NEW RABIES VIRUS IN NEW RABIES VIRUS IN BATS IN SOUTH-WEST BATS IN SOUTH-WEST

SIBERIASIBERIA

Rassadkin Y., Axionov V., Ternovoi Rassadkin Y., Axionov V., Ternovoi V., Tomilenko A., Scharf B., V., Tomilenko A., Scharf B.,

Shestopalov A.Shestopalov A.

OBJECTIVESOBJECTIVES

1.1. Rhabdovirus ReminderRhabdovirus Reminder

2.2. Details about Expeditions and Details about Expeditions and ResultsResults

Rabies VirusRabies Virus Family – RhabdoviridaeFamily – Rhabdoviridae Order – MononegaviralesOrder – Mononegavirales Genus – LyssavirusGenus – LyssavirusViruses of the family Rhabdoviridae known to infect Viruses of the family Rhabdoviridae known to infect

humans are classified into 2 genera:humans are classified into 2 genera:

1. Vesiculovirus1. Vesiculovirus

2. Rabies2. Rabies

Rhaboviruses that cause rabies and some diseases of Rhaboviruses that cause rabies and some diseases of fish appear to havefish appear to have

life cycles confined to vertebrates. life cycles confined to vertebrates.

All other rhabdoviruses are thought to be transmitted by All other rhabdoviruses are thought to be transmitted by arthropods which may be the original hosts from which all arthropods which may be the original hosts from which all rhabdoviruses evolved (Shope & Tesh)rhabdoviruses evolved (Shope & Tesh)

Genome StrandGenome Strand

____________________________________________________________

N - Nucleocapsid (single stranded RNA)N - Nucleocapsid (single stranded RNA)

L & P – Minor polymerase proteinsL & P – Minor polymerase proteins

G – integrated glycosylated proteinG – integrated glycosylated protein

M – Peripheral Matrix ProteinM – Peripheral Matrix Protein

Only 1 Serogroup has been established within the Lyssavirus Only 1 Serogroup has been established within the Lyssavirus genus.genus.

Placement within the genus is determined by serological Placement within the genus is determined by serological cross-reaction of viral cross-reaction of viral

antigens (e.g. Complement fixation, immunoflourescence antigens (e.g. Complement fixation, immunoflourescence test, particularly test, particularly

antigenic sites on the N protein. antigenic sites on the N protein.

Placement of a virus as rabies or rabies – related is Placement of a virus as rabies or rabies – related is determined by anti-genic sites determined by anti-genic sites

on the glycoprotein (as recognized in virus neutralization on the glycoprotein (as recognized in virus neutralization tests) along with tests) along with

phylogenetic analysis of limited N gene sequences.phylogenetic analysis of limited N gene sequences.

Rabies SerogroupRabies SerogroupRabies (RAB) genotype I Duvenage (DUV) A IV BatsRabies (RAB) genotype I Duvenage (DUV) A IV BatsV European bat type I (EB – 1)V European bat type I (EB – 1)VI European bat type II (EB - 2)VI European bat type II (EB - 2)Kotonkan (KOT) A – midges & mosquitosKotonkan (KOT) A – midges & mosquitosLagos Bat (LB) A II – midges & mosquitosLagos Bat (LB) A II – midges & mosquitos

Rodents – Mokola (MOK) A III – midges & mosquitosRodents – Mokola (MOK) A III – midges & mosquitosObodhiang (OBD) A – midges & mosquitosObodhiang (OBD) A – midges & mosquitosRochambeau (RBU)Rochambeau (RBU)

VII Australian bat LyssavirusaVII Australian bat LyssavirusaNew strain of Duvenage virus, two red fox variants, Irkut New strain of Duvenage virus, two red fox variants, Irkut

virus, WCBV (Western Caucaus Bat Virus), Khujand, and virus, WCBV (Western Caucaus Bat Virus), Khujand, and Aravan viruses arguably have brought the total genotype to Aravan viruses arguably have brought the total genotype to 13.13.

All rabies virus isolates from hematophagous, insectivorous All rabies virus isolates from hematophagous, insectivorous and frugivorousand frugivorous

bats from the Americas belong to the rabies virus species.bats from the Americas belong to the rabies virus species.

The two Lyssavirus species from European The two Lyssavirus species from European insectivorous bats (EBL insectivorous bats (EBL

type I & II) are classified as rabies - related viruses type I & II) are classified as rabies - related viruses because of the because of the

antigenic differences from rabies virus. Although antigenic differences from rabies virus. Although rabies-related rabies-related

viruses are pathogenic for mammals (including viruses are pathogenic for mammals (including humans) and infectionhumans) and infection

with these viruses can result in a rabies like with these viruses can result in a rabies like encephalitis the encephalitis the

importance of these agents as disease entities is still importance of these agents as disease entities is still a matter of a matter of

controversy.controversy.

Rabies virus G protein is the only antigen capable of inducing and Rabies virus G protein is the only antigen capable of inducing and reacting with virusreacting with virus

neutralizing antibodies.neutralizing antibodies.

G Protein has been cloned and sequenced. All sequences contain a G Protein has been cloned and sequenced. All sequences contain a single open – single open –

Reading frame encoding a 524 amino acid protein. There is Reading frame encoding a 524 amino acid protein. There is approximately a 90% approximately a 90%

homology homology

at the amino acid level of G proteins of rabies virus strains at the amino acid level of G proteins of rabies virus strains sequenced so far. sequenced so far.

Mokola virus is 2 a.a. shorter than rabies virus G protein. Only Mokola virus is 2 a.a. shorter than rabies virus G protein. Only 54.3% homology w/rabies.54.3% homology w/rabies.

Nucleoprotein (N)Nucleoprotein (N)

450 Residues long and there is a high degree of homology (98% - 450 Residues long and there is a high degree of homology (98% - 99.6%) amongst fixed 99.6%) amongst fixed

rabiesrabies strains. strains.

Sequencing of rabies related virus strains (Lagos bat, Duvenage, Sequencing of rabies related virus strains (Lagos bat, Duvenage, EBL I & II) showed 77% EBL I & II) showed 77%

homology at the N protein.homology at the N protein.

M protein contains 202AA M protein contains 202AA

L protein is the longest and contains 2,142 a.a in rabies virus.L protein is the longest and contains 2,142 a.a in rabies virus.

Why Look Into Rabies Virus in Why Look Into Rabies Virus in Bats?Bats?

All insectivorousAll insectivorous

10 Siberian species10 Siberian species

Biological and genetic drift?Biological and genetic drift?

Reservoir SpeciesReservoir Species

Map of Russia Highlighting Map of Russia Highlighting the Novosibirsk and Altay the Novosibirsk and Altay

RegionsRegions

Late May 2002 Expedition Highlighting the Late May 2002 Expedition Highlighting the Maslanino Territory where the Barsookovskya Maslanino Territory where the Barsookovskya

and Yegorievskya caves are locatedand Yegorievskya caves are located

March 2003 Expedition Highlighting the Altay March 2003 Expedition Highlighting the Altay Region Region

where four caves were searched for bats. where four caves were searched for bats.

Our TeamOur Team

Andrei Tomilenko, bat Andrei Tomilenko, bat specialistspecialist

Bruce Scharf, US Bruce Scharf, US collaboratorcollaborator

Alexander Alexander Shestopalov, Shestopalov, Laboratory HeadLaboratory Head

Vasily Axionov, Vasily Axionov, Novosibirsk Novosibirsk Diagnostic LaboratoryDiagnostic Laboratory

Vasily’s Son, our Vasily’s Son, our driverdriver

Bruce with Shestopalov Laboratory Bruce with Shestopalov Laboratory personnel, Sergei and Alexandra.personnel, Sergei and Alexandra.

Andrei Tomilenko, Andrei Tomilenko, our bat specialist, is our bat specialist, is completing his completing his doctorate in bat doctorate in bat biology and ecology biology and ecology at Novosibirsk State at Novosibirsk State University. University.

Enjoying the Enjoying the Beautiful Beautiful Siberian Siberian Spring,Spring,May 2002 May 2002 ExpeditionExpedition

Preparing To Enter the Preparing To Enter the CavesCaves

On the May 2002 On the May 2002 expedition we collected expedition we collected ten bats from the ten bats from the Barsookovskya and Barsookovskya and eight bats from the eight bats from the Yegorievskya caves. Yegorievskya caves. Two out of eight were Two out of eight were positive for antigen positive for antigen from the Yegorievskya from the Yegorievskya cave and four out of ten cave and four out of ten bats were positive for bats were positive for viral antigen from the viral antigen from the Barsookovskya cave.Barsookovskya cave.

Entering the Entering the Yegorievskya CaveYegorievskya Cave

This cave was This cave was extremely wet, extremely wet, cold and difficult cold and difficult to move within to move within due to the due to the necessity of necessity of ropes to go into ropes to go into the cave’s shaft. the cave’s shaft.

Myotis DaubentoniMyotis Daubentoni

During the May During the May 2002 expedition all 2002 expedition all eighteen bats eighteen bats collected from both collected from both caves were this caves were this species of bat. All species of bat. All of the bats of the bats appeared healthy. appeared healthy. It is very It is very interesting that interesting that bats seem to have bats seem to have active rabies virus active rabies virus and not get sick. and not get sick. They are thought They are thought to be a reservoir to be a reservoir for rabies virus.for rabies virus.

Myotis DaubentoniMyotis Daubentoni These bats tend to These bats tend to

huddle together in cave huddle together in cave crevices. When we crevices. When we collected our May 2002 collected our May 2002 samples the bats were in samples the bats were in torpor since it was mid-torpor since it was mid-day and temperatures day and temperatures are at their hottest. are at their hottest. They were hanging They were hanging upside down with their upside down with their wings folded in at the wings folded in at the time of collection. These time of collection. These bats were easy to put bats were easy to put into a bag with adequate into a bag with adequate space for oxygenation for space for oxygenation for the journey back to the journey back to Vector.Vector.

Myotis DaubentoniMyotis Daubentoni This species of This species of

bat is primarily bat is primarily insectivorous. insectivorous. Many insects are Many insects are inside the caves. inside the caves. Despite -30 Despite -30C C temperatures. temperatures. Mosquitoes were Mosquitoes were observed inside observed inside the Altay caves the Altay caves close to where close to where the bats roost.the bats roost.

Murina LeucogasterMurina Leucogaster

In the March In the March 2003 expedition 2003 expedition a total of nine a total of nine bats were bats were collected. Three collected. Three species of bats species of bats were observed were observed in the caves, one in the caves, one of which is this of which is this species.species.

Myotis BrandtiMyotis Brandti All of the species of All of the species of

bats observed are bats observed are primarily primarily insectivorous. Bats insectivorous. Bats were again collected were again collected in torpor during the in torpor during the March 2003 March 2003 expedition and expedition and placed in breathable placed in breathable bags. They were bags. They were separated, packaged separated, packaged and placed into and placed into liquid nitrogen in liquid nitrogen in Biisk for Biisk for transportation back transportation back to Vector where to Vector where tissue processing tissue processing occurred.occurred.

Successful Exit from a Cave, Successful Exit from a Cave, May 2002May 2002

One of our subjects, May One of our subjects, May 20022002

Bats heads were painlessly Bats heads were painlessly cut off with scissors, blood cut off with scissors, blood was drained, lungs, was drained, lungs, salivary glands and heads salivary glands and heads were then individually were then individually wrapped and placed in wrapped and placed in liquid nitrogen. Bruce liquid nitrogen. Bruce should be wearing a mask should be wearing a mask and gloves, but he likes to and gloves, but he likes to look good for his photos. look good for his photos. Bruce is vaccinated Bruce is vaccinated against rabies. Bear in against rabies. Bear in mind that the vaccine only mind that the vaccine only protects against genotypes protects against genotypes 1 and 6 out of a total of 7 1 and 6 out of a total of 7 found in the world. CVS found in the world. CVS strain is in genotype 1.strain is in genotype 1.

March 2003, Processing of Tissues at March 2003, Processing of Tissues at VectorVector

Dr. Rassadkin and laboratory Dr. Rassadkin and laboratory personnel processed the personnel processed the

tissuestissues The cerebellum and The cerebellum and

posterior spinal posterior spinal cord was removed. cord was removed. Half of the tissue Half of the tissue was properly was properly packaged and sent packaged and sent for fluorescent for fluorescent antibody antibody microscopy microscopy analysis by Dr. analysis by Dr. Axionov and the Axionov and the other half was other half was delivered to Dr. delivered to Dr. Ternovoi for direct Ternovoi for direct viral sequencing.viral sequencing.

Dr. Dr. Rassadkin Rassadkin

at workat work

Tissues harvested were Tissues harvested were brain, lungs, salivary glands brain, lungs, salivary glands and blood. Appropriate and blood. Appropriate tissues were also checked tissues were also checked for presence of for presence of paramyxovirus (distemper), paramyxovirus (distemper), orthomyxovirus (influenza) orthomyxovirus (influenza) and hanta virus.and hanta virus.

Processing the TissuesProcessing the Tissues

In May 2002, we In May 2002, we carried the live bats carried the live bats back to the back to the laboratory where we laboratory where we immediately immediately processed the processed the tissues. Dr. Sergei tissues. Dr. Sergei Koryenev, from Koryenev, from Novosibirsk State Novosibirsk State University, is shown University, is shown with Dr. Scharf with Dr. Scharf labeling a tube with labeling a tube with blood.blood.

Viral Tree showing sequential position of bat Viral Tree showing sequential position of bat rabies virus closest to classical rabies virus rabies virus closest to classical rabies virus

CVS_2 strain CVS_2 strain

In the March In the March 2003 2003 expedition we expedition we drove as far as drove as far as we could on we could on the snow the snow covered, icy, covered, icy, mountainous mountainous roads prior to roads prior to hiking to each hiking to each cave. cave.

The Altay The Altay Mountains are Mountains are similar to the similar to the Catskill Mountains Catskill Mountains located in New located in New York State. The York State. The biggest Altay biggest Altay mountain is called mountain is called Belucha and is Belucha and is 4,500 meters high. 4,500 meters high. The mountains The mountains the caves we the caves we visited were visited were approximately approximately 1,500 meters high.1,500 meters high.

Bruce, Alexander and Andrei ready to hike to Bruce, Alexander and Andrei ready to hike to the first cave.the first cave.

Bruce, Andrei and Bruce, Andrei and our guide, our guide, Vladimir Vladimir Alexandrovich Alexandrovich Kaminsky prior to Kaminsky prior to our hike to the our hike to the first cave. first cave. Vladimir (Valodya) Vladimir (Valodya) is a very is a very experienced guide experienced guide who has traveled who has traveled with Dr. with Dr. Shestopalov on Shestopalov on many expeditions many expeditions in Siberia, in Siberia, Kazakstan and Kazakstan and Mongolia.Mongolia.

We were lucky that the 2002 to 2003 Winter We were lucky that the 2002 to 2003 Winter was very mild in Siberia. The snow levels were was very mild in Siberia. The snow levels were between my knees to my pelvis. In other years between my knees to my pelvis. In other years

the snow could be over my head.the snow could be over my head.

I was not used to walking up what I was not used to walking up what ordinarily is a moderate incline in such ordinarily is a moderate incline in such

deep levels of snow.deep levels of snow.

You can see how You can see how tired I am. tired I am. Luckily, the Luckily, the others made the others made the path in the snow path in the snow easier for me. easier for me. Alexander Alexander particularly made particularly made sure that I kept sure that I kept my two hats on my two hats on and did not lie in and did not lie in the snow.the snow.

I finally made it to the second cave. On I finally made it to the second cave. On average, it took me five minutes longer on average, it took me five minutes longer on

each hour to hike to each cave than the more each hour to hike to each cave than the more experienced snow walking members of our experienced snow walking members of our

team.team.

Valodya’s hat is Valodya’s hat is made of mink. made of mink. He would be He would be happy to take happy to take you hunting, you hunting, fishing or hiking fishing or hiking in Siberia.in Siberia.

After finally reaching After finally reaching one of the caves, one of the caves, Bruce had to enjoy Bruce had to enjoy the moment with the moment with collapse. I was collapse. I was surprised how much surprised how much I sweat and how I sweat and how easy it was for me to easy it was for me to take my outer take my outer garments off to cool garments off to cool down in spite of the down in spite of the frigid temperatures frigid temperatures after reaching each after reaching each cave. Soon I was cave. Soon I was back on my feet back on my feet because there was because there was much work to be much work to be done.done.

While two While two members of our members of our team would team would explore the cave explore the cave the others would the others would make a fire to help make a fire to help keep us warm. We keep us warm. We would take turns would take turns first exploring the first exploring the caves, locating the caves, locating the bats and deciding bats and deciding which bats, if any, which bats, if any, to collect for to collect for terminal sampling.terminal sampling.

Because we would Because we would get very dirty inside get very dirty inside the caves we the caves we brought extra gear brought extra gear to put on top of our to put on top of our normal outerwear. normal outerwear. Since Andrei was Since Andrei was the youngest he had the youngest he had the honors of the honors of carrying the carrying the backpack with extra backpack with extra gear up to the caves gear up to the caves and back down to and back down to the truck.the truck.

CDC Recently confirmed the first case of rabies transmission CDC Recently confirmed the first case of rabies transmission through whole organ donation. through whole organ donation.

Rabies testing is not a part of routine organ donation screening.Rabies testing is not a part of routine organ donation screening.

I hope to see beautiful Siberia I hope to see beautiful Siberia again!again!