Devendra T. Mourya

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): selection pressure and molecular clock analyses.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Crimean-Congo haemorrhagic fever in India

On Jan 13, 2011, a 25-year-old woman was admitted to our hospital in Ahmedabad, Gujarat State, with a high fever for the previous 3 days and vomiting. She had high concentrations of alanine (93 U/L) and aspartate (394 U/L) aminotransferases, serum lactate dehydrogenase (8194 U/L) and ferritin (307210 pmol/L). Her platelet count fell from 80·4×109/L on Jan 13, to 34·6×109/L on Jan 15. Tests for all probable infections—malaria, leptospirosis, infections with bacteria and dengue, Hantaan, Nipah, and Kyasanur forest disease viruses— were negative. Serum and urine tests were negative for Marburg but positive for Crimean-Congo haemorrhagic fever (CCHF) viral RNA in real-time PCR, confi rmed by PCR and sequencing. On Jan 16, our patient was isolated and given oral ribavarin (30 mg/kg followed by 15 mg/kg) according to WHO recommendations. She developed haemoptysis, bleeding from the lips, haematuria, palatal petechiae, haematemesis, and diffi culty in breathing. On Jan 18, she became drowsy and disoriented; on examination she had mild ascites, melaena, vaginal bleeding, pleural eff usion, moderate hepatomegaly, and a contracted gall bladder with a diff use oedematous wall. She died from multi-organ failure and disseminated intravascular coagulation. Our patient was the fi rst laboratory-confi rmed case of CCHF from India. Our patient (case C) had been the attending nurse to a 32-year-old woman (case A), from a village 20 km from Ahmedabad. Case A had been admitted to a local hospital on Dec 27, 2010, with clinical and laboratory fi ndings similar to those of our patient and was moved to the intensive-care unit in our hospital on Dec 31. Case A was given only supportive treatment with no ribavarin and died on Jan 3, 2011. She was probably the index case in this outbreak. A 42-year-old physician (case B), who had treated the index case was admitted to a local hospital on Jan 6, and was treated symptomatically with no ribavarin. Clinical and laboratory fi ndings were similar to those of the index case and our patient. He died from a cardiac arrest on Jan 13. The husband of the index case (case D) was also admitted to the same local hospital on Jan 16, and was positive for CCHF virus; he was treated with recommended oral ribavarin and discharged after 10 days. Case D looked after the index case and was her household contact. Another confi rmed case of CCHF was a doctor in our hospital (case E), admitted on Jan 29 with symptoms of CCHF; he was treated with recommended oral ribavarin but died on Jan 31. Case E was exposed to cases B and D in the same local hospital, which suggests hospital-acquired infection. CCHF is reported in many countries of Africa, the Middle East, Eastern Europe, Russia, and China, Although presence of antibodies to CCHF virus in India has been reported, human cases of CCHF were restricted to the countries north and west of India. Our patient’s case is the fi rst report of nosocomial infection with CCHF virus in India. Infl uenza, dengue, Kyasanur forest disease, chikungunya, and leptospirosis are also causes of fever in India. Diff erential diagnosis at the early stage of the illness is diffi cult. Many severely ill patients with haemorrhagic fevers require admission to intensive care facilities. To avoid infections in hospital settings, stringent infection control practices, proper air handling in intensive-care units, isolation of patients, and correct handling of clinical specimens are essential. Tick bites and contact with infected animals are the main modes of infection to people. Disinfection of domestic animals and their accommodation can help reduce the risk of human infection. Febrile patients with haemorrhagic symptoms, who are negative for dengue virus, should be considered as possible cases of CCHF for the purpose of hospital infection control and isolation of patients in India.

Detection, Isolation and Confirmation of Crimean-Congo Hemorrhagic Fever Virus in Human, Ticks and Animals in Ahmadabad, India, 2010–2011

Background In January 2011, human cases with hemorrhagic manifestations in the hospital staff were reported from a tertiary care hospital in Ahmadabad, India. This paper reports a detailed epidemiological investigation of nosocomial outbreak from the affected area of Ahmadabad, Gujarat, India. Principal Findings Samples from 3 suspected cases, 83 contacts, Hyalomma ticks and livestock were screened for Crimean-Congo hemorrhagic fever (CCHF) virus by qRT-PCR of which samples of two medical professionals (case C and E) and the husband of the index case (case D) were positive for CCHFV. The sensitivity and specificity of indigenous developed IgM ELISA to screen CCHFV specific antibodies in human serum was 75.0% and 97.5% respectively as compared to commercial kit. About 17.0% domestic animals from Kolat, Ahmadabad were positive for IgG antibodies while only two cattle and a goat showed positivity by qRT-PCR. Surprisingly, 43.0% domestic animals (Buffalo, cattle, sheep and goat) showed IgG antibodies in the adjoining village Jivanpara but only one of the buffalo was positive for CCHFV. The Hyalomma anatolicum anatolicum ticks were positive in PCR and virus isolation. CCHFV was isolated from the blood sample of case C, E in Vero E-6 cells and Swiss albino mice. In partial nucleocapsid gene phylogeny from CCHFV positive human samples of the years 2010 and 2011, livestock and ticks showed this virus was similar to Tajikistan (strain TAJ/H08966), which belongs in the Asian/middle east genetic lineage IV. Conclusions The likely source of CCHFV was identified as virus infected Hyalomma ticks and livestock at the rural village residence of the primary case (case A). In addition, retrospective sample analysis revealed the existence of CCHFV in Gujarat and Rajasthan states before this outbreak. An indigenous developed IgM ELISA kit will be of great use for screening this virus in India.

Detection of Nipah Virus RNA in Fruit Bat (Pteropus giganteus) from India

The study deals with the survey of different bat populations (Pteropus giganteus, Cynopterus sphinx, and Megaderma lyra) in India for highly pathogenic Nipah virus (NiV), Reston Ebola virus, and Marburg virus. Bats (n = 140) from two states in India (Maharashtra and West Bengal) were tested for IgG (serum samples) against these viruses and for virus RNAs. Only NiV RNA was detected in a liver homogenate of P. giganteus captured in Myanaguri, West Bengal. Partial sequence analysis of nucleocapsid, glycoprotein, fusion, and phosphoprotein genes showed similarity with the NiV sequences from earlier outbreaks in India. A serum sample of this bat was also positive by enzyme-linked immunosorbent assay for NiV-specific IgG. This is the first report on confirmation of Nipah viral RNA in Pteropus bat from India and suggests the possible role of this species in transmission of NiV in India.

Malsoor Virus, a Novel Bat Phlebovirus, Is Closely Related to Severe Fever with Thrombocytopenia Syndrome Virus and Heartland Virus

ABSTRACT During a survey in the year 2010, a novel phlebovirus was isolated from the Rousettus leschenaultii species of bats in western India. The virus was identified by electron microscopy from infected Vero E6 cells. Phylogenic analysis of the complete genome showed its close relation to severe fever with thrombocytopenia syndrome (SFTS) and Heartland viruses, which makes it imperative to further study its natural ecology and potential as a novel emerging zoonotic virus.

Kyasanur Forest disease, India, 2011-2012.

To determine the cause of the recent upsurge in Kyasanur Forest disease, we investigated the outbreak that occurred during December 2011–March 2012 in India. Male patients >14 years of age were most commonly affected. Although vaccination is the key strategy for preventing disease, vaccine for boosters was unavailable during 2011, which might be a reason for the increased cases.

Diagnosis of Kyasanur forest disease by nested RT-PCR, real-time RT-PCR and IgM capture ELISA.

Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases.

Salivary gland transcriptome analysis during Plasmodium infection in malaria vector Anopheles stephensi

BACKGROUND Understanding the tissue-specific molecular cross-talk mechanism during the mosquito-parasite interaction is of prime importance in the design of new strategies for malaria control. Because mosquito salivary glands are the final destination for the parasite maturation and transmission of vector-borne diseases, identification and characterization of salivary genes and their products are equally important in order to access their effect on the infectivity of the parasite. During the last five years there have been several studies on the sialomes of Anopheles mosquitoes, however very limited information is available on the changes in the salivary gland transcriptome in the presence of Plasmodium, and this information is limited to the mosquito Anopheles gambiae. METHODS In this study we aimed to explore and identify parasite-induced transcripts from the salivary glands of Anopheles stephensi, using a subtractive hybridization protocol. RESULTS Ninety-four percent of expressed sequence tags (ESTs) showed close homology to previously known families of mosquito salivary gland secretary proteins, representing the induced expression of alternative splicing and/or additional new members of the protein family. The remaining 6% of ESTs did not yield significant homology to any known proteins in the non-redundant database and thus may represent a class of unknown/novel salivary proteins. Primary analysis of the ESTs also revealed identification of several novel immune-related transcripts, including defensin and cecropins, probably involved in counter-activation of the antagonistic defense system. A comprehensive description of each family of proteins has been discussed in relation to the tissue-specific mosquito-parasite interaction. CONCLUSION This is the first report on the identification of new putative salivary genes, presumably activated during parasite infection.

Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010–2011

A nosocomial outbreak of Crimean Congo hemorrhagic fever (CCHF) was reported among humans in Ahmadabad district, Gujarat, India during January, 2011. In the present study we provide the complete genomic sequences of four CCHFV isolates derived from two human patients and two pools of Hyalomma anatolicum ticks during the period of this outbreak and the complete S segment sequence of two retrospective human serum samples, positive for CCHFV in 2010. Sequence-based molecular characterization of the Indian CCHFV showed that they possessed the functional motifs known to occur in the S, M and L gene segment products as in other CCHF viruses. The S segment of the six Indian CCHF viruses showed 99.8% nucleotide identity. Notably both tick isolates shared 100% nucleotide identity with one of the Indian human isolates of 2011. Phylogenetic analysis based on the S segment demonstrated that the Indian CCHFV isolates formed a distinct cluster in the Asian-Middle East group IV of CCHF viruses. The S segment was closest to a Tajikistan strain TADJ/HU8966 of 1990 (98.5% nucleotide identity) and was of South-Asia 2 type while the M segment was of type M2. Both M and L segments were closest to an Afghanistan strain Afg09-2990 of 2009 (93% and 98% nucleotide identity, respectively). The Indian isolates were thus identified as a South-Asia 2/M2 far-east virus combination and the differing parental origin in the S and L/M segments is suggestive that it may be an intra-genotypic reassortant. Molecular clock studies further revealed that the ancestry of the viruses was not very recent and dated back to about 33years on the basis of the S segment while it was about 15years based on the M segment. Thus though the 2011 outbreak may not have resulted from a very recent introduction, considering that so far there is no evidence of multiple circulating strains in the country, the possibility of a recent re-introduction of the virus from any of the neighboring countries cannot be ruled out. The study thus warrants the need for continued surveillance and increased sampling of CCHFV in different parts of the country.

Buffalopox outbreak in humans and animals in Western Maharashtra, India.

An outbreak of febrile illness with rash was reported in humans and buffaloes with pox lesions in some villages of Solapur and Kolhapur districts of Maharashtra state, India. Detailed clinico-epidemiological investigations were done with collection of blood, vesicular fluid and scab from humans and animals. A total of 166 suspected human cases from Kasegaon village in Solapur district and 185 cases were reported from 21 different villages from Kolhapur district. The attack rate in humans in Kasegaon village was 6.6% while in Kolhapur district the attack rate for buffaloes was 11.7%. Pox-like lesions were associated with fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy in the humans. Infected buffaloes had lesions on teats, udders, external ears and eyelids. Laboratory investigations included detection of Buffalopox virus (BPXV) by electron microscopy (EM), virus isolation and polymerase chain reaction (PCR). Presence of BPXV was confirmed in 7 human cases and one buffalo in Kasegaon and 14 human cases from Kolhapur. The virus was isolated from 3 clinical specimens and Orthopoxvirus (OPXV) particles could be observed in EM. Thus, BPXV was identified as the etiological agent of the outbreak among both humans and buffaloes. Phylogenetic analysis based on the ATI and C18L gene revealed that a single strain of virus is circulating in India. Re-emergence of OPXV like BPXV is a real danger and contingency planning is needed to define prophylactic and therapeutic strategies to prevent or stop an epidemic. Considering the productivity losses caused by buffalopox infection and its zoonotic impact, the importance of control measures in reducing the economic and public health impact cannot be underestimated.