Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

Infectious dose-dependent accumulation of live highly pathogenic avian influenza H5N1 virus in chicken skeletal muscle-implications for public health.

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross-boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non-fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (102 , 103 and 104 EID50 ) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 102 EID50 ) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post-infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses.

Survivability of low pathogenic (H9N2) avian influenza virus in water in the presence of Atyopsis moluccensis (Bamboo shrimp).

Low pathogenic avian influenza virus (LPAIV) exhibits an ecological climax with the aquatic ecosystem. The most widely prevalent subtype of LPAIV is H9N2. Wild aquatic birds being the natural reservoirs and ducks, the "Trojan horses" for Avian Influenza Virus (AIV), can contaminate the natural water bodies inhabited by them. The virus can persist in the contaminated water from days to years depending upon the environmental conditions. Various aquatic species other than ducks can promote the persistence and transmission of AIV; however, studies on the role of aquatic fauna in persistence and transmission of avian influenza virus are scarce. This experiment was designed to evaluate the survivability of H9N2 LPAIV in water with and without Atyopsis moluccensis (bamboo shrimp) for a period of 12 days. The infectivity and amount of virus in water were calculated and were found to be significantly higher in water with A. moluccensis than in water without A. moluccensis. The study also showed that A. moluccensis can accumulate the virus mechanically which can infect chicken eggs up to 11 days. The virus transmission potential of A. moluccensis requires further studies.

Survivability of Highly Pathogenic Avian Influenza H5N1 Virus in Poultry Faeces at Different Temperatures.

Highly pathogenic avian Influenza (HPAI) is an important zoonotic disease and is becoming a great threat to poultry industry. India has experienced continual outbreaks of H5N1 HPAI virus since February, 2006 especially in Eastern India. Survivability in poultry faeces is an important determinant in evaluating the persistence of the virus in the poultry sheds and their vicinity. In this paper, survivability of Indian H5N1 HPAI virus in dry and wet poultry faeces at 42, 37, 24 and 4 °C, respectively is reported. The effect of different temperatures was determined by linear regression model and defined in terms of linear equation. The virus survived up to 18 h at 42 °C, 24 h at 37 °C, 5 days at 24 °C and 8 weeks at 4 °C in dry and wet faeces, respectively. The coefficients of determination (R(2)) values for dry and wet faeces revealed that the difference in viral persistence in dry and wet faeces at all temperatures was not very marked. Results of the present study indicated that H5N1 HPAI virus may remain viable for extended periods of time in faeces at low temperatures and may act as a long term source of influenza virus in the environment.

Nipah virus infection: current scenario.

The emergence of Nipah virus (NiV) infection into the pig population and subsequently into the human population is believed to be due to changes in ecological conditions. In Malaysia, A major NiV outbreak occurred in pigs and humans from September 1998 to April 1999 that resulted in infection of 265 and death of 105 persons. About 1.1 million pigs had to be destroyed to control the outbreak. The disease was recorded in the form of a major outbreak in India in 2001 and then a small incidence in 2007, both the outbreaks in West Bengal only in humans without any involvement of pigs. There were series of human Nipah incidences in Bangladesh from 2001 till 2013 almost every year with mortality exceeding 70 %. The disease transmission from pigs acting as an intermediate host during Malaysian and Singapore outbreaks has changed in NIV outbreaks in India and Bangladesh, transmitting the disease directly from bats to human followed by human to human. The drinking of raw date palm sap contaminated with fruit bat urine or saliva containing NiV is the only known cause of outbreak of the disease in Bangladesh outbreaks. The virus is now known to exist in various fruit bats of Pteropus as well as bats of other genera in a wider belt from Asia to Africa.

Isolation and characterization of influenza A virus (subtype H5N1) that caused the first highly pathogenic avian influenza outbreak in chicken in Bhutan.

We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other "Qinghai-like" H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids ((222)Q and (224)G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1-100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds.

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.

Genetic characterization of vaccine and field strains of serotype A foot-and-mouth disease virus from India.

Extreme antigenic and genetic heterogeneity of serotype A foot-and-mouth disease virus (FMDV) population has resulted in change of vaccine strains in India twice in the last decade. In such a situation, complete characterization of the vaccine strains is imperative. With regard to the frequent outbreaks of this disease, FMDV field strains are also of interest. Therefore three vaccine strains and two field strains of type A FMDV from India were completely sequenced and the obtained sequences were subjected to sequence and phylogenetic analyses. Based on the complete coding region, all the Indian strains clustered in the Asia topotype and exhibited a more than 11% nt divergence from the other Asian strains. The 5'-UTR of some Indian strains revealed block deletions of 43 and 86 nt corresponding to the pseudoknot region. Amino acids S44 in VP2 and F164 in VP1 were found to be the exclusive signatures for the Asia topotype. The vaccine strains differed at 65 aa positions in the capsid region, 13 of them antigenically critical. Variability at such positions is likely to affect the antigenic profile of these strains. Complete genome sequences of the vaccine strains presented here could serve as the reference for any comparative genomics in future.

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.

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.

Influence of dose of inocula on outcome of clinical disease in highly pathogenic avian influenza (H5N1) infections--an experimental study.

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.

Constraints on adaptation: explaining deviation from optimal sex ratio using artificial neural networks.

Determining processes constraining adaptation is a major challenge facing evolutionary biology, and sex allocation has proved a useful model system for exploring different constraints. We investigate the evolution of suboptimal sex allocation in a solitary parasitoid wasp system by modelling information acquisition and processing using artificial neural networks (ANNs) evolving according to a genetic algorithm. Theory predicts an instantaneous switch from the production of male to female offspring with increasing host size, whereas data show gradual changes. We found that simple ANNs evolved towards producing sharp switches in sex ratio, but additional biologically reasonable assumptions of costs of synapse maintenance, and simplification of the ANNs, led to more gradual adjustment. Switch sharpness was robust to uncertainty in fitness consequences of host size, challenging interpretations of previous empirical findings. Our results also question some intuitive hypotheses concerning the evolution of threshold traits and confirm how neural processing may constrain adaptive behaviour.

Isolation and molecular characterization of a H5N1 virus isolated from a Jungle crow (Corvus macrohynchos) in India.

In 2008, India experienced widespread outbreaks of H5N1 virus in West Bengal, Tripura, and Assam. The virus was detected in Kamrup district of Assam in November 2008 and subsequently spread to eight more districts. Two Jungle or Large billed crows (Corvus macrohynchos) were found dead in a hospital campus at about 8 km from the foci of initial detection of the virus in the same district. One of the crows was positive for H5N1 avian influenza virus by virus isolation, real time RT-PCR, and RT-PCR tests. Full length sequencing of all the eight segments of the virus was carried out. The phylogenetic analysis indicated that all the eight genes grouped with clade 2.2 viruses and were closely related to the human isolate of Bangladesh and avian isolates from India, Bangladesh, Kuwait, Germany, and Saudi Arabia. The molecular analysis indicated avian receptor (alpha 2,3 sialic acid) specificity, susceptibility to oseltamivir and amantadine group of antivirals and lower pathogenicity to mice.

Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

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.

Analysis of the PB2 gene reveals that Indian H5N1 influenza virus belongs to a mixed-migratory bird sub-lineage possessing the amino acid lysine at position 627 of the PB2 protein.

Outbreaks of highly pathogenic avian influenza (HPAI) H5N1 virus were reported for the first time in India during February 2006. Herein, we have sequenced and analyzed the PB2 genes of five influenza virus isolates obtained from three affected states (Gujarat, Madhya Pradesh and Maharashtra) in India during the outbreaks. In the phylogenetic analysis, the Indian isolates were grouped in the mixed-migratory bird sub-lineage of the Eurasian lineage. From the phylogenetic tree, it is evident that viruses were probably introduced to India from China via Europe because they share a direct ancestral relationship with the Indian isolates. The virus might have spread through migratory waterfowls that survived the HPAI H5N1 infection. These viruses were able to replicate in cultured cells of avian and mammalian hosts and posses lysine at position 627 of the PB2 protein, indicating that they might be able to cross the host barrier to infect mammals.