Genetic characterisation of field and attenuated rabies viruses and molecular epidemiology of rabies in Finland and Russia

Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian 7 group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies

Genetic characteristics of field and attenuated rabies viruses and molecular epidemiology of rabies in Finland and Russia

Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies

Genetic characteristics of field and attenuated rabies viruses

Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian 7 group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies

Transmission experiments support clade-level differences in the transmission and pathogenicity of Cambodian influenza A/H5N1 viruses

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more “avian-like,” with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings

Estimating the global burden of endemic canine rabies

Background: Rabies is a notoriously underreported and neglected disease of lowincome countries. This study aims to estimate the public health and economic burden of rabies circulating in domestic dog populations, globally and on a country-by-country basis, allowing an objective assessment of how much this preventable disease costs endemic countries.<p></p> Methodology/Principal Findings: We established relationships between rabies mortality and rabies prevention and control measures, which we incorporated into a model framework. We used data derived from extensive literature searches and questionnaires on disease incidence, control interventions and preventative measures within this framework to estimate the disease burden. The burden of rabies impacts on public health sector budgets, local communities and livestock economies, with the highest risk of rabies in the poorest regions of the world. This study estimates that globally canine rabies causes approximately 59,000 (95% Confidence Intervals: 25- 159,000) human deaths, over 3.7 million (95% CIs: 1.6-10.4 million) disability-adjusted life years (DALYs) and 8.6 billion USD (95% CIs: 2.9-21.5 billion) economic losses annually. The largest component of the economic burden is due to premature death (55%), followed by direct costs of post-exposure prophylaxis (PEP, 20%) and lost income whilst seeking PEP (15.5%), with only limited costs to the veterinary sector due to dog vaccination (1.5%), and additional costs to communities from livestock losses (6%).<p></p> Conclusions/Significance: This study demonstrates that investment in dog vaccination, the single most effective way of reducing the disease burden, has been inadequate and that the availability and affordability of PEP needs improving. Collaborative investments by medical and veterinary sectors could dramatically reduce the current large, and unnecessary, burden of rabies on affected communities. Improved surveillance is needed to reduce uncertainty in burden estimates and to monitor the impacts of control efforts.<p></p&gt

Estimating the global burden of endemic canine rabies

Rabies is a fatal viral disease largely transmitted to humans from bites by infected animals —predominantly from domestic dogs. The disease is entirely preventable through prompt administration of post-exposure prophylaxis (PEP) to bite victims and can be controlled through mass vaccination of domestic dogs. Yet, rabies is still very prevalent in developing countries, affecting populations with limited access to health care. The disease is also grossly underreported in these areas because most victims die at home. This leads to insufficient prioritization of rabies prevention in public health agendas. To address this lack of information on the impacts of rabies, in this study, we compiled available data to provide a robust estimate of the health and economic implications of dog rabies globally. The most important impacts included: loss of human lives (approximately 59,000 annually) and productivity due to premature death from rabies, and costs of obtaining PEP once an exposure has occurred. The greatest risk of developing rabies fell upon the poorest regions of the world, where domestic dog vaccination is not widely implemented and access to PEP is most limited. A greater focus on mass dog vaccination could eliminate the disease at source, reducing the need for costly PEP and preventing the large and unnecessary burden of mortality on at-risk communities.S1 Text. Supporting bibliography.S1 Table. Estimates by country of rabies deaths, exposures, PEP use, prevented deaths, dog vaccination coverage, probability that a dog is rabid (RP), of bite victims receiving PEP (PP), DALYs, costs and 95% confidence intervals of estimates. Clusters to which countries are assigned are shown and inputs used for estimating parameters including the human development index and whetehr a country s rabies-free or endemic (RISK). Estimates of years of life lost (YLL) and DALYs (due to rabies and to adverse events from the use of nerve tissue vaccines) are shown under different assumptions (estimates under the assumption of no time discounting or age-weighting should be directly comparable to the 2010 Global Burden of Disease study).S1 Fig. Division of costs associated with rabies, prevention and control across sectors by cluster. Inset shows proportional expenditure in different clusters. Full details of countries by cluster are given in S1 Table. Asia 4 comprises: Philippines, Sri Lanka, Thailand (High PEP use); Asia 3 comprises Bhutan, Nepal, Bangladesh, Pakistan (Himalayan region); Asia 2 comprises Cambodia, Myanmar, Laos, Vietnam and Democratic People’s Republic of Korea; SADC comprises countries in the Southern African Development Community, Eurasia comprises Afghanistan, Kazakhstan, Kyrgyzstan, Mongolia, the Russian Federation, Turkmenistan, Tajikistan, and Uzbekistan.S1 Dataset. Model code and input data files including references, rationale and detail of Delphi process. The code folder contains seven R scripts: burden_model.R runs the model using data compiled in burden_1.R, after estimating parameters using: FitCovInc.R, FitPP.R, and creating Fig 2 (RabiesBurdenFig2.R). The script burden_results.R summarizes findings using the output of burden_model.R and burden_sensitivity.R runs the sensitivity analyses. The data folder contains 12 csv files called by the R code for the analyses, and one excel file (Vet. xlsx) with additional details about the data sources in vcountry2.csv and vcluster2.csv and with Delphi process estimates for dog vaccination coverage. Data sources are detailed in the relevant data sources and the details of the sources of data used in the analysis are in the supporting bibliography, S1 text.This study was funded by the UBS Optimus Foundation (http://www.ubs.com/optimusfoundation) and the Wellcome Trust (095787/Z/11/Z).http://www.plosntds.orgam201

Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia

This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998–2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia