal cord was removed. Half of the tissue was properly packaged and sent for fluorescent antibody microscopy analysis by Dr. Axionov and the other half was delivered to Dr. Ternovoi for direct viral sequencing.
Tissues harvested were brain, lungs, salivary glands and blood. Appropriate tissues were also checked for presence of paramyxovirus (distemper), orthomyxovirus (influenza) and hanta virus.
Processing the Tissues • In May 2002, we carried the live bats back to the laboratory where we immediately processed the tissues. Dr. Sergei Koryenev, from Novosibirsk State University, is shown with Dr. Scharf labeling a tube with blood.
Viral Tree showing sequential position of bat rabies virus closest to classical rabies virus CVS_2 strain
In the March 2003 expedition we drove as far as we could on the snow covered, icy, mountainous roads prior to hiking to each cave.
The Altay Mountains are similar to the Catskill Mountains located in New York State. The biggest Altay mountain is called Belucha and is 4,500 meters high. The mountains the caves we visited were approximately 1,500 meters high.
Bruce, Alexander and Andrei ready to hike to the first cave.
Bruce, Andrei and our guide, Vladimir Alexandrovich Kaminsky prior to our hike to the first cave. Vladimir (Valodya) is a very experienced guide who has traveled with Dr. Shestopalov on many expeditions in Siberia, Kazakstan and Mongolia.
We were lucky that the 2002 to 2003 Winter was very mild in Siberia. The snow levels were between my knees to my pelvis. In other years the snow could be over my head.
I was not used to walking up what ordinarily is a moderate incline in such deep levels of snow.
You can see how tired I am. Luckily, the others made the path in the snow easier for me. Alexander particularly made sure that I kept my two hats on and did not lie in the snow.
I finally made it to the second cave. On average, it took me five minutes longer on each hour to hike to each cave than the more experienced snow walking members of our team.
Valodya’s hat is made of mink. He would be happy to take you hunting, fishing or hiking in Siberia.
After finally reaching one of the caves, Bruce had to enjoy the moment with collapse. I was surprised how much I sweat and how easy it was for me to take my outer garments off to cool down in spite of the frigid temperatures after reaching each cave. Soon I was back on my feet because there was much work to be done.
While two members of our team would explore the cave the others would make a fire to help keep us warm. We would take turns first exploring the caves, locating the bats and deciding which bats, if any, to collect for terminal sampling.
Because we would get very dirty inside the caves we brought extra gear to put on top of our normal outerwear. Since Andrei was the youngest he had the honors of carrying the backpack with extra gear up to the caves and back down to the truck.
CDC Recently confirmed the first case of rabies transmission through whole organ donation. • Rabies testing is not a part of routine organ donation screening.
