Evidence of hepatitis A virus infection in the patients with acute encephalitis syndrome in Gorakhpur region, North India.

The etiological agent remained unidentified in a large number of patients hospitalized for acute encephalitis syndrome (AES) in 2008-2009 in Uttar Pradesh and Bihar, north India. All patients were found to present with fever and altered sensorium, while 28%, 19% and 13% showed hepatomegaly, splenomegaly and meningeal signs, respectively. Involvement mostly of children with abnormal hepatic features prompted us to undertake an exploratory study on viral hepatitis A to determine its association, if any, with hepatic derangements. AES patients (n = 2515) and healthy children (n = 167) were investigated for the presence of serum anti-hepatitis A virus (anti-HAV) IgM and anti-Japanese encephalitis (anti-JE) virus IgM by ELISA. Cerebrospinal fluids (CSFs, n = 595) and rectal swabs (n = 182) were examined for anti-HAV IgM and/or HAV RNA. Anti-HAV IgM was detected in the sera of 14.6% patients as against 6.6% of healthy children (p = 0.0042). Anti-JE virus IgM positivity was Keywords: acute encephalitis syndrome; cerebrospinal fluid; hepatitis A virus; anti-HAV IgM; non-Japanese encephalitis.

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae) from Sergentomyia species of sandflies from Nagpur, Maharashtra, India.

BACKGROUND & OBJECTIVES: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra s0 tate, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. METHODS: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. RESULTS: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. INTERPRETATION & CONCLUSIONS: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Antiviral drug profile of human influenza A & B viruses circulating in India: 2004-2011.

BACKGROUND & OBJECTIVES: Recent influenza antiviral resistance studies in South East Asia, Europe and the United States reveal adamantane and neuraminidase inhibitor (NAIs) resistance. This study was undertaken to evaluate antiviral resistance in influenza viruses isolated from various parts of India, during 2004 to 2011. METHODS: Influenza viruses were analyzed genetically for known resistance markers by M2 and NA gene sequencing. Influenza A/H1N1 (n=206), A/H3N2 (n=371) viruses for amantadine resistance and A/H1N1 (n=206), A/H3N2 (n=272) and type B (n=326) for oseltamivir resistance were sequenced. Pandemic (H1N1) (n=493) isolates were tested for H274Y mutation by real time reverse transcription (rRT)-PCR. Randomly selected resistant and sensitive influenza A/H1N1 and A/H3N2 viruses were confirmed by phenotypic assay. RESULTS: Serine to asparagine (S3IN) mutation was detected in six isolates of 2007-2008. One dual-resistant A/H1N1 was detected for the first time in India with leucine to phenylalanine (L26F) mutation in M2 gene and H274Y mutation in NA gene. A/H3N2 viruses showed an increase in resistance to amantadine from 22.5 per cent in 2005 to 100 per cent in 2008 onwards with S3IN mutation. Fifty of the 61 (82%) A/H1N1 viruses tested in 2008-2009 were oseltamivir resistant with H274Y mutation, while all A/H3N2, pandemic A/H1N1 and type B isolates remained sensitive. Genetic results were also confirmed by phenotypic analysis of randomly selected 50 resistant A/H1N1 and 40 sensitive A/H3N2 isolates. INTERPRETATION & CONCLUSIONS: Emergence of influenza viruses resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for continuous monitoring of antiviral resistance.

Influenza virus genotypes circulating in and around Lucknow, Uttar Pradesh, India, during post pandemic period, August 2010--September 2012.

BACKGROUND & OBJECTIVES: During the post influenza pandemic period, continuous surveillance of influenza virus and its subtypes is mandatory to help the policy makers to take effective and appropriate decisions. Therefore, this study was planned to determine the pattern of influenza virus activity in context to various meteorological and clinical parameters in and around Lucknow, Uttar Pradesh, India, during post pandemic period August 2010 - September 2012. METHODS: Nasal swabs/throat swabs/nasopharyngeal aspirates of 2669 patients were collected. One-step real time PCR for detection of influenza virus was done according to the Centers for Disease Control and Prevention (CDC) protocol. RESULTS: Influenza positivity was 15.8 per cent (423/2669) in symptomatic patients. Of the 423 total positives, 192 (7.2%) were influenza A and 231 (8.7%) were influenza B. Positivity for influenza virus was significantly (P=0.001, OR=2.9, CI=1.9-4.3) higher in patients with Influenza like illness (ILI) (17.4%, 396/2271) than those with severe acute respiratory illness (SARI) (6.8%, 27/398). Influenza A positive samples were subtyped as; pdmH1N1 (67.2%, 129/192) and seasonal H3N2 (32.8%, 63/192). It significantly correlated with monthly mean rainfall, humidity and dew point while atmospheric pressure was inversely related. No significant association was found with temperature and wind speed. Clinical variations were observed between different strains of Influenza virus. INTERPRETATION & CONCLUSIONS: The findings provide a clear picture of different clinical presentations of various strains of influenza A and B viruses and epidemiology of influenza infection from Lucknow (UP), India. The seasonality of influenza virus infection showed variation in relation to different environmental factors. Pandemic H1N1 caused more systemic infection than seasonal influenza A/H3N2 virus.

Isolation of a novel adenovirus from Rousettus leschenaultii bats from India.

Surveillance work was initiated to study the presence of highly infectious diseases like Ebola-Reston, Marburg, Nipah and other possible viruses that are known to be found in the bat species and responsible for causing diseases in humans. A novel adenovirus was isolated from a common species of fruit bat (Rousettus leschenaultii) captured in Maharashtra State, India. Partial sequence analysis of the DNA polymerase gene shows this isolate to be a newly recognized member of the genus Mastadenovirus (family Adenoviridae), approximately 20% divergent at the nucleotide level from Japanese BatAdV, its closest known relative.

Molecular characterization of Chittoor (Batai) virus isolates from India.

BACKGROUND & OBJECTIVES: Chittoor virus (CHITV) belongs to genus Orthobunyavirus, family Bunyaviridae. It has been isolated from various species of mosquitoes and pig from different parts of India. Five isolates of CHITV were characterized at the molecular level and compared with other Batai viruses (BATV) to find out any kind of reassortment in their genome. METHODS: Complete nucelocapsid (S), glycoprotein (M) and partial RNA polymerase (L) segments of CHITV were amplified and sequenced. These sequences were compared with those of Batai viruses, isolated from different geographical locations in Asia, Africa and Europe. RESULTS: Phylogenetic analysis revealed CHITV as a variant of BATV. High level of conservation was seen among the CHITV isolates studied. The CHITV sequences showed clustering in one lineage with the sequences from Japan and Malaysia, however, BATV sequences from Europe and Africa formed a separate phylogenetic lineage. INTERPRETATION & CONCLUSIONS: The study indicates the presence of a single genotype of CHITV circulating in India, despite the involvement of different hosts in the natural cycle by this virus. Analysis of the sequences of the S, M and L segments of genome indicated that the virus has not undergone any reassortment. This virus has not caused any epidemic involving humans, however, replication of the virus in different mosquito and vertebrate hosts species suggests that it is a cause of concern.

Molecular epidemiology of measles in India, 2005-2010.

Measles is a childhood disease that causes great morbidity and mortality in India and worldwide. Because measles surveillance in India is in its infancy, there is a paucity of countrywide data on circulating Measles virus genotypes. This study was conducted in 21 of 28 States and 2 of 7 Union Territories of India by MeaslesNetIndia, a national network of 27 centers and sentinel practitioners. MeaslesNetIndia investigated 52 measles outbreaks in geographically representative areas from 2005 through June 2010. All outbreaks were serologically confirmed by detection of antimeasles virus immunoglobulin M (IgM) antibodies in serum or oral fluid samples. Molecular studies, using World Health Organization (WHO)-recommended protocols obtained 203 N-gene, 40 H-gene, and 4 M-gene sequences during this period. Measles genotypes D4, D7, and D8 were found to be circulating in various parts of India during the study period. Further phylogenetic analysis revealed 4 lineages of Indian D8 genotypes: D8a, D8b, D8c, and D8d. This study generated a large, countrywide sequence database that can form the baseline for future molecular studies on measles virus transmission pathways in India. This study has created support and capabilities for countrywide measles molecular surveillance that must be carried forward.

Investigation of a Chikungunya-like illness in Tirunelveli district, Tamil Nadu, India 2009-2010.

OBJECTIVE: To identify the aetiological agent/s of an outbreak of chikungunya-like illness with high morbidity and several fatalities in Tamil Nadu, India, 2009-2010. METHODS: Two hundred and seventeen serum samples were collected from the affected areas and screened for chikungunya virus (CHIKV), dengue virus (DENV) and Japanese encephalitis virus (JEV) IgM antibodies using MAC-ELISA kits. A few selected samples were also tested for Ross River, Sindbis, and Murrey Valley viruses by RT-PCR and Hantan virus by serology. Twelve acute serum and mosquito samples were processed for virus isolation in C6/36 cells. CHIKV isolate was characterised by RT-PCR and sequencing. RESULTS: Diagnostic levels of IgM antibodies were detected in 107 (49.3%) CHIKV samples and 22 (10.1%) DENV samples. IgM antibodies against JEV were not detected (n=46). Characterisation of the CHIKV isolate at genetic level demonstrated it as ECSA (E1: 226A). Thirty-six selected samples were also negative for Ross River, Sindbis, Murrey Valley and Hantan viruses. CONCLUSION: High prevalence of CHIKV IgM antibody positivity, clinical symptoms, virus isolation and the presence of vector mosquitoes clearly suggest CHIKV as the aetiological agent responsible for the outbreak.

Retrospective analysis of necropsy findings in patients of H1N1 and their correlation to clinical features.

India reported its first case of H1N1 in July 2009 in Pune and since then, the number of reported cases and deaths exploded in India. Since very little data is available about histopathological findings in patients of H1N1 fatal cases in India, a retrospective chart analysis of necropsy findings of 15 cases of 2009 H1N1 fatal cases was performed. Common clinical features were fever, cough, and breathlessness followed by sore throat and rhinorrhea. Common lung findings were mononuclear cell infiltration, thick alveolar septae, intraalveolar hemorrhage. The other findings were congested pulmonary blood vessels, pulmonary edema, cytomegaly, fibrin accumulation and formation of eosinophilic membrane. These findings are suggestive of diffuse alveolar damage (DAD) and DAD with hemorrhage. All patients who underwent necropsy had radiographic findings suggestive of unilobar or multilobar pneumonia. This clinical finding can be correlated pathologically in these patients as all of them had either polymorphonuclear or mononuclear infiltrate. Furthermore, necrotizing pneumonitis pattern seen on these patients is the likely cause of mortality in these patients. Although clinical ARDS pattern was noted in all these patients, it was well correlated in lung pathology in all these cases.

Complete genome characterization and evolutionary analysis of dengue viruses isolated during 2016-2017 in Pune, India.

Dengue is the most common mosquito-borne viral infection in tropical and sub-tropical countries. In the recent years, frequent dengue outbreaks are being reported in many parts of India. DENV circulates as four independent serotypes posing a major public health threat around the globe. Phylogenetic and full genome sequence analyses of 19 complete DENV genome sequences presenting all the four serotypes in Pune, India (2016-2017) revealed no change in the circulating genotypes i.e., genotype V clade C (D1), genotype IVB (D2), genotype III lineage III (D3) and genotype I clade D (D4). Additionally, unique amino acid substitutions that may potentially influence viral fitness and virulence in host cells were identified. Mapping of the unique amino acid substitutions onto the T cell epitopes of the reference strains revealed that 8/10 (D1), 14/15 (D2), 3/4 (D3) and 21/74 (D4), amino acids were involved in T-cell epitope presentation for a maximum number of HLA alleles associated with disease outcome. Selection pressure analysis documented a positive selection pressure to be acting on few amino acid sites indicating continuous evolutionary changes in the viral RNA. Overall, the evolutionary and selection pressure data generated during this study may help in better understanding of DENV evolution and epidemiology.

Immunomodulatory cytokines determine the outcome of Japanese encephalitis virus infection in mice.

Japanese encephalitis virus (JEV) induces an acute infection of the central nervous system, the pathogenic mechanism of which is not fully understood. To investigate host response to JEV infection, 14-day-old mice were infected via the extraneural route, which resulted in encephalitis and death. Mice that received JEV immune splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of mice that either succumbed to JEV infection or were protected from infection by JEV immune cell transfer. Mice undergoing progressive JEV infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN) pathway. In contrast, mice receiving immune cell transfer had increased production of the Th2 cytokine IL-4, and of IL-10, with subdued expression of IFN-gamma. We observed IL-10 to be an important factor in determining clinical outcome in JEV infection. Data obtained by microarray analysis were further confirmed by quantitative RT-PCR. Together, these data suggest that JEV infection causes an unregulated inflammatory response that can be countered by the expression of immunomodulatory cytokines in mice that survive lethal infection.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

BACKGROUND & OBJECTIVES: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. METHODS: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). RESULTS: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. INTERPRETATION & CONCLUSIONS: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Ganjam virus.

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratory-acquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent.

Global distribution of novel rhinovirus genotype.

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

Venereal transmission of Chandipura virus by Phlebotomus papatasi (Scopoli).

Experiments were conducted in the laboratory on Phlebotomus papatasi to determine the possible role of males in maintaining or sustaining the Chandipura virus (CHPV) activity in nature. This study indicated that infected males are capable of passing on the virus to female sand flies while mating. The infection rate was found to be 12.5% in uninfected females when mated with infected males. The occurrence of venereal transmission of this virus may have epidemiologic importance in the natural cycle of CHPV.

A large outbreak of acute encephalitis with high fatality rate in children in Andhra Pradesh, India, in 2003, associated with Chandipura virus.

BACKGROUND: An outbreak of acute encephalitis of unknown origin with high case fatality (183 of 329 cases) was reported in children from Andhra Pradesh state in southern India during 2003. We investigated the causative agent. METHODS: Cell lines and peripheral blood lymphocyte co-cultures were used to isolate the causative agent from clinical samples. Identity of the agent was established by electron microscopy and serological and molecular assays. FINDINGS: Clinical samples tested negative for IgM antibodies to Japanese encephalitis, West Nile, dengue, and measles viruses, and for RNA of coronavirus, paramyxovirus, enterovirus, and influenza viruses. Virus was isolated from six patients with encephalitis and was identified as Chandipura virus by electron microscopy, complement fixation, and neutralisation tests. Chandipura virus RNA was detected in clinical samples from nine patients. Sequencing of five of these RNA samples showed 96.7-97.5% identity with the reference strain of 1965. Chandipura viral antigen and RNA were detected in brain tissue of a deceased child by immunofluorescent antibody test and PCR. Neutralising, IgG, and IgM antibodies to Chandipura virus were present in some patients' serum samples. Serum samples obtained after 4 days of illness were more frequently positive for IgM to Chandipura virus than were those obtained earlier (p<0.001). A similar trend was noted for neutralising antibodies. INTERPRETATION: Our findings suggest that this outbreak of acute encephalitis in Andhra Pradesh was associated with Chandipura virus, adding to the evidence suggesting that this virus should be considered as an important emerging pathogen.

An outbreak of Chandipura virus encephalitis in the eastern districts of Gujarat state, India.

An outbreak of encephalitis with a case fatality rate of 78.3% was investigated among children in Gujarat State, India. Twenty-six cases were reported. Three patients had IgM antibodies to Chandipura virus. Virus was isolated from one patient with rhabdomyosarcoma in porcine stable cell lines and in suckling mice. Chandipura virus RNA was present in 9 of 20 acute-phase serum samples, and virus sequences from the present outbreak were closely related to prototype strain (1965) and Andhra Pradesh, India (2003) isolates. Serologic and molecular assays documented the absence of Japanese encephalitis virus, West Nile virus, dengue virus, and paramyxoviruses in clinical samples. The etiologic agent was Chandipura virus, which has become an important encephalitis-causing virus in India.

Detection of chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India.

A total of 191 adult sand flies belonging to the genus Sergentomyia were collected from seven villages in Karimnagar and Warangal districts of Andhra Pradesh State, India, after an outbreak of encephalitis due to Chandipura virus (CHPV). Fifteen pools, each containing two specimens, were tested by reverse transcriptase-polymerase chain reaction and sequencing. One pool of Sergentomyia from Kolanur village in Karimnagar District was positive for CHPV.

Vertical and venereal transmission of Chandipura virus (Rhabdoviridae) by Aedes aegypti (Diptera: Culicidae).

Experiments in the laboratory documented vertical and venereal transmission of Chandipura virus (CHPV) in Aedes aegypti (L.). The minimum filial infection rate among the progeny of infected females was 1.2%; the rate among male and female progeny was 0.9 and 1.4%, respectively. The venereal infection rate of CHPV among inseminated females was 32.7%. Our study indicates the possible occurrence of vertical and venereal transmission of CHPV in insect vectors.