H. V. Murugkar

Avian Influenza (H5N1) Virus of Clade 2.3.2 in Domestic Poultry in India

South Asia has experienced regular outbreaks of H5N1 avian influenza virus since its first detection in India and Pakistan in February, 2006. Till 2009, the outbreaks in this region were due to clade 2.2 H5N1 virus. In 2010, Nepal reported the first outbreak of clade 2.3.2 virus in South Asia. In February 2011, two outbreaks of H5N1 virus were reported in the State of Tripura in India. The antigenic and genetic analyses of seven H5N1 viruses isolated during these outbreaks were carried out. Antigenic analysis confirmed 64 to 256-fold reduction in cross reactivity compared with clade 2.2 viruses. The intravenous pathogenicity index of the isolates ranged from 2.80–2.95 indicating high pathogenicity to chickens. Sequencing of all the eight gene-segments of seven H5N1 viruses isolated in these outbreaks was carried out. The predicted amino acid sequence analysis revealed high pathogenicity to chickens and susceptibility to the antivirals, amantadine and oseltamivir. Phylogenetic analyses indicated that these viruses belong to clade 2.3.2.1 and were distinct to the clade 2.3.2.1 viruses isolated in Nepal. Identification of new clade 2.3.2 H5N1 viruses in South Asia is reminiscent of the introduction of clade 2.2 viruses in this region in 2006/7. It is now important to monitor whether the clade 2.3.2.1 is replacing clade 2.2 in this region or co-circulating with it. Continued co-circulation of various subclades of the H5N1 virus which are more adapted to land based poultry in a highly populated region such as South Asia increases the risk of evolution of pandemic H5N1 strains.

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003–2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R–S–S–R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R–S–N–R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.

Emergence of amantadine-resistant avian influenza H5N1 virus in India

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008–2009) and avian and human isolates from Bangladesh (2007–2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.

Outbreak of avian influenza virus H5N1 in India

SIR, — The highly pathogenic avian influenza (hpai) virus subtype h5n1 has become endemic in poultry populations in south-east Asia since 2003. During the second week of July 2007, an unusual mortality of 82 per cent was reported in a flock of 132 chickens on a poultry farm in Manipur, northeast

Novel Reassortant Highly Pathogenic Avian Influenza (H5N8) Virus in Zoos, India

Highly pathogenic avian influenza (H5N8) viruses were detected in waterfowl at 2 zoos in India in October 2016. Both viruses were different 7:1 reassortants of H5N8 viruses isolated in May 2016 from wild birds in the Russian Federation and China, suggesting virus spread during southward winter migration of birds.

Avian influenza virus (H5N1) in chickens in India

In India, outbreaks of avian influenza H5N1 in chickens were previously confirmed in February 2006 (in commercial and backyard units), July 2007 (in a single backyard unit) and January 2008 (mainly in backyard units). All these outbreaks were recorded in different geographical areas and were

Phylogenetic evidence of multiple introduction of H5N1 virus in Malda district of West Bengal, India in 2008

Outbreaks of H5N1 avian influenza virus were reported in 15 districts of West Bengal State in India in early 2008 and subsequent re-occurrence in 5 districts in December, 2008 to May, 2009. We have sequenced complete genome of 12 viruses isolated from early 2008 outbreak and from recurrent outbreak and determined the phylogenetic relationship between the viruses isolated from the two outbreaks. One of the virus isolated in early 2008 from Malda district (A/chicken/West Bengal/81760/2008) clustered with Korean and Russian isolates of 2006 in European-Middle Eastern-African (EMA) 3 sub-lineage of sub-clade 2.2, whereas other viruses showed close genetic relationship with 2007-2009 isolates of Bangladesh. Nucleotide sequence analysis revealed that the PB1-F2 protein expression might be completely abolished due to mutated start codon ((95)ATG(97)→(95)ACG(97)) in this isolate but in all other isolates it was completely expressed. Hence, we conclude that there were two separate introductions of H5N1 viruses in Malda district and this H5N1 virus was not epidemiologically dominant as the viruses isolated subsequently from the same district and region did not share close relationship with this virus. The failure of this virus to spread to adjoining areas suggests that the culling and disposal operations initiated by Government of India were effective.

Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.

A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 μl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.

Influence of Dose of Inocula on Outcome of Clinical Disease in Highly Pathogenic Avian Influenza (H5N1) Infections—An Experimental Study

Abstract Twelve-week-old Vanaraja (an Indian native dual purpose breed) chickens were inoculated intranasally with different doses (100, 1000, and 10,000 mean embryo infective dose [EID50]) of H5N1 virus, and the clinical disease and pathologic changes were compared. Although the overall severity of clinical signs was more severe in the 100 EID50 group, the progression of the clinical disease was slower with delayed onset of mortality when compared with the other two groups. The mean death time of the 100 EID50 group (4.57 days) differed significantly from that of the 10,000 EID50 group (3.60 days) and from that of the 1000 EID50 group (3.33 days). Similarly, overall severity of gross lesions was expressed more in the 100 EID50 group. The histopathologic lesions were of a more hemorrhagic and necrotic nature in the 100 EID50 group, histopathologic lesions were of an inflammatory/proliferative nature in the 1000 EID50 group, and a tendency for intravascular coagulopathy was observed in the 10,000 EID50 group. These differences may be assigned to the influence of dose in the outcome of disease.

The Efficiency of HEPA Filters in the Air-handling System of a Bio-containment Laboratory in India

The High Security Animal Disease Laboratory (HSADL) in Bhopal, India is a BSL-4 laboratory that was constructed following the Labystad (The Netherlands) model and was commissioned in 1997 as a bio-containment facility to undertake research and diagnostic work on exotic and emerging diseases of animals in the country. The laboratorys air-handling system is comprised of 23 air-handling units (AHUs) with 97 HEPA filters (Anfilco, India) fitted in 92 filter housings. Preventing environmental contamination is achieved by maintaining graded negative pressure ranging between −50 to −200 Pascals. This paper discusses the efficiency of the HEPA filters following the dioctylphthalate (DOP) testing carried out over a decade-long period. Out of the 81 (83.5%) filters replaced during this period, HEPA filters of 59 (72.83%) housings were replaced once; of 17 housings (20.98%) were replaced twice, and of 6 housings (7.4%) were replaced three times. Major reasons for the replacement of these filters include blockage (48.18%) and lowered efficiency (51.85%) of the filter medium. The majority of the filter changes at HSADL can be attributed to the routine ageing of the filters, resulting in the degraded mechanical strength of the filter medium or choking due to aerosols and dust. Some areas of the laboratory encountered a higher rate of filter change and the reasons thereof are discussed. These observations regarding HEPA filter efficiency in AHUs are probably the first in a tropical environment. The possible roles of factors such as the availability of a dense tree canopy in the direction of the air flow, the close location of the lab to a large water body, and the labs distance from the dusty highways in the longevity of this laboratorys HEPA filters are also discussed. This study re-emphasizes the need for regular monitoring of the HEPA filter function in the AHUs and their prompt replacement when damaged to ensure effective bio-containment.