during april 928 , 2007 , five persons became ill and died within a few days at village belechuapara , nadia district , west bengal , india , which borders bangladesh .
the index case - patient ( case - patient 1 ) was a 35-year - old male farmer addicted to country liquor derived from palm juice .
hundreds of bats were observed hanging from the trees around his residence , which strongly suggested association with the infection of the index case - patient and possibility of contamination of the liquor with bat excreta or secretions .
three patients were close relatives of case - patient 1 : his 25-year - old brother ( case - patient 2 ) , his 30-year - old wife ( case - patient 3 ) , and his 39-year - old brother - in - law ( case - patient 4 ) .
they became ill 12 , 14 , and 14 days , respectively , after contact with case - patient 1 . in another person , a 28-year - old man ( case - patient 5 ) who collected blood samples from and performed a computed tomography scan of the brain of case - patient 1 , symptoms developed 12 days after contact
brain and lung tissues from case - patient 3 , cerebrospinal fluid ( csf ) from case - patient 4 , and urine and csf from case - patient 5 were collected .
blood samples were obtained from case - patients 4 and 5 and from 34 asymptomatic contacts from the village .
serum samples from these persons were tested for immunoglobulin ( ig ) m and igg antibodies to niv ( igm / igg anti - niv ) with elisa by using reagents provided by the centers for disease control and prevention ( atlanta , ga , usa ) . to detect niv rna , urine ( 250 l ) , csf ( 100 l ) , or autopsied brain or lung tissue ( 100 mg ) were used , and rna was extracted by using trizol ls and trizol reagents ( invitrogen life technologies , carlsbad , ca , usa ) . nested reverse transcription 
pcr ( rt - pcr ) was conducted by using nucleocapsid ( n ) gene  based primers ( 8) .
the full - length genomic sequence was obtained from the lung of case - patient 3 by using 36 sets of primers ( table a1 ) , superscript ii rnase reverse transcriptase for reverse transcription ( invitrogen ) , and pfx polymerase for amplification ( invitrogen ) .
the pcr products of predicted molecular size were gel eluted ( qiaquick pcr purification kit ; qiagen , hilden , germany ) and sequenced by using bigdye terminator cycle sequencing ready reaction kit ( applied biosystems , foster city , ca , usa ) and an automatic sequencer ( abi prism 3100 genetic analyzer ; applied biosystems ) .
phylogenetic analysis was conducted by using partial n gene and full niv genome sequences with the kimura 2-parameter distance model and neighbor - joining method available in mega version 3.1 software ( www.megasoftware.net ) .
the reliability of phylogenetic groupings was evaluated by the bootstrap test with 1,000 bootstrap replications .
patients signs and symptoms included high fever ( 103f105f [ 39.4c49.6c ] ) with and without chills , severe occipital headache , nausea , vomiting , respiratory distress , pain in calf muscles , slurred speech , twitching of facial muscles , altered sensorium , ( focal ) convulsions , unconsciousness , coma , and death .
the first 3 case - patients died within 23 days after symptom onset ; case - patients 4 and 5 died after 5 and 6 days , respectively .
results of serologic tests for malaria parasite , typhoid , anti - dengue igm , hiv , and hepatitis b surface antigen were negative .
alanine aminotransferase , aspartate aminotransferase , creatine phosphokinase , and c - reactive protein were elevated . blood gas analysis in case - patient 4 ( case - patient 5 values in parentheses ) showed oxygen saturation 49.4% ( 71% ) , po2 36.1 mm hg ( 44.8 mm hg ) , pco2 44.4 mm hg ( 44.1 mm hg ) , hco3 15.7 mmol / l ( 19.1 mmol / l ) , and ph 7.166 ( 7.255 ) .
lumbar puncture of case - patient 5 showed opening pressure within reference range ( 1 drop / second ) , 2 cells / cm ; all cells observed were lymphocytes .
chest radiograph indicated pulmonary edema , which suggested acute respiratory distress syndrome . at the time of admission , computed tomography and magnetic resonance imaging scans of the brain showed no abnormality .
serum samples from case - patients 4 and 5 were positive for igm anti - niv .
of the clinical samples screened , brain and lung tissues of case - patient 3 , csf of case - patient 4 , and urine of case - patient 5 were niv rna positive .
of the 34 asymptomatic contacts , 1 was positive for igg anti - niv and negative for igm anti - niv and did not report any major illness in the past .
this positivity may reflect a previous subclinical infection or cross - reactivity in elisa needing further follow - up . before this report
partial n gene sequences confirmed niv in the clinical specimens from all 3 case - patients .
phylogenetic analysis showed that similar to findings from the 2001 outbreak study ( 8) , viruses from bangladesh and india clustered and diverged from the viruses from malaysia .
fj513078 ) was closer to the virus from bangladesh ( figure , panel b ) , with 99.2% ( 151 nt substitutions ) and 99.80% ( 17 aa substitutions ) identity at nucleotide and amino acid levels respectively .
of the 151 nt substitutions , 9 occurred in the n open reading frame ( orf),11 in the phosphoprotein orf , 8 in the matrix orf , 11 in the fusion glycoprotein orf , 7 in the attachment protein orf , and 47 in the large polymerase orf .
fifty - eight substitutions occurred in nontranslated regions at the beginning and the end of each orf .
the intergenic sequences between gene boundaries were highly conserved in the isolate from india , compared with the isolate from bangladesh , which showed 1 change ( gaa to uaa ) between the attachment protein and large polymerase genes .
a ) phylogenetic analysis based on partial nucleocapsid ( n ) gene nucleotide sequences ( 159 nt , according to nipah virus [ niv ] bangladesh sequence , genbank accession no .
ay988601 , 168327 nt ) of the 4 nivs sequenced during this study ( boldface ) .
five sequences of the viruses from siliguri ( 8) and from representative niv sequences obtained from genbank indicated by the respective accession numbers .
b ) full genome  based phylogenetic analysis of the niv sequenced from the lung tissue of a patient ( boldface ) .
the table compares amino acid substitutions in the different regions of the genome of the isolate from india with those of the viruses from bangladesh and malaysia .
of the 17 aa substitutions , 7 were unique to the isolate from india , and 10 were similar to the isolates from malaysia .
overall , however , the isolate from india was closer to the isolate from bangladesh , although distinct differences were observed .
* genbank accession numbers of isolates examined : india ( fj513078 ) , bangladesh ( ay988601 ) , and malaysia ( ay029767,ay029768 , and aj564623 ) .
boldface indicates unique amino acids in the isolate from india . to our knowledge , this is the second report of an niv outbreak in india , identified within 1 week of the investigation .
the first outbreak affected mainly hospital staff or persons visiting hospitalized patients ; the 74% case - fatality rate strongly suggested person - to - person transmission ( 8) . both outbreaks ( 2001 and 2007 ) occurred in the state of west bengal bordering bangladesh wherein several outbreaks of the disease have been reported ( 7,913 ) .
however , fruit bats from west bengal have not been screened for evidence of niv infection .
this state needs to create awareness about niv and obligatory testing of suspected case - patients .
niv caused an intrafamilial outbreak with a 100% case - fatality rate , which confirmed person - to - person transmission .
the niv strains from india and bangladesh were closer than the malaysian viruses . although the outbreaks occurred in neighboring geographic areas , niv outbreaks in bangladesh and india were not caused by the same virus strain or by spillover .
 an intrafamilial outbreak in west bengal , india , involving 5 deaths and person - to - person transmission was attributed to nipah virus . 
 full - genome sequence of nipah virus ( 18,252 nt ) amplified from lung tissue showed 99.2% nt and 99.8% aa identity with the bangladesh-2004 isolate , suggesting a common source of the virus . 
The Study
Conclusions