Rattus norvegicus and Rattus tanezumi, the predominant rodents caught in urban Singapore.Hantaviruses cause human disease in endemic regions around the world. Outbreaks of hantaviral diseases have been associated with changes in rodent population density and adaptation to human settlements leading to their proliferation in close proximity to human dwellings. In a parallel study initiated to determine the prevalence of pathogens in Singapore's wild rodent population, 1206 rodents were trapped and screened. The findings established a hantavirus seroprevalence of 34%. This paper describes the molecular characterization of hantaviruses from Rattus norvegicus and Rattus tanezumi indicated that 27 (2.24%) of the animals were positive. sequence analysis of the S and M segments established that two different hantavirus strains circulate in the rodent population of Singapore. Notably, the hantavirus strains found in Rattus norvegicus clusters with other Asian Seoul virus sequences, while the virus strains found in Rattus tanezumi had the highest sequence similarity to the Serang virus from Rattus tanezumi in Indonesia, followed by Cambodian hantavirus isolates and the Thailand virus isolated from Bandicota indica.Pan-hanta RT-PCR performed on samples of Rattus norvegicus (Seoul virus strain Singapore) and Rattus tanezumi (Serang virus strain Jurong TJK/06) revealed that two genetically different hantavirus strains were found in rodents of Singapore. Evidently, together with Serang, Cambodian and Thailand virus the Jurong virus forms a distinct phylogroup. Interestingly, these highly similar virus strains have been identified in different rodent hosts. Further studies are underway to analyze the public health significance of finding hantavirus strains in Singapore rodents.Sequence analysis of the S and M segments of hantavirus strains found in  This group of viruses includes the etiological agents of hemorrhagic fever with renal syndrome (HFRS), largely seen in Europe and Asia, and hantaviruses causing (cardio) pulmonary syndrome (HCPS) in the Americas. The clinical severity of hantavirus infections ranges from asymptomatic infections to fulminate hemorrhagic shock and death. Hantaan virus (HTNV) and Dobrava viruses (DOBV) are causative agents of severe forms of HFRS and mortality rates of up to 15% have been reported. About 20 - 30% of HTNV infected patients develop hemorrhages [The Hantaviruses are enveloped and contain genomes composed of three negative-stranded RNA segments; small (S), medium (M) and large (L) segment, named according to the size of the individual RNAs . The L sTransmission of hantavirus to humans occur mainly through inhalation of aerosolized rodent excreta and hantavirus infections are therefore limited to the geographic regions inhabited by the infected animal hosts. Today, a wide array of hantaviruses has been detected in numerous rodent or insectivore species -6.Bandicota (B.) indica in 1994 [Rattus (R.) tanezumi and R. norvegicus in Indonesia [Hantaviruses are endemic in many countries of the world, and the trend in recent years indicates that the natural foci have extended from rural to more urban areas. The mainland China accounts for the majority of all cases reported worldwide and HTNV and Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS in Asia -9. Speci in 1994 -12, and ndonesia -15.Rattus species are difficult to distinguish morphologically and there are many changes over the years in both genus and assignments [cytochrome oxidase I [cytochrome b gene [R. rattus group and the R. norvegicus [R. rattus group comprises about 21 species including R. rattus and R. tanezumi. The R. rattus species is further divided into two subspecies based on the chromosome number [R. rattus and an Asian type that was named R. tanezumi.Many ignments . Recentlxidase I  or the ce b gene . Accordirvegicus , and thee number : an OceaR. norvegicus [R. norvegicus and one-fifth of the R. tanezumi rodents were seropositive towards the SEOV nucleocapsid protein. Of the seropositive rodents, 5.5 and 26% were also tested positive by PCR, respectively.In the late 80's in Singapore, Wong and co-workers found evidence of hantavirus infections in both rodents and humans and one hantavirus strain (R36) was isolated from rvegicus . They alrvegicus . In a paR. norvegicus and R. tanezumi, respectively. Phylogenetic analysis, nucleotide and amino acid sequence identity were also determined.This paper describes the subsequent screening of the animals using PCR, and the characterization of two genetically different hantavirus strains denoted Seoul virus strain Singapore and Serang virus strain Jurong TJK/06 of samples from All animals were handled in strict accordance with good animal practice, as defined by The Animal Research Ethics Committee of DSO National Laboratories, 20 Science Park Drive, Singapore 118230.cytochrome b gene fragments obtained by using the primers mcytb1 and mcytHb  before phylogenetic-based species identification was conducted. Obtained rodent sequences are available from the authors or accessible from GenBank (GQ274946 - GQ274949).The rodents were identified through PCR based amplification and sequencing of R. norvegicus).Indirect Enzyme-linked immunosorbent assay (ELISA) was performed using a recombinant truncated nucleocapsid protein of the Seoul virus (M34881) and serum samples of collected rodents. The analysis was done as described earlier . The cal++ free PCR buffer supplemented with 2 mM of MgCl2, 0.2 mM of dNTP, 1 μM of the primers and 1.25 U of AmpliTaq® , in a total volume of 25 μl. After a preincubation for 5 min at 95°C, 35 cycles at 53°C for 30 sec, 72°C for 45 sec and 45 sec at 95°C were done. After a final incubation for 10 min at 72°C the result was examined by agarose gel electrophoresis. A resulting PCR product of 170 bp was considered PCR positive.Primers with the potential to detect all known rodent-borne hantaviruses were designed towards conserved regions identified from multiple alignments of hantavirus L segment sequences by the BioEdit program package . Using tGQ274936 - GQ274945).RNA was extracted from tissue samples of kidneys and/or lungs of individual rodents using Qiagen RNeasy Mini kit  or the TRIzol reagent , according to manufacturer's instructions. Reverse transcription of RNA was performed with SuperScript III  and random hexamers. For direct sequencing of overlapping amplimers, numerous PCR primers were constructed using the PriFi software . SpecifiThe resulting sequences were aligned by ClustalW , as implcytochrome b gene identifies the rodent hosts of the Seoul virus strain Singapore (RN41 and 46) as R. norvegicus. Furthermore, the rodent hosts carrying the virus strain Jurong TJK/06 (RT49 and 50), (in this paper denoted R. tanezumi) clearly clusters with R. rattus diardi, R. tanezumi and R. kandianus of the diardii clade of the R. rattus complex  of R. tanezumi were PCR positive for hantavirus RNA. An observation was made when comparing the optical density OD values of the ELISA with the PCR results. It was noticed that rodents with the highest OD values were more likely to be PCR positive too. Among the rodents with OD values more than 1.0, 70% (7/10) were also found PCR positive  and one ELISA negative but PCR positive R. norvegicus. It is likely that the latter rodent was infected close to the time of trapping.RT-PCR screening of kidney samples from rodents with Pan-hantavirus primers for the L segment, together with a succeeding PCR targeting the S segment revealed that 2.1% (21/996) of R. norvegicus and R. tanezumi) were selected: rodent # 41, 46 and 49, 50, respectively. When comparing the complete open reading frames of the S segments from the two rodent species of Singapore with other complete or partial hantavirus S segment sequences  showed the highest nucleotide sequence identity (95.0%) to the Serang virus, obtained of R. tanezumi from Indonesia [B. indica [For hantavirus sequence analysis, two PCR positive lung samples of each of the two rodent species  to the Jurong strain. However, M and L segment of the Cambodian hantavirus strains are not available in public databases and only 343 bp and 412 bp of the Serang virus are currently accessible for sequence comparisons. As shown in Additional file R. norvegicus. The nucleotide sequence identity between hantavirus strains of Seoul Singapore and Cambodian Seoul virus strains was found to be between 97 and 99% and the highest identity was observed towards the Cambodian strain 41 (Camb 41) with 99.1% identity over the overlapping S segment sequences.An analogous high nucleotide sequence identity was also noticed when comparing hantavirus S segment sequences of hantaviruses of SimPlot comparisons of the complete open reading frames of the Jurong virus with the Seoul/Hantaan/Dobrava types of viruses demonstrated a significant non-conserved region of approximately 300 nt near the middle of the S segment . HoweverDue to the lack of available sequences in public databases, a comprehensive SimPlot comparison of the M segment could only be performed towards the Thailand virus along with a subset of less related hantaviruses. As expected, the M segment of the Jurong strain was most similar to the Thailand virus, especially when comparing the amino acid sequence of the glycoprotein Gn. In addition, two highly conserved regions consisting of approximately 300 amino acids each were found near the middle of the glycoproteins of the Jurong and Thailand viruses (data not shown).Cricetidae family, Arvicolinae, Neotominae or Sigmodontinae subfamilies  or hantaviruses carried by family Muridae subfamily Murinae, such as Seoul virus, Hantaan virus and Dobrava-Belgrad virus are genetically more distant . Interestingly, the four closely related viruses; Jurong, Cambodia, Serang, and Thailand are carried by two or more different rodent species. Hantavirus strains of R. norvegicus, such as the Seoul Singapore and corresponding Cambodian isolates  cluster closely together with other Seoul virus sequences, see Fig As indicated by the phylogenetic trees for the viral S and M segments  and R. norvegicus. The viral strains were denoted Jurong TJK/06 (RT49 and 50) and Seoul Singapore (RN41 and 46), respectively. Nucleotide sequences of the S and M segments as well as partial L cDNA sequences confirmed that these two virus strains are genetically distinct from each other. The nucleotide sequence identity of the S and M segments between the Jurong and Seoul Singapore strains was only 78% and 75%, whereas the amino acid sequence identity were 87% and 83%, respectively. These levels are much lower than the officially accepted 93% amino acid identity required for defining hantaviruses as same serotypes, or 7% divergence required for defining hantaviruses as different serotypes [In this study, we have identified and characterized two hantaviruses from the lungs of B. indica [R. tanezumi [Phylogenetic analysis of hantaviruses of the Asian region revealed a close genetic relationship of the Jurong virus with the Thailand hantavirus carried by . indica  and Camb. indica . By analtanezumi  showed tet al [SimPlot analysis of S segment sequences from genetically and geographically different hantaviruses revealed a highly variable region in the middle of the S segment. A similar observation was made by Schmaljohn et al  after coSimPlot analysis of open reading frames of M segments shows that the Gn protein is more variable than the carboxy terminal protein (Gc). As for the S segment, the Thailand virus shows a similar amino acid sequence of the Gn and Gc proteins as the Jurong virus. Unfortunately, due to limited sequence information available, with regards to the Cambodian and Serang hantavirus strains, a proper comparison of the M segment sequences could not be performed. However, after analyzing the phylogenetic relationships, it is evident that the Serang, Cambodian, Thailand and the Jurong viruses are genetically related and form a distinct phylogroup of hantaviruses. As these genetically related viruses are harboured by different rodent species, this might suggest the possibility of a host-switch event ,30.R. tanezumi were trapped at the same location, a shipyard in the Jurong area. However, no such clustering of hantavirus PCR positive rats to specific locations was observed among the 21 hantavirus RNA positive R. norvegicus carrying the Seoul Singapore virus.Even though the trapping sites were scattered over most of the Singapore island, all PCR positive B. indica or R. tanezumi is still unclear and further studies of epidemiological and epizootiological studies are required. Work is underway to evaluate the significance of our findings in public health context by establishing the seroprevalence of rodent borne diseases in Singapore human population.In conclusion, by genetic identification of the different rodent hosts, sequence analysis of the corresponding hantaviruses nucleotide sequences in context with geographical distribution we are able to learn more of ancestral viruses, virus evolution, host-switch events and virus migration routes. Other interesting question, which regards to the genetic diversity of hantaviruses in Southeast Asia is the impact on human health. The pathogenicity of hantaviruses of The authors declare that they have no competing interests.PJ participated in the study design, participated in fieldwork, preparation and analysis of rodent samples, performed (phylo)genetic analyses and contributed to writing of the manuscript. GY participated in the study design, fieldwork, preparation and analysis of rodent samples and contributed to writing of the manuscript, HTL and CCS participated in fieldwork preparation and analysis of rodent samples. RK performed virus isolation, RNA preparation, sequencing, cloning and writing. LCN participated in the study design, fieldwork and coordination, and contributed to writing. GB participated in the coordination of virus characterization, sequencing and drafting of the manuscript. All authors read and approved the final manuscript.Figure S1 - Rodent identification. The rodents from Singapore's wild rodent population were identified through PCR based amplification and sequencing of cytochrome b gene fragments, as described in Materials and Methods. The obtained sequences (red text), along with rodent sequences found in public databases were used for phylogenetic-based species identification and a few clades of interest are high-lighted. Rodents belonging to this study are indicated by arrows. Obtained rodent sequences are available from the authors or accessible from GenBank (GQ274946 - GQ274949).Click here for fileTable S1 - Sequence pair distances. S segment: (1) Jurong TJK/06 (This study); (2) Serang virus (AM998808); (3) Camb117 and Camb132 (AJ427511.1); (4) Thailand virus (Nakhon Ratchasima/Bi0017/2004) (AM397664.1); (5) Seoul Singapore (This study); (6) Seoul SR11 (M34881.1); (7) Hantaan virus (AB027111.1); (8) DOBV, strain Dobrava-Belgrade (L41916.1). The nucleotide and the amino acid sequence identity comparisons were made using the complete open reading frames of the S-segment except for Cambodia (nt 329-961) or Serang virus (nt 1-815). Comparisons between the partial sequences of Serang virus and Cambodian sequences (2) and (3) were made using the 486 nt overlapping coding region. M segment: (1) Jurong TJK/06 (This study); (2) Serang virus (AM998807); (3) Thailand virus L08756); (4) Seoul Singapore (This study); (5) Seoul virus  Hantavirus Gou3 (AF145977); (7) Hantaan virus (NC_005219); (8) Dobrava-Belgrade virus (L33685). The nucleotide and the amino acid sequence identity comparisons were made using the complete open reading frames of the M-segment except for the Serang virus when the nucleotides 1990-2332 were used.Click here for file


From April 2007 to March 2008, cloacal swabs were obtained from 246 casualty raptors recovered by various wildlife rehabilitation centers in the United States. The swabs were placed in a virus transport medium and transported to the laboratory on ice packs. At the laboratory, the samples were pooled with each pool consisting of five samples. All pools (n = 50) were screened for the presence of avian influenza virus (AIV) using a real time reverse transcription-polymerase chain reaction (rRT-PCR); one of the pools was found positive. All five samples in this pool were tested individually by rRT-PCR; one sample from a bald eagle was found positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Complete genome sequencing of the isolate revealed a mixed infection with H1N1 and H2N1 subtypes. Further analysis revealed that the PB1-F2 gene sequence of H1N1 virus had the N66S virulence-associated substitution. Further studies on ecology and epidemiology of AIV in raptors are needed to help understand their role in the maintenance and evolution of AIV. Wild birds are considered natural reservoirs of avian influenza virus (AIV) and at least 105 species of wild birds have been reported to harbor these viruses -5 althouHaliaeetus leucocephalus).A number of surveillance programs have been undertaken to isolate and identify the subtypes of AIV present in wild bird species e.g., waterfowl and geese ,7,8 withFrom April 2007 to March 2008, under an NIH funded surveillance program on avian influenza, cloacal swabs were collected from 246 casualty raptors  recovered by various wildlife rehabilitation centers in the U.S. The swabs were collected in brain heart infusion broth containing penicillin (500 IU/mL), streptomycin (500 μg/mL), neomycin (150 μg/mL), fungizone (1.5 μg/mL), and gentamicin (50 μg/mL). After collection, the swabs were transported on ice to the laboratory in Saint Paul by overnight shipping. At the laboratory, these samples were stored frozen at -80°C until tested (within six months of collection). The experimental plan was to thaw the samples and pool them in pools of five samples each followed by a matrix gene based real time reverse transcription-polymerase chain reaction (rRT-PCR) to detect the presence of AIV . From thCY043315 - CY043330). Using standard BLAST algorithm, GenBank databases were queried for the most closely related isolates. Sequences of A/green-winged teal/Ohio/430/1987(H1N1) (CY011040) and A/mallard/Ohio/37/1986(H2N1) (CY021125) were homologous to those of the bald eagle isolate. Sequences of all eight segments from these two subtypes were used as a reference for subsequent alignments performed with GenMapper . The alignments yielded full genome sequences for two subtypes, H1N1 and H2N1. This was further confirmed by manual alignment of all contigs obtained from GenAssembler and GenMapper using Sequencher .One of the 50 pools was found positive for AIV by rRT-PCR. All five cloacal samples in the positive pool were then tested individually by rRT-PCR and a bald eagle sample was positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Whole genome sequence of this isolate was obtained using the 454-pyrosequencing technique that enables resolution beyond what is possible by standard Sanger sequencing. Contigs of all eight segments of influenza A virus were first identified using GenAssembler software  that detected two distinct hemagglutinin subtypes - H1 and H2 (GenBank Accession nos. CY043317) [On further analysis, the matrix gene of H1N1 was found to be more closely related to A/sanderling/DE/1258/1986(H6N6). The nucleotides of HA and NA genes of H2N1 subtype had 99% and 100% identity, respectively, with those of A/mallard/Ohio/37/1986(H2N1). The nucleotide identities between each segment of the two subtypes were also determined (Table Y043317) .Bubo bubo), buzzards (Buteo lagopus), and peregrine falcon  (ProMED).All AIV subtypes (except certain H5 and H7 subtype combinations) are considered to be LPAI in nature and generally do not cause apparent disease in avian species. Even infection with HPAI H5N1 is not fatal to some waterfowl species . Most ofThis is the first report on isolation of AIV in a Bald eagle from the U.S. The bald eagle in question was recovered from Gloucester County, Virginia in March, 2008. In general, samples from injured raptors are collected immediately after admission to the Rehabilitation Center and before mixing with raptors already residing at the Rehabilitation Center. These procedures were in place when the bald eagle was brought to the Rehabilitation Center. In addition, the raptors residing at the Rehabilitation Center were AIV negative, thus ruling out the possibility of the bald eagle acquiring AIV infection at the Rehabilitation Center.The bald eagle, a bird of prey, is a protected and revered species in the US. In addition to fish, these birds prey on ducks and geese , resultiThe genome sequences of this bald eagle isolate revealed a mixture of H1N1 and H2N1 subtypes. Phylogenetic analysis identified a high degree of sequence similarity of all segments of these two subtypes to influenza A viruses circulating in wild waterfowl populations suggesting that the bald eagle possibly acquired the viruses while preying on infected wild birds. Our results indicate the need to study the raptor population in detail to gain better understanding of AIV ecology and epidemiology.AIV: avian influenza virus; HPAI: highly pathogenic avian influenza virus; LPAI: low pathogenic avian influenza virus; NIH: National Institutes of Health; rRT-PCR: real time reverse transcriptase polymerase chain reaction; RT-PCR: reverse transcriptase polymerase chain reactionThe authors declare that they have no competing interests.PTR was responsible for collection of samples from bald eagles. SMG and SS were responsible for overall coordination and planning of the study. NJ, YC, and MAR contributed in virus isolation, RT-PCR, sequencing and sequence analysis. NJ, SMG, and SS drafted the manuscript. All authors' have read and approved final manuscript.


Chromatin immunoprecipitation (ChIP) has become a very popular technique to study epigenetic regulation because it can be used to identify proteins and protein modifications present at specific locations in chromatin. While techniques have been developed to investigate epigenetic modifications present in chromatin during a specific biological function such as transcription, they depend upon the ability of the ChIP to analyze two epitopes on the same chromatin and are generally time consuming, difficult to perform, and not very sensitive. The Dual Agarose Magnetic (DAM) ChIP procedure described here is designed to address these shortcomings.Protein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.The DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin. Chromatin Immunoprecipitation (ChIP) has become an extremely powerful tool for the characterization of biologically functional chromatin. Specific antibodies bound to either protein A agarose or protein G magnetic beads are used to immune-select fragments of chromatin containing the target epitope of the antibody followed by PCR amplification to identify and quantitate the DNA present in the chromatin -4. A carWe have taken advantage of the distinct properties of agarose and magnetic beads and developed a procedure which allows chromatin immunoprecipitation with two antibodies present in the same tube. A schematic representation of this procedure is shown in Figure We first determined whether normal rabbit IgG and antibody to hyperacetylated histone H4 (Hyp H4) when bound to different carriers in the same ChIP would still function as they do in typical ChIP assays. We have extensively studied the chromatin structure of SV40 minichromosomes and showed that they contain hyperacetylated histones -7. All CFor standard ChIPs the resuspended agarose and magnetic beads carrying either IgG or antibody to Hyp H4 were added to 650 μl of CDB in an Eppendorf tube. Purified SV40 minichromosomes  {75 μl} As expected from our previous studies -7, thereFor DAM ChIPs 100 μl of agarose containing IgG and 100 μl of magnetic beads containing Hyp H4 were added to an Eppendorf tube containing 550 μl of CDB. Similarly, in a separate tube 100 μl of magnetic beads containing IgG and 100 μl of agarose containing Hyp H4 were added to 550 μl of CDB. SV40 minichromosomes {150 μl} were added to each of the tubes and the assay incubated as above. Following incubation overnight, the agarose and magnetic beads were separated. The tubes were placed adjacent to a magnet stand for 2 minutes. The supernatant including the agarose was then removed and transferred to a new tube also in the magnet stand. The magnetic beads were washed once with 100 μl of CDB and the wash liquid added to the new tube. Following a further 2 minutes to remove any remaining magnetic beads the supernatant containing the agarose was again transferred to a new tube. The agarose was sedimented by low speed centrifugation and the liquid removed. The tubes containing magnetic beads and agarose were then washed and the chromatin eluted and prepared as described above.The results of this analysis are shown in Figure Next, we investigated whether DAM ChIPs could be used to determine whether two different proteins or protein modifications can be observed in the same SV40 minichromosomes. The DAM ChIP strategy that we used to demonstrate this is shown in Figure In the first step, a pool of SV40 minichromosomes isolated from infected cells was immune selected with antibody to X bound to magnetic beads and the magnetic beads containing the selected minichromosomes washed according to the Millipore protocol to remove the contaminating SV40 minichromosomes lacking this epitope (Part B). In the second step antibody to Y bound to agarose was added and incubated overnight with end over end rotation. We hypothesized that if a minichromosome bound to the magnetic beads also contained the protein target for the second antibody in an accessible location, that portion of the fragment containing the antibody target would be bound by the antibody on the second carrier and would ultimately be transferred to it as a result of shearing of the chromatin (Part C). Following the incubation, the magnetic beads were separated from the agarose and the agarose was subsequently washed according to the Millipore protocol. The bound chromatin was eluted from the agarose using the Millipore lysis buffer and prepared for PCR analysis.Typical results of this analysis are also shown in Figure Finally, we analyzed replicating and transcribing SV40 minichromosomes for the presence of modified histones ; (iii) Extensive sonication of chromatin is not required in the DAM ChIP procedure; (iv) Finally, we show similar results using the DAM ChIP technique regardless of which carrier is used for the first or second antibody. Using this technique we also observed no cross contamination with either of the carriers. Considering the significant advantages of the DAM ChIP protocol we anticipate this modified technique will be a useful tool for the study of epigenetic modification.We have demonstrated the feasibility of using two antibodies which are individually bound to different carriers in a single ChIP assay and present evidence that this strategy can be used either to include an internal negative control during an assay or to determine whether two proteins or protein modifications can be located in a large fragment of chromatin. The advantages of the former procedure are that only half as much input chromatin is required, the washes can be performed while the two carriers are together, and the binding conditions for the negative control and test antibody are identical. The major advantage of DAM ChIPs is its simplicity. The procedure is relatively rapid and unlike most other procedures like ReChIP  does notThe authors declare that they have no competing interests.LB and BM contributed equally to this work in the design and execution of experiments, and the preparation of the manuscript.


West Nile virus epidemic were analyzed by reverse transcription-polymerase chain reaction. Products corresponding to E, NS3, and NS5 sequences were amplified from cerebellar but not from cortical samples. Phylogenetic analyses indicated a closer relationship of this isolate to 1996 Romanian and 1999 Russian than to 1998-99 Israeli or 1999 New York isolates.Specimens from a patient of the 2000 Israel  West Nile virus (WNV), is a member of the genus Flavivirus (family Flaviviridae), which is grouped together with Alfuy virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, and Yaoundevirus in the Japanese encephalitis virus antigenic complex  analysis of cerebrospinal fluid (CSF) showed no evidence of herpes simplex virus infection, and WNV antibodies were detected in serum and CSF, acyclovir was discontinued and ribavirin was initiated at an oral dosage of 2.4 g per day. The patient’s clinical status continued to deteriorate with aspiration pneumonia and intermittent generalized seizures. Intravenous immunoglobulin was added (35 g/d for 2 days) without improvement. Approximately one month after onset of illness, the patient died of respiratory failure.Postmortem examination of the brain showed multiple meningiomas, generalized atrophy, and surgical resection of the right parietal lobe. Histology was remarkable for neurofibrillary plaques consistent with Alzheimer’s disease, and scattered microglial nodules and perivascular lymphocytic inflammation in the medulla, pons, and midbrain were consistent with viral encephalitis.. Sequences were submitted to GenBank .RNA was extracted from frontal cortex and cerebellum with TRI-Reagent . Four micrograms of total RNA from each brain region was used as a template for reverse transcription-polymerase chain reaction (RT-PCR) with primer sets representing three regions of sequence conservation in flavivirus genomes: NS3-1 , NS5-1.1 , and NS5-2  . The reSequence analysis of the cloned NS3 and NS5 gene fragments indicated similarity to completely sequenced Romanian and Russian WNV isolates WNV-RO97-50-1996 and WNV-RUS-VLG4-1999, respectively; thus, to facilitate detailed phylogenetic analysis, E gene sequence from the Israel human brain sample was amplified. An E gene sequence of 1509 nucleotides (GenBank Accession Number AF394217) was amplified from total RNA by using GeneChoice UNIPOL polymerase  and primers EDL/E-U1006 (5´- GGA GTG TCT GGA GCA ACA TGG GT) or EDL/E-U1476 (5´- TCC TGC GGC GCC TTC AT) and EDL/E-L2244 (5´- CCC CTC CAA CTG ATC CAA AGT CC) or EDL/E-L2538 (5´- TCC ATC CAA GCC TCC ACA TCA), respectively. Sequence analysis of this fragment confirmed data from NS3 and NS5 sequence analyses, indicating a closer relationship of WNV-ISR-hISR2000 sequence to Romanian and Russian isolates than to the 1997/98/99 Israeli and the WNV-NY1999 isolates . The extent to which this WNV genotype contributed to human disease in the 2000 epidemic remains undetermined. WNV-ISR-hISR2000 may have been carried into Israel by migrating birds from reservoirs in southeastern Europe or reservoirs in northeastern Africa, where a highly related virus was isolated in 1998 (WNV-KEN-KN3829-1998)


This pathogen likely has wider distribution than is currently recognized. Cryptococcus gattii and C. neoformans cause pulmonary and systemic cryptococcosis. Recently, C. gattii was recognized as a distinct pathogen of humans and animals. We analyzed information from 400 publications (1948–2008) to examine whether the fungus occurs globally. Known distribution of C. gattii is possibly limited because specialized reagents for differentiation from C. neoformans are not readily available and not always used, and environmental surveys are patchy. However, autochthonous reports of C. gattii cryptococcosis have now been recognized from tropical and temperate regions. An ongoing outbreak in western Canada strengthens the case that the range of the pathogen has expanded. A few studies have highlighted differences in cryptococcosis between C. gattii and C. neoformans. More than 50 tree species have yielded C. gattii especially from decayed hollows suggesting a possible ecologic niche. This pathogen merits more attention so its environmental occurrence and role in cryptococcosis can be accurately determined. Cryptococcus has been recognized for >125 years, first from fruit juice, milk, humans, soil, and pigeon droppings and from roosting areas  by agglutination (C. neoformans antibodies in body fluids and development of a latex agglutination test (Guizotia abyssinica (Nigerseed) creatinine agar medium to distinguish pigment-producing C. neoformans from other Cryptococcus spp., which facilitated rapid screening of clinical and environmental samples for pathogenic C. neoformans isolates. A major advance in the classification and taxonomy of C. neoformans occurred with the discovery of a heterothallic, bipolar mating involved in the production of the perfect state for C. neoformans var. gattii (serotypes B and C). It was termed Filobasidiella bacillispora and differentiated from F. neoformans by production of smooth, elongate cylinder- to rod-shaped basidiospores (Evans described and differentiated C. neoformans is recognized as a species complex comprising C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D), which have distinct clinical manifestations and biological characteristics (C. gattii (serotypes B and D) was recognized as a species distinct from C. neoformans because of differences in basidiospore morphology, environmental niches, morphologic features in vivo, limited molecular identity (55%–61% relatedness of DNA), multiple gene genealogies, unique random amplified polymorphic DNA typing patterns, and inefficient cross-species mating with the production of sterile progeny and no recombination  and Google Scholar.We comprehensively searched for published reports using the PubMed database  for 1948–2008. The keywords used in the search were Cryptococcus isolates, serotype, mating type, molecular type, geographic location, and other relevant details were entered into a master spreadsheet. All publications with adequate documentation of C. gattii by >1 valid laboratory methods were regarded as acceptable for inclusion.One of us (D.J.S.) independently examined the title, abstract, methods, data tables and figures of publications identified in the literature search. Information about C. gattii from clinical, veterinary, and environmental sources. Geographically, the reports originated from a total of 48 countries, although most reports concentrated on few areas  medium. This work built on the discovery that C. neoformans can assimilate creatinine as sole source of carbon and nitrogen. Further modification in CDB medium led to development of canavanine-glycine-bromthymol blue agar, which has since become the differential medium of choice  have been recognized by PCR amplification of genomic DNA by using bacteriophage M13 single-stranded primers. Genotypes VGI and VGII are prevalent worldwide, and VGIII and VGIV are less common , New Zealand (kiwi), Africa (cheetah), and Canada (squirrel)  was reported by Ellis and Pfeiffer from Eucalyptus trees in 1990  are angiosperms; gymnosperms account for 23% of positive species  and Cupressus lusitanica in Colombia; and Cedrus deodara and Cupressus sempervirens in Argentina. Angiosperms other than Eucalyptus spp. have been reported positive for C. gattii from North America, South America, Africa, and India. Like Eucalyptus spp., other angiosperm tree species reported as hosts for C. gattii have been extensively exported from their native areas  trees. Ficus spp. are widely distributed in the tropics and subtropics, and many are exported as ornamentals. Ficus spp. have been recorded as C. gattii hosts in Brazil and Colombia but not in other regions  indicate because of extensive exportation and cultivation, which suggest further expansion of the known range of C. gattii.We suggest that C. gattii cryptococcosis in native and domestic animals in Europe, Africa, Australia and New Zealand, and the Western Hemisphere, suggesting that habitats of many of these animals overlap ecologic niches with the fungus  thus supporting the universal dispersal hypothesis, which suggests that most of the free-living microbial eukaryotes are likely to be globally distributed  Escandon demonstrated that C. gattii can survive in live almond trees and can contaminate the soil in immediate surrounding  have been recognized as C. gattii hosts; these trees have been widely exported from their native ranges as ornamental or commercially valuable trees. The evidence for C. gattii dispersal by wind and air currents is limited, but fungal isolations from air samples have been obtained around positive trees in Canada and India.C. gattii in water or water currents: 1) naturally infected porpoises and dolphins have been identified, 2) the fungus has been isolated from natural freshwater and saltwater samples in British Columbia and from contaminated water in habitats of captive animals, and 3) C. gattii can survive in water in vitro for long periods (C. gattii to geographic areas presently uninhabited by C. gattii. Isolation of the fungus from psittacine bird excrement in South America is suggestive because many of these birds fly long distances and are migratory or are exported as exotic pets or exhibit items for zoos (C. gattii spread include koalas in Australia (The following observations suggested dispersal of C. gattii is likely to be acquired in areas where mature trees are abundant either in forested or rural to semiurban settings. We derived this conclusion from published clinical reports on C. gattii cryptococcosis in Aborigines in Australia, native Africans in the Congo River basin, Canadians who visited parks and forests on Vancouver Island, and a Spanish farmer and Italian farmer (C. gattii strains from environmental sources from Canada and human clinical specimens from Italy strongly suggest that the point source of infection is the immediate vicinity of patients’ residences (C. gattii with decayed woody hollows, bark, and tree debris also suggests a role for mature trees (C. gattii acquisition (Blastomyces dermatitidis, another elusive primary pathogen, which causes blastomycosis. Some similarities in characteristics include clustered infection patterns in humans and mammals; increases in exposure risk from outdoor activities, and restricted and infrequent environmental isolations (Infections reported in domestic and wild animals in Australia, New Zealand, Africa, Spain, the United States, and Canada provide another important clue to risk areas for C. gattii is a globally established primary fungal pathogen with a specialized ecologic niche on trees and in hollows of trees. Future epidemiologic studies and environmental surveys are likely to reveal the extent of C. gattii prevalence in different environments especially in areas with known incidence of cryptococcosis but no reported isolations of C. gattii. Such information will be helpful in devising strategies to manage potential outbreaks of cryptococcosis. More clinical studies are also needed to follow up the course and outcome of C. gattii cryptococcosis, the salient point by which this fungus can be differentiated from the disease caused by C. neoformans, and any changes in patient management strategies.


Recently, our group reported the isolation of the Melaka virus from a patient with acute respiratory disease and provided data suggesting that this new orthoreovirus is capable of human-to-human transmission and is probably of bat origin. Here we report yet another Melaka-like reovirus (named Kampar virus) isolated from the throat swab of a 54 year old male patient in Kampar, Perak, Malaysia who was suffering from high fever, acute respiratory disease and vomiting at the time of virus isolation. Serological studies indicated that Kampar virus was transmitted from the index case to at least one other individual and caused respiratory disease in the contact case. Sequence analysis of the four small class genome segments indicated that Kampar and Melaka viruses are closely related. This was confirmed by virus neutralization assay, showing an effective two-way cross neutralization, i.e., the serum against one virus was able to neutralize the other. Although the exact origin of Kampar virus is unknown, epidemiological tracing revealed that the house of the index case is surrounded by fruit trees frequently visited by fruit bats. There is a high probability that Kampar virus originated from bats and was transmitted to humans via bat droppings or contaminated fruits. The discovery of Kampar virus highlights the increasing trend of emergence of bat zoonotic viruses and the need to expand our understanding of bats as a source of many unknown viruses.First discovered in the early 1950s, reoviruses ( Although orthoreoviruses have been identified as the causative agents of diseases in animals, infections in humans are generally benign with very rare cases of mild upper respiratory tract illness or enteritis in infants or children Reoviruses , was isolated from a human patient suffering acute upper respiratory disease Pteropus poliocephalus) in New South Wales, Australia Pteropus hypomelanusBats have been shown to be the reservoir hosts of many recently emergent zoonotic viruses, including Hendra virus, Nipah virus, Menangle virus, and potentially SARS and Ebola viruses Here, we report the discovery and characterization of Kampar virus (KamV), the fourth member in the NBV species group and its isolation from a human patient with fever and acute respiratory illness. Although there is no direct evidence to suggest that KamV originated from bats, the close relationship of KamV with other members of the NBV group and preliminary epidemiological data suggest that KamV is most likely a bat-borne orthoreovirus.To mitigate the potential health as well as socio-economic impact of emerging diseases, in particular, the potential emergence of pandemic influenza, the Ministry of Health Malaysia undertook nation-wide influenza-like illness surveillance for early detection, identification and control of these emerging diseases. In August 2006, as part of the surveillance process, a patient with acute influenza-like illness was investigated in Kampar, a town situated in the north-western part of peninsular Malaysia, about 36 kilometres south of Ipoh, the capital city of the state Perak.Subject 1 (S1), a 54-year old Chinese man, was well until 19 August 2006 when he developed sudden onset of high fever with chills and rigor. This was associated with cough, sore-throat and headache. There was no associated dyspnoea, tachypnoea, haemoptysis or chest pain on coughing. Besides the severe frontal throbbing headache, he had generalized body aches, myalgia and severe malaise. On the following day, he developed nausea, vomiting and diarrhoea. The vomitus consisted of food taken and was not bile-stained. The stool was described as watery without excessive mucous and it was non-malenic. The gastrointestinal symptom was associated with abdominal pain and loss of appetite. His illness was not relieved with self-medication of antipyretics. On review, there was no associated giddiness, blurring of vision, photophobia, skin bleeding or arthritis.He sought medical treatment at the government health clinic in Kampar on 21 August 2006. At the outpatient clinic, he was noted to be febrile (an axillary temperature of 40.1 degree Celsius), ill-looking with a generalized body erythema that blanched on pressure and was more prominent over the face and upper trunk. He had mild conjunctivitis but there was no jaundice. His tonsils were enlarged and injected but there was no white exudate noted over the tonsils. He was not in any respiratory distress and his lungs were clear with good air entry on auscultation. Other systemic examination was essentially normal and there was no significant lymphadenopathy noted. A provisional diagnosis of influenza-like illness was made at which he was given a higher dose of anti-pyretic. His illness was noted to resolve on 23 August 2006 although he still appeared weak and lethargic.Venous blood samples were taken from the patient for full blood count analysis, and the results are shown in His throat swab was taken on August 21 for virus isolation as described below.Subject 2 (S2) is a 28 year old female Chinese medical officer who attended to the index patient in the government health clinic in Kampar on 21 August 2006. On 25 August, she developed nasal obstruction with mild runny nose. This was associated with a mild sore-throat and hoarseness of voice. There was no associated cough or breathing difficulty. Apart from the subjective feeling of mild lethargy and general unwellness, she did not experience fever, headache or myalgia. Her upper respiratory symptoms resolved within three days.Subject 3 (S3), a 46 year old female, is the wife of the index patient. She regularly stays and takes care of the family home. Due to the nature of their work in other parts of the country, her husband and their 17-year old son only come back intermittently to stay in the same house. She was unable to re-call having a similar illness as her husband previously, although she did remember experiencing incidents of mild headache and fever in late August to early September, which resolved with self-medication. Since she did not seek medical help, there was no record of medical examination at the time of suspected infection.On August 21, during the examination by the local doctor, a throat swab was taken from the index case patient (S1) and sent in viral transport medium (VTM) to the National Public Health Laboratory for virus isolation. After 3 days of culturing, a syncytial cytopathic effect (CPE) was noted in MDCK and Vero cells, but not in Hep-2 cells. After 2 passages in MDCK cells, the virus was able to replicate and cause syncytial CPE in all types of mammalian cell-lines available in the laboratory inclusive of C6/36 (ATCC CRL-1660) cell-line which is of mosquito cell in origin (data not shown). The virus was named Kampar virus (KamV) after the location of the index case. Due to the similar CPE morphology  and cellThe small township of Kampar is bordered by forested limestone hills on the eastern side and a scattering of abandoned old tin mining ponds on the other side. S1 is a part-time carpenter and lives with his wife and their youngest son (17 years old) in an old wooden house with metal zinc roof and plain cement floor. His house is situated on reclaimed old tin mining land which is about 80 metres from the central market of Kampar town. Many tall trees, comprising of mango trees, coconut palms and natural wild fruiting trees , are found surrounding his house and between neighbouring houses. A tall fruiting mango tree is planted close to the house which is of less than three feet from the side wall of the house and directly adjacent to the window of the living room. At night, fruit bats were noted to use this mango tree to feed on the “sea-almond” fruits which they obtained from the nearby Ketapang trees. Partially eaten fruits and leftover stones of the fruits abandoned by the fruit bats were noted on the ground just outside the window of the living room or on the roof of the house. Only occasionally, the fruit bats were noted to feed on the ripened mangoes. During night time, S1 and his wife often rested in the living room while watching television. There was no recollection of fruit bats flying into his house or roosting inside the house. There was also no record of dead fruit bats being found in the house compound.Pteropus vampyrus (1/55) and Pteropus hypomelaunus (2/77). Considering that the closely related PulV virus was isolated from Pteropus hypomelaunusPteropus poliocephalusEonycteris spelaean and Cynopterus brachyotis, are more commonly found in the areas where KamV and MelV were discovered. Further field study is required to elucidate the bat species mainly responsible for the spill over of these viruses into human population in Malaysia.It is not clear at the present time whether KamV is carried by a specific fruit bat species or by multiple bat species circulating in the region. A preliminary survey of bat sera collected in peninsular Malaysia from our previous studies indicated a low prevalence of KamV-specific antibodies in at least two different bat species, The family keeps a couple of chickens in the backyard and a few pet birds in the patio. A week prior to the onset of S1's illness, his wife bought 6 chickens from a neighbour and one of them died on day 2 of illness.On follow-up investigation, venous blood samples were taken from S1, his wife (S3) and the doctor (S2) who examined him on August 21. All sera were tested for the presence of antibodies against KamV by immunofluorescent antibody testing using KamV-infected MDCK cells. As indicated by the results in To exclude the possibility that KamV was just a passenger virus which might be commonly present in the human population in this region and was not the cause of the observed sickness in S1 and S2, we have since conducted serological surveillance of 31 human serum samples collected in the same region for an unrelated study in early 2008. None of these sera had any specific IgM antibodies against KamV and only one serum sample contained low level (1∶80) of KamV-reactive IgG antibodies.To further establish the antigenic relationship between KamV and three other known orthoreoviruses in the NBV species group, a four way cross-neutralization was conducted as described in the The close genetic relationship between KamV and MelV was confirmed by the comparison of genome segment mobility or electropherotypes using SDS-PAGE. The two viruses have similar but not identical RNA genome segment profiles . The genRespiratory tract infections remain the main infectious disease of humans and account for a large proportion of public health spending worldwide. Despite recent success in discovery of novel respiratory pathogens during the last decade Isolation of MelV from a patient suffering acute respiratory disease marked a new beginning of linking orthoreoviruses to acute human RTIs In this context, the isolation of KamV confirms the observed trend of zoonotic virus emergence out of bat populations in the pacific region. Although there is no direct evidence to prove the origin of KamV, the close genetic relationship of KamV with MelV, PulV and NBV and the location of the house of the index case in close proximity to fruit trees frequently visited by fruit bats indicated a high probability that KamV is also a bat-borne virus. Subsequent serological surveillance confirmed low levels of antibodies to KamV circulating in two bat species commonly found in peninsula Malaysia.Retrospective serological studies indicated that the index has transmitted the virus to at least one other person, his doctor, presumably via contact droplet nuclei or aerosol. It is more difficult to assess how his wife was infected by KamV although serological results suggested she could have been infected earlier in the same manner as her husband.The risk of virus spillover from bats as a result of increasing encroachment by animals and humans into bat habitats is enhanced by the wide distribution of a large number of bat species and the seemingly great genetic diversity of newly emergent bat-borne viruses. This is true for henipaviruses During the preparation of this manuscript, the partial S segment sequences of a new orthoreovirus were made available in the GenBank by a group in Hong Kong (GenBank accession numbers EU165526 and EU170365-7). This virus, tentatively named reovirus strain HK23629/07, was isolated from a patient suffering an acute respiratory infection. Phylogenetic analysis of the available sequences confirmed that the HK strain is very closely related to other members of the NBV group. A closer look at the phylogenetic tree based on the most variable protein, cell attachment protein sigma C , indicatIn conclusion, the discovery and characterization of KamV corroborate our previous work on MelV and demonstrate the increasing risk posed by unknown bat viruses which are capable of infecting and causing disease in humans. This further highlights the urgent need to systematically survey bat-borne viruses in the international community so as to enable us to conduct more effective risk assessment, to provide forecast for potential future outbreaks and to devise better prevention and control strategies.2 and examined daily for the presence of CPE in cultured cells. Supernatant from cultures with visible syncytial cytopathic effect (CPE) after 3 days was taken for further analysis by serial passage in different cell lines available in the laboratory.A throat swab was taken from the patient at the time of his first clinical examination and transported in viral transport medium (VTM) to the National Public Health Laboratory for virus isolation. The sample was treated with antibiotics  for an hour before being inoculated in duplicate  into freshly confluent monolayers of MDCK , Vero  and Hep-2  cells cultured in a 24-well tissue culture plate. The plate was incubated at 37°C in 5% COThe investigation conducted in this study was approved by the ethics committee of the Malaysian National Public Health Laboratory. All patients (subjects) in this manuscript have given written informed consent (as outlined in the PLoS consent form) to publication of their case details. No identification of the subjects is to be revealed in any publication.50 was added to the diluted sera and incubated for 30 min. The pre-incubated virus/serum mix was added to confluent cell monolayers and incubated for 1 h. The inoculum was removed, monolayers washed three times with PBS and cell media replaced. Ability of sera to neutralize virus was determined by scoring the extent of CPE observed in duplicate wells three days later.Immunofluorescence antibody testing (IFAT) and virus neutralization assay were conducted as previously described Virion from culture supernatant was harvested and concentrated by centrifugation and viral RNA was extracted and purified using the QIamp viral RNA kit . For each virus, an aliquot of 15 µg RNA was run on a 10% SDS-polyacrylamide/Bis gel under denaturing and reducing conditions at 150 V for 4 hrs at room temperature. The gel was washed with distilled water, stained with ethidium bromide before the photo was taken.Extraction and purification of dsRNA and synthesis of randomly primed cDNA were carried out as previously described


Northern blot analysis is a powerful research tool for discovery, validation and expression of genes, and is currently widely used to detect microRNA (miRNA) accumulation. However, the traditional Northern blot procedure, which is based on a support membrane, is overly elaborate and time-consuming, although it is unsurpassed in accuracy for determining the sizes and amounts of multiple small RNAs sharing high sequence identity. Here we present an alternative method derived from plant miRNAs, liquid Northern hybridization, using fluorescently labeled oligonucleotide probes and characterized by simple and specific miRNA determination and quantitation. The entire detection process is completed within a few hours, and multiple miRNAs can be simultaneously detected in a single experiment. To date, traditional Northern blot is the gold standard for the direct study of gene expression at the mRNA level, although sensitive RT-PCR and high-throughput microarray techniques have been developed. Traditional Northern blot has absolute advantages in accurately determining the sizes of small RNAs and simultaneously displaying the sizes and amounts of multiple small RNAs . The trawith 32P ,14. Thiswith 32P ,16. In awith 32P . This pahttp://microrna.sanger.ac.uk) (We first designed a series of 20-nt oligo-DNAs derived from the rice miR156 sequence (found at r.ac.uk)  and FITCr.ac.uk) . The 0.0To evaluate the specificity of the fluorescently labeled oligonucleotide probe in liquid Northern hybridization, we conducted a series of hybridizations with Osa-miD156* and Osa-miD156s  that conTo screen for the appropriate hybridization buffer, we used small RNA samples from one-week-old rice seedlings to detect the five designed buffers: buffer 1 contained DEPC (diethylpyrocarbonate)- treated water + 10 mmol/L EDTA (ethylenediaminetetraacetic acid); buffer 2 contained 30 mmol/L Sodium phosphate buffer + 10 mmol/L EDTA; buffer 3 contained 0.3 mol/L NaCl + 30 mmol/L phosphate buffer + 10 mmol/L EDTA; buffer 4 contained 2 × SSC + 10 mmol/L EDTA; and buffer 5 contained Northern blotting solution . Small RWe conducted further quantitative analyses of the electrophoresis results of liquid Northern hybridization of Osa-miR156 from rice seedlings using a Chemi Doc XRS System camera . A series of hybridizations of different concentrations of Osa-miD156* with synthesized Osa-miR156 was used to create a standard curve. The analysis showed that a good linear relationship exists between fluorescence intensity and miR156 concentration , and the expression of Osa-miR156 in rice seedlings is 22 ng/mg fresh weight .wheat, maize, tobacco and Arabidopsis  from one-week-old rice seedlings using 4 pmol of other miRNA probes . The resbidopsis , by liqubidopsis , suggestbidopsis  was lessbidopsis , suggestOryza sativa L., Zea mays L. and Triticum aestivum L. were immersed in distilled water for two days and then germinated at room temperature. Two weeks later, seedlings that were approximately 5 cm high were used for RNA extraction. Some seedlings were grown in pots under natural conditions. Leaves, stems, roots and inflorescences or panicles were collected for RNA extraction when the plants reached maturity. Nicotiana tabacum plants, the leaves of which were used to isolate RNA, were grown in pots under natural conditions, while Arabidopsis thaliana (L.) Henyh.(Columbia ecotype) plants were grown in controlled environment chambers with a 16-h photoperiod at 24 °C. Whole plants were collected for RNA extraction when they grew to the 8-leaf stage.Seeds of et al. [2 and 2.5 volume of absolute ethanol were added. The mixture was incubated at −20 °C for at least 2 hr, followed by centrifugation of 15,000 g at 2 °C for 30 min. The resulting small RNA pellets were washed with 75% (v/v) ethanol twice, air-dried and dissolved in DEPC-H2O and stored at −70 °C.Total RNA and small RNA were extracted from plant tissues using RNA extraction buffer , 20 mmol/L sodium citrate, pH 7.0, 100 mmol/L NaCl, 10 mmol/L EDTA, 0.5% (w/v) SDS, and 1% (v/v) 2-mercaptoethanol), according to the method described by Chun et al.  with minhttp://microrna.sanger.ac.uk). Their complementary probe sequences, which were labeled with fluorescein FITC or Cy3, and other series of oligonucleotides were synthesized by TaKaRa Biotechnology Co., Ltd., Dalian, China  and synthesized probes were added to a 200-μL Eppendorf tube. Hybridization buffer (30 mmol/L Sodium phosphate buffer (pH 8.0), 0.3 mol/L of NaCl, 10 mmol/L of EDTA) was added up to 16 μL. After mixing thoroughly, the reaction mixture was heated to 94 °C for 5 min and incubated in a water bath at 42 °C for 60 min. When the hybridization reaction completed, non-hybridized single-strand sequences, including the hybridization probe, were digested with 10 U Exonuclease I at 37 °C for 30 min. Exonuclease I  catalyzes the removal of nucleotides from single-stranded DNA in the 3prime;–5prime; direction.A 0.8-mm thick, non-denaturing 10% TBE-polyacrylamide gel or 10% TAE-polyacrylamide gel was prepared according to Sambrook and Russell . After gHybridization of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 or 0.078 pmol Osa-miD156* probes with an excess of Osa-miR156 and Osa-miD156* probes were performed with 6 μL small RNA from rice seedlings. After non-denaturing PAGE electrophoresis, the gel was placed on a citaBlue conversion screen in a Chemi Doc XRS System camera  equipped with a 560DF50nm, 62 mm Emssion filter and auto-exposure. The imaging data were analyzed with Quantity One software.These experiments have clearly confirmed that the replacement of solid-phase hybridization by liquid-phase hybridization greatly shortens the time of detection. In addition, the utilization of fluorescently labeled probes instead of radioisotope labeled probes can further shorten the time. Thus, the entire process is simple and easy, and takes only a few hours (approximately three hours in this study) rather than several days as for traditional Northern blot hybridization.Moreover, for hybridization on support membranes, the effect of hybridization is largely determined by the ability of nucleic acids to attach to the membrane, which is closely related to the sequence length. miRNAs are difficult to firmly absorb on the solid support because their sequences are too short. They are therefore easily washed away from the membrane during or after hybridization. The resulting hybridization signal from some low abundance miRNAs cannot be detected. Such a problem, however, is avoided in liquid Northern hybridization. Hence, through liquid hybridization, it is possible to detect small RNAs that cannot be easily detected by solid-phase membrane hybridization.http://www.mirbase.org/cgi-bin/browse.pl). Our experiments indicated that liquid hybridization could distinguish one base variation if the hybridization is conducted at Tm  and osa-miR160f  have a two-base difference; Another example can also be found in the human miRNA family, where only one-base difference exists between hsa-let-7b  and hsa-let-7c  .Besides, the method described here can determine the expression level of miRNA in RNA sample through the standard curve method , nat-siRNA , heterochromatic small RNA and pi-RNAs. They are less than 31 nt in length and broadly present in cells of plant, animal and microbes –28, whic


The genus contains the species Aichi virus and Bovine kobuvirus, and a candidate porcine kobuvirus has been identified. Aichi virus, the type species, was first recognized in 1989 as the cause of oyster-associated nonbacterial gastroenteritis in persons in the Aichi Prefecture in Japan.The Japanese term


Routine us of vancomycin for perioperative prophylaxis is discouraged, principally to minimize microbial resistance to it. However, outcomes and costs of this recommendation have not been assessed. We used decision-analytic models to compare clinical results and cost-effectiveness of no prophylaxis, cefazolin, and vancomycin, in coronary artery bypass graft surgery. In the base case, vancomycin resulted in 7% fewer surgical site infections and 1% lower all-cause mortality and saved $117 per procedure, compared with cefazolin. Cefazolin, in turn, resulted in substantially fewer infections and deaths and lower costs than no prophylaxis. We conclude that perioperative antibiotic prophylaxis with vancomycin is usually more effective and less expensive than cefazolin. Data on vancomycin's impact on resistance are needed to quantify the trade-off between individual patients' improved clinical outcomes and lower costs and the future long-term consequences to society.


Human rabies still continues to be a significant health problem in India and other developing countries where dogs are the major vectors of transmission. Rabies in humans can present in two clinical forms, i.e., furious and paralytic. While diagnosis of furious rabies can be made based on the typical symptoms and signs, paralytic rabies poses a diagnostic dilemma to the neurologists who may encounter these cases in their practice. Although there are certain clinical features that distinguish this disease from other forms of Guillain-Barre syndromes, confirmation of diagnosis may require laboratory assistance. Conventional techniques such as antigen detection, antibody assays and virus isolation have limited success. The recently introduced molecular techniques show more promise in confirming the cases of paralytic rabies. There has not been much success in the treatment of confirmed rabies cases and recovery from rabies is extremely rare. Therefore, preventive measures of this dreaded disease after an exposure become extremely important. The present article reviews the current status of human rabies with regard to antemortem diagnosis, disease management and post-exposure prophylaxis. Rabies - the oldest and most feared human disease known to man - causes an acute, progressive incurable encephalomyelitis caused by highly neurotropic ssRNA virus, taxonomically classified in the genus Lyssa virus and family Rhabdoviridae. Rabies in humans can present in two clinical forms, i.e., furious and paralytic. While the diagnosis of furious rabies can be made on distinctive symptoms and signs, paralytic rabies poses a diagnostic dilemma to the neurologists. Although there are certain clinical features that distinguish this disease from various forms of Guillain-Barre syndromes, confirmation of diagnosis may require extensive laboratory assistance. Neurologists in developing countries are likely to encounter cases of rabies encephalitis, particularly atypical and paralytic forms and a few with psychiatric manifestations for many years to come. An array of laboratory tests has been carried out in laboratories for the diagnosis of rabies, each having its own merits and de-merits. Conventional techniques such as antigen detection, antibody assays and virus isolation are time consuming and have limited success. In the present era, the antemortem diagnosis of rabies is gaining tremendous attention. For instance, the survival of patients presenting with hydrophobia are being reported from various geographical locations around the globe and in developing countries such as India.2 There iRabies virus is a negative sense single stranded RNA virus belonging to the genus Lyssavirus and family Rhabdoviridae. It is a bullet-shaped enveloped virus measuring 180 nm × 75 nm. The envelope is surrounded by numerous spikes made up of glycoprotein (G), which are necessary for viral attachment to receptors and is also a major protein for the induction of neutralizing antibodies. The other important proteins are the ribonucleoprotein (N), which is intimately associated with the helical RNA, the phosphoprotein (P) and a matrix protein (M) . The rabHuman infection by the rabies virus almost always occurs through the bite of an infected animal, dogs being the major vector of transmission. Direct transmission through the nervous system from the site of bite can occur in which case the incubation period tends to be short, sometimes as short as a week, if there are sufficient nerve endings exposed at the wound site. However, in majority of the cases, the virus undergoes a period of replication in the exposed muscles before entering the motor or sensory nerves supplying the muscle. NicotiniThe development of rabies encephalitis in an exposed individual depends on several factors viz. the severity of bite, site of bite, virus content in the saliva and probably some non-specific host immune factors. Transdermal bites on the head, neck and hands carry the highest risk and are usually associated with shorter incubation period. However, bites on the lower extremities are more common, and as revealed in the recent Indian survey, 80% of all human rabies cases had bites on the legs. It is also known that not every rabid bite results in clinical rabies. Rabies mortality varies from 35 to 57% in unvaccinated bite victims.[The incubation period for rabies is the most variable of all acute CNS infections. The average incubation period is around 1-3 months but may range from less than 7 days to 6 years. It is not still clear why there is such a variable and prolonged incubation period. The incubation period may depend on several factors including density of rabies virus receptors in the affected tissue, the degree of innervation in tissues, the quantity of virus inoculated and the properties of the rabies virus stain. It is asExcept for the local symptoms at the site of bite or bitten extremity, these are usually vague and nonspecific. There may be fever, malaise, generalized mayalgia and a sense of “not well-being.” In nearly 50% cases of paralytic rabies and 30% cases of furious rabies, local manifestation in the form of itching, pain or paresthesia at the site of bite may be the earliest symptom, which is often neglected by the patient and ignored by the physician. This symptom may later involve the entire bitten extremity. The sensory function is usually intact, and at this stage, there is no demonstrable weakness of the bitten extremity.Within hours or a few days after the prodrome, rabies patients enter an acute neurological phase. Two-thirds of patients suffer from an encephalitic or furious form and the remaining present with paralysis or a condition resembling Guillain-Barre syndrome (GBS). Patients with encephalitic form of illness usually die within 3-4 days; however, there may be a prolonged course of illness, sometimes up to 15 days in cases of paralytic rabies, especially if these patients are treated in intensive care units. A case of paralytic rabies at the neurological services of national institute of mental health and neurosciences (NIMHANS), Bangalore, India survived up to 45 days under these conditions.The earliest manifestation of furious rabies is nervousness and hyperactivity resembling an acute anxiety reaction, often associated with moderate to high-grade fever. Initially, mentation is preserved, but the attention span is shortened and within hours three typical features of furious rabies develop: phobic spasms, fluctuating consciousness and signs of autonomic dysfunction.Typical phobic spasms include hydrophobia and aerophobia. These are seen in almost all patients with furious rabies, although they may not be present in later stages of the disease when drowsiness and coma supervene. The term “hydrophobia” is derived form the Greek word meaning “fear of water.” This term is almost synonymous with rabies in humans. Aerophobia and hydrophobia can be demonstrated by blowing or fanning of air on the face or chest wall and by asking the patient to swallow water or mere offering a glass of water. Intense startling reaction results from spasms of the accessory respiratory muscles of the neck, pharyngeal muscles and diaphragm followed by extension of the neck and a feeling of dyspnoea. During these episodes, they are extremely aroused and exhibit fearful facial expressions. The pathophysiological mechanism of hydrophobia, which is only observed in humans and not in rabid animals, is still not clear. The mental status alternates between periods of agitation and apparent normal mental status. As the disease progresses, confusion becomes severe and patients can become wild and aggressive. The period of agitation is gradually followed by impaired consciousness and coma.The signs of autonomic dysfunction include hyper salivation, pupillary abnormalities such as constricted or dilated pupils, anisocoria, generalized piloerection, excessive sweating, priapism and rarely spontaneous ejaculations. Occasionally patients may present with typical features of mania, increased sexual arousal, attempts to rape, all of which can make the patients to land in psychiatric clinics.This form of rabies is seen in approximately 20% of patients, often with diagnostic problems. The major cardinal signs of furious rabies may never appear or appear very late in the disease and can be very mild. Weakness usually starts in the bitten extremity and then progressively involves all limbs and pharyngeal and laryngeal muscles. Facial weakness is a common feature. Some of the features that may differentiate paralytic rabies from Guillain Barre Syndrome are:Fever is almost always present in paralytic rabies but usually absent in cases of GBS once weakness develops, unless followed by complications such as aspiration pneumonia.Sensory function is intact in paralytic rabies, except for the initial paresthesia at the site of bite.Quadriparesis with the predominant involvement of proximal muscles and urinary incontinence are always found in the early course of paralytic rabies. In cases of GBS, urinary incontinence is rare. In GBS, especially in the early phase, both proximal and distal muscles are affected.Percussion myoedema may be present in both early and later stages of paralytic rabies. This is best elicited by the percussion of the chest, deltoid or thigh region and consists of mounding of a part of the muscle at the percussion site, which then flattens and disappears over a few seconds. It is not observed in patients with GBS, encephalitic form of rabies or in patients with neurological complications following Semple type of sheep brain rabies vaccines.Generalized or focal fasciculations are frequently present in paralytic rabies patients.Hydrophobia is unusual and can be terminal manifestation in the paralytic form of the disease. Survival is usually longer than in the furious form. Eventually, bulbar and respiratory muscles are paralyzed resulting in death due to cardio-respiratory arrest. Although the hallmark of paralytic rabies is ascending paralysis, sometimes the features of furious rabies such as anxiety, aerophobia, inspiratory spasms and altered sensorium may be observed - particularly in the terminal stages - thereby resulting in the overlap of symptoms between the two forms. These features, if present, provide a clue to the diagnosis of rabies rather than GBS.The pathogenetic basis for the two different clinical forms of rabies is not known. There is no difference in the virus strains that cause furious and paralytic forms of the disease and has no correlation with the incubation period or the site of bite. However, paralytic rabies is more frequently observed in patients who take partial course of either Semple or cell culture rabies vaccines. It is possible that paralytic rabies results from an immunopathologic attack on virus infected cells in the brain and spinal cord.Computed tomographic (CT) studies of the brain are usually normal in both forms of rabies, although hypodense lesions in cortex and basal ganglia have been described. On MagneThe electroencephalogram (EEG) may be normal or show nonspecific abnormalities in human rabies. Slow wave activities as well as periodic epileptic form activities have been observed. HematoloSerum, CSF, saliva and tissue such as skin, from highly innervated anatomical areas are the appropriate biological specimens for antemortem diagnosis and all these samples should be considered as potentially infectious see see . The apppost mortem to establish a definitive diagnosis [Viral isolates can be obtained by animal inoculation or cell culture passage of saliva or oral swabs, although of limited utility in clinical setting. These samples can be probed for viral nucleic acid. Viral antigen can be detected by Direct Fluorescent Antibody (DFA) test on brain biopsy material collected usually during iagnosis , cornealIntra vitam diagnosis of rabies by Fluorescent antibody test (FAT) in corneal impression was first described by Schneider in animals and by Cifuentes et al. in humans.[et al. showed that the reliability of corneal impressions for rabies diagnosis is limited, especially when sampling is performed under field conditions and a negative result could not rule out the diagnosis of rabies.[Rabies virus antigen can be detected by a simple FAT test on corneal epithelium during the terminal stages of the disease. n humans.19 The mef rabies.intra vitam diagnosis in animal and humans.[The examination of skin biopsy material was shown to be a valuable technique for d humans.22et al. found that 25-50% of patients showed immunofluorescence positive results for rabies viral antigen, and a proportion of positive results during the early phase of clinical illness increased as the disease progressed.[post mortem diagnosis in countries, where the opening of skull of the dead person is not accepted on cultural and religious grounds.Blenden ogressed. The testet al. have reported the use of hemi-nested RT-PCR for rapid ante mortem diagnosis within 36 h of sample submission in case of suspected rabies, where Fluorescent antibody test (FAT), Mouse inoculation test (MIT) and Rabies Tissue Culture Infection Test (RTCIT), have yielded a negative results. Smith et al. standardized hemi-nested RT-PCR, followed by automated sequencing and confirmed the presence of classical rabies virus (genotype1) in both saliva and skin specimens with in 36 h.[et al. evaluated the utility of conventional RT-PCR and SYBR Green Real time PCR for the ante mortem diagnosis of rabies using saliva samples. Real-time PCR assay was found to be more sensitive than conventional RT-PCR assay, highlighting the utility of molecular diagnostic tests in establishing ante mortem diagnosis of rabies using saliva samples within a few hours.[et al. conducted a study to find presence of rabies virus in saliva and cerebrospinal fluid (CSF) of suspected live rabid dogs at the time of quarantine by using a SYBR Green real-time RT-PCR based assay for the detection of rabies virus RNA. Positive CSF samples were found in 4 out of 15 dogs (27%), whose saliva samples were tested. The time interval from sample collection to result was less than 5 h. Their study suggested that because the virus may be absent or present at very low levels in clinical fluids, samples taken for ante-mortem diagnosis cannot definitively rule out rabies in the animal.[The demonstration of viral nucleic acid in saliva, CSF, corneal smear, skin biopsy by Polymerase Chain Reaction (PCR) appears to be quite promising for the antemortem diagnosis of human rabies . Both co in 36 h. Nagaraj ew hours. As the se animal. In 2001, 15 dogs %, whose et al. proposed the tissue culture technique (BHK 21 cells) as a routine method for the isolation of street strain of rabies virus.[intra vitam includes saliva samples, throat swabs, swabs of the nasal mucosa, corneal smears and cerebrospinal fluid.[Virus isolation from animals (or humans) is one of the most reliable methods for the diagnosis of rabies. Virus isolation from infected tissue can be carried out either in mice (mouse inoculation test - MIT) or in cell culture (rabies tissue culture isolation test-RTCIT). Rudd es virus. The specal fluid.Mouse inoculation test (MIT)The Swiss albino mouse is preferred for rabies diagnosis, but other strains can also be used since all are equally susceptible to intracerebral inoculation with street rabies virus. The Swiss albino mice are the most susceptible and common animals used for virus isolation. While weanling mice {21 to 30 days old, (weighing 9-15g)} are preferred, suckling mice less than 4 days are more susceptible. The homogenized brain tissue sample in buffered diluent containing protein stabilizer and antibiotics are intracerebrally inoculated (0.03 ml) into a minimum of six weanling mice for each specimen.The technical simplicity and high sensitivity are favored attributes of MIT. The main drawback of this test is the technical expertise, environment and ethical issues related to the usage of large number of animals.Rapid tissue culture infection test (RTCIT)in-vivo virus isolation is considerably reduced with the cell culture inoculation. Several cell lines may be used for the isolation such as BHK 21, the CER and mouse neuroblastoma.[2', the cells are fixed, stained with an antinucleocapsid antibody conjugated to fluorescein isothiocyanate (FITC) and observed under a fluorescent microscope.[The inoculation of the cell culture is a recognized method for the isolation of street virus for rabies diagnosis at present. The unpredictable and problematic delay with blastoma.31 The spcroscope. In many An indirect evidence for rabies infection is demonstrating a high titer of antibodies in CSF in nonvaccinated individuals. The following are the WHO -recommended tests based on this principle: Mouse Neutralization Test (NT) and Rapid Fluorescent Focus Inhibition Test (RFFIT).Mouse Neutralization Test (MNT)50', is determined by the Reed-Muench or Spearman-Karber methods. To each serum dilution from a suspected case of rabies, 32 to100 times the LD50 virus is added and the mixture is incubated for 90 min at 37°C and then inoculated intracerebrally into weanling mice (5-10 mice for each serum dilution). The mice are observed for 14 days, and the number of deaths occurring in each serum dilution is noted. The dilution of serum at which 50% of the mice are protected  is calculated by the Reed-Muench or Spearman-Karber methods. The precision of the assay is limited by the number of mice inoculated for each serum dilution and the increment between dilutions to be used. Tediousness and economics limit the numbers of mice, to be inoculated and dilutions to be evaluated. The individual resistance and nonspecific mortality associated with any animal test also adversely affects the precision of the test.[Webster and Atanasiu developed virus neutralization test in mice  in 1935.33 This ithe test.The adaptation of the “Challenge virus standard (CVS) strain” of rabies virus (attenuated virus) to grow in cell culture has led to the development of alternative assays in which the replication of non-neutralized virus in culture is detected by the observation of plaques, fluorescent antibody-stained infected cells or the colour developed with enzyme-labeled anti-rabies antibodyRapid Fluorescent Focus Inhibition Test (RFFIT)et al.[50  of virus and incubating the serum-virus mixture at 37°C. Subsequently, suspensions of BHK-21 cells are added. After a 20-h incubation period at 37°C in a 5% CO2 incubator, the cells are stained with an anti-rabies antibody conjugated to FITC. The antibody titer is defined as the reciprocal of the serum dilution, which reduces the challenge virus to 1FFD50. The titers obtained are approximately equal to those measured by the MNT. The rapidity with which the test can be carried out and the ease with which several samples may be tested at a time make it suitable for routine diagnosis. The demonstration of antibody in the serum in the absence of a history of vaccination for rabies or in CSF offers indirect evidence of rabies infection. The RFFIT has been shown to be slightly more sensitive than the MNT for detecting Virus Neutralizing antibodies (VNAs) in post-vaccinal sera.[The RFFIT is one of the most widely used substitutes to the Mouse Neutralization Test (MNT) and was developed by Smith et al. It is a et al.. The sernal sera.Fluorescence Antibody Virus Neutralization Test (FAVN)et al. in 1998.[50 live virus in cell line and each well is scored as having virus present or no virus after a 40-h incubation period. Results obtained with this test showed good agreement with the MNT and RFFIT. According to the authors, distinguishing negative sera from the positive with low titers of antibody is easier with the FAVN as compared to RFFIT. Hostnik has described a modification of this test in which the monoclonal anti-rabies antibodies and a peroxidase anti-mouse conjugate were used instead of a fluorescein-conjugated anti-rabies antibody.[This test, which is an adaptation of RFFIT, was developed by Cliquet  in 1998. In this antibody.The Indirect immunofluorescence assay (IFA)et al. and Gispen and Sathof reported the IFA to be more sensitive than NT, several other investigators found NT to be better.[et al., from South India, reported a sensitivity of 97.2% and specificity of 97.9% for IFA.[The IFA is based on the interaction between antibody and viral antigen present in smears of infected mouse brain or cell cultures. The bound antibody is visualized using a fluorescein-labeled species-specific antiserum directed against one or more immunoglobulins classes. The slide is examined under a fluorescent microscope. The reaction is graded by the amount and brilliance of fluorescence. The IFA is reported to be more sensitive than the neutralization test (NT) in detecting antibody early in the immune response, making it valuable for diagnosis.40 The IFe better.–44 Madhu for IFA. A compar for IFA. The IFA Membrane staining is not easily observed in acetone-fixed preparations and it would be difficult to detect by the customary IFA technique. Relative insensitivity in detecting antiglycoprotein antibody may lead to an inaccurate estimate of the protection afforded by immunization, making the IFA unsuitable for vaccine efficacy trials.ante mortem diagnosis of rabies using clinical specimens may not be always successful, and negative tests do not rule out the possibility of rabies. In contrast to ante mortem diagnosis of rabies, post mortem diagnosis is more sensitive and specific. Negri bodies are present in more than 70% of infected human or animal brains [ante-mortem diagnosis. Rabies virus antigen could be detected by agglutination on a glass slide using latex particles coated with gamma globulin. Recently Kasempimolporn et al. developed a simple and inexpensive test based on latex agglutination (LA) for rabies virus antigen detection in the saliva of live dogs.[The attempts for l brains  Inset. Ml brains  The detel brains  or recenl brains 48 The del brains  Studies ive dogs.Human rabies is still considered as cent percent fatal, although some cases of recovery have been reported form time to time.–53 The mAlthough rabies is virtually 100% fatal once the symptoms develop, it can be effectively managed if appropriate post-exposure treatment is instituted immediately after exposure. The post-exposure prophylaxis consists of three important ingredients viz. local wound treatment, passive immunization with rabies immunoglobulins and active immunization with rabies vaccines. All these are equally important and treatment failures have occurred due to neglecting any of the procedures, particularly passive immunization.Local wound treatment should be instituted soon after the exposure. The bite wound(s) should be flushed with running tap water and washed with plenty of soap or detergent for at least 10 min. This removes the virus mechanically as well as inactivates the virus to a great extent. This should be followed with applying a local antiseptic such as Povidone Iodine. Suturing should be avoided as far as possible and if deemed necessary for surgical reasons, should be done only after infiltrating the wound with anti rabies immunoglobulin.www.apcri.org.The further management of the bite depends on the degree or severity of exposures and risk of developing rabies. The present categorization of exposures and the required treatment procedures that is universally followed is as per the national guideline, the details of which can be accessed from the web site of Association for Prevention and Control of rabies in India (APCRI): et al. assessed the safety of post-exposure rabies immunization and immunogenicity of purified vero cell rabies vaccine (PVRV) during pregnancy and found it to be safe and immunogenically efficacious. None of the women experienced any adverse side effects to the vaccine.[The manufacture and use of outdated nerve-tissue-derived Semple vaccine has been discontinued in India since January 2005. Highly potent and safe cell culture vaccines are available for post-exposure treatment for almost 3 decades. These are the Human Diploid Cell Vaccine (HDCV), Purified Chick Embryo Cell vaccine  and Purified Vero cell Rabies vaccine . Recently, a highly purified duck embryo vaccine  was introduced in India. Five doses of vaccine must be administered on day 0, 3, 7, 14 and 28 intramuscularly over the deltoid region. The dosage schedule is same for all age groups and there is no contraindication for rabies vaccination. Sudarshan  vaccine. As cell  vaccine. Recently vaccine.This is one aspect of post-exposure prophylaxis (PEP) that is often neglected by the medical practitioners. The presently available rabies vaccines induce protective levels of antibodies usually 10-14 days after initiating the course. Some cases of category III exposures may have very short incubation period and in such cases the mere administration of vaccine will not prevent rabies, as the virus would have reached CNS before the formation of sufficient quantities of neutralizing antibodies. Therefore, WHO advocates passive immunization with rabies immunoglobulin (RIG) in all category III exposures There are two types of RIGs presently available viz. highly purified and enzyme-refined equine rabies immunoglobulin (ERIG) and human rabies immunoglobulin (HRIG). The recommended dosage is 40 IU/kg body weight of ERIG and 20 IU/kg body weight of HRIG. The calculated dose should be infiltrated as much as possible locally into and around the wound and remaining if any should be administered IM in the gluteal region. The ERIGs should be administered only after a proper sensitivity test, although the incidence of anaphylactic reactions following the administration of present day ERIGs is very rare, (around 0.001%). Other adThis is recommended to people who are at continued risk of exposure to rabies such as veterinarians, laboratory workers handling rabies virus, dog catchers and forest rangers, medical and paramedical people treating rabies patients in ICU. Three doses of any modern rabies vaccine can be administered on days 0, 7 and 28. A booster dose is recommended to people at continued risk.et al. recommended the pre-immunization to travelers visiting areas where canine rabies is enzootic and access to appropriate medical care is limited.[As per the WHO recommendation, 2 doses of any modern vaccine is required on days 0 and 3 for people who are re-exposed to rabies and have taken a course of complete post-exposure treatment with any of the modern cell culture vaccine any time in the past. There is no need for administration of rabies immunoglobulin. Incorporation of intradermal vaccine into childhood immunization schedules has been proposed for developing countries. Hatz et  limited.The intramuscular route is recommended for rabies pre exposure vaccination if the antimalarial Chloroquine phosphate is being administered concurrently. This recommendation was made after a Peace Corps Volunteer died of rabies despite having completed a full intradermal (ID) pre-exposure series 6 months previously.In the era of wide international travel, neurologists, internists and medical practitioners in critical care management are confronted with cases of rabies masquerading as other neurological illness such as peripheral neuropathy. Similarly, in psychiatric practice, a case diagnosed as manic psychosis could in reality be one of the types of rabies. The knowledge of the technical modalities for antemortem diagnosis may assist in planning the hospital management and post-exposure prophylaxis of the treating medical team. Although the clinical outcome of rabies still remains grim, the pre-exposure and post-exposure vaccination in combination with passive immunization is likely to change the clinical outcome favorably.