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.

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.

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.

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.

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.

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.

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.

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.

Genetic and antigenic characterization of bovine viral diarrhea virus type 2 isolated from Indian goats (Capra hircus).

Recent studies have shown that bovine viral diarrhea virus (BVDV) type 1 is widely prevalent in Indian cattle. In a surveillance of randomly collected 562 blood samples from seven states during 2004-2006, BVDV type 2 was detected in two native Indian goats by nested reverse transcription polymerase chain reaction (nRT-PCR). The virus isolated from them was classified antigenically as BVDV 2 on the basis of virus neutralization test and reactivity with monoclonal antibodies. Phylogenetic analysis of three different genomic regions, 5' un-translated region (5' UTR), E(rns) structural coding region and NS5B nonstructural coding region typed Indian goat isolate as BVDV 2a having close similarity with strains from North America and Europe suggesting its probable introduction through trade. It was placed in a separate clade within the 2a branch having unique mutations in E(rns) and NS5B region. This is the first report of BVDV 2 in India and only second time recorded in goat species. The isolation of BVDV 2 from goat warrants intensive surveillance in cattle and sheep.

Sequence analysis of the non-structural 3A and 3C protein-coding regions of foot-and-mouth disease virus serotype Asia1 field isolates from an endemic country.

A total of 18 foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates belonging to two different lineages (including the divergent group) as delineated earlier in VP1-based phylogeny were sequenced in the non-structural 3A and 3C protein-coding regions. The phylogenetic trees representing the regions coding for the non-structural proteins were very similar to that of the structural VP1 protein-coding region. Phylogenetic comparison at 3C region revealed clustering of Asia1 viruses with the isolates of serotypes O, A and C in the previously identified clade. Comparison of amino acid sequences identified lineage-specific signature residues in both the non-structural proteins. Overall analysis of the amino acid substitutions revealed that the 3A coding region was more prone to amino acid alterations than 3C region.

Genetic heterogeneity of Indian field isolates of foot-and-mouth disease virus serotype O as revealed by partial sequencing of 1D gene.

The sequence of 165 nucleotides at the 3' end of the 1D gene, determined from RT PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987 1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/ field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3 20.6%) from the exotic type O strains of O1BFS, O1K, and O1Campos. The type O strains analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 71% divergent (7.3-15.2%) from the field strains which revealed significant ( > 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of India, the viruses/ sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149-->E and another from P208-->L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogeneous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it.

Antigenic characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates (n = 100) were compared using a panel of 11 monoclonal antibodies (Mab) in sandwich ELISA. The majority (over 89%) of the isolates showed either homologous (76% and above reactivity) or reduced affinity (20-75% reactivity) for the Mabs 2A, 13, 40, 34 and 81, suggesting that these Mab binding epitopes are conserved, whereas a more variable reactivity was observed for the Mabs B3, 1A, 24, 72, 82 and 89. Polyclonal relationship ('r' value) of the field isolates in liquid phase blocking (LPB) ELISA was examined, and the mean 'r' value was 0.62 relative to vaccine virus IND 63/72. Some of the field isolates (n = 34) were tested in virus neutralization test (VNT) and showed an 'r' value of >0.40. Although a minor antigenic difference was observed in the Mab profiling study, there has not been large antigenic divergence between reference virus and field viruses, thereby providing evidence of wide antigenic coverage of the vaccine strain.

Serotype C foot-and-mouth disease virus isolates from India belong to a separate so far not described lineage.

Complete 1D gene sequences of 13 Indian foot-and-mouth disease virus (FMDV) type C field isolates and a vaccine strain (C-Bombay/64) were determined. All the field isolates showed a greater genetic homogeneity (95-100%) among themselves and were 19.7-21.2% divergent from the vaccine strain. In the phylogenetic analysis, the Indian field isolates formed a separate lineage (lineage VII) different from the previously identified six lineages (lineage I-VI) in type C FMDV [J. Virol. 66 (1992) 3557]. The vaccine strain was grouped with European lineage (lineage II). Comparison of the deduced amino acid sequences of antigenic sites A and C of field isolates showed no significant variation from the vaccine strain. One-way serological relationship determined in ELISA showed antigenic closeness of the field isolates with C-Bombay/64.

Emergence of a novel subgroup within the widely circulating lineage of foot-and-mouth disease virus serotype Asia 1 in India.

The complete VP1 encoding (1D) gene of 54 foot-and-mouth disease (FMD) virus serotype Asia1 field isolates, most of which were isolated during 2000 and 2001, was sequenced. The phylogenetic analysis identified a novel subgroup (>10% nucleotide divergence) within the widely circulating lineage of this serotype. The newly emerged viruses were responsible for disease outbreaks in both cattle and buffaloes and were present in six different states in the country. Amino acid sequence comparison of these isolates revealed significant sequence divergence at many of the amino acid positions in comparison to those of lineage VI-A and C. Emergence of such viruses may affect the efficacy of vaccine strain currently used for protection against FMD in India.

Sequence variability in the structural protein-encoding region of foot-and-mouth disease virus serotype Asia1 field isolates.

A total of 30 field isolates of foot-and-mouth disease virus (FMDV) serotype Asia1 belonging to two different lineages and five isolates belonging to a divergent group as delineated earlier in 1D (encodingVP1 protein) gene-based phylogeny were sequenced in the structural protein (P1) coding region. Phylogenetic comparison of these isolates along with some of the published exotic sequences revealed the presence of five different lineages around the world. Similar grouping pattern was observed for the P1 region and 1D gene-based phylogeny, where the Indian isolates were clustered in two genetic lineages. The recently identified divergent group of virus falls into a separate sub-cluster. Similar grouping was also observed in L gene-based phylogeny. Comparison of amino acid sequences identified lineage-specific signature residues in all the structural proteins. Comparison of Asia1 field isolates at the identified key residues of other FMD viruses involved in the formation of the heparan sulfate-binding ligand confirmed many of them to be conserved and the presence of VP3(56) Arg suggested their cell culture adaptation. Although a considerable genetic variation was observed among the isolates of present study, all of them tested in micro-neutralization test were serologically related to the vaccine strain.

Comparison of amino acid sequences at the amino acid 130-160 region of VP1 polypeptide of Indian field isolates of foot-and-mouth disease virus serotype Asia 1.

The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.

One-tube and one-buffer system of RT-PCR amplification of 1D gene of foot-and-mouth disease virus field isolates.

A method of reverse transcription (RT) and polymerase chain reaction (PCR) amplification of 1D (VP1) gene of foot-and-mouth disease (FMD) virus using one reaction mixture containing both avian myeloblastosis virus (AMV) reverse transcriptase (RTase) and Tfl DNA polymerase is described. The procedure was time saving, made use of a single buffer for both RT and subsequent amplification and performed better than the two-step procedure usually conducted with Moloney murine leukemia virus (MMLV) RTase and Taq DNA polymerase for amplification of the VP1 gene of field isolates of FMD virus serotypes O,-A, C and Asia 1. The failure to amplify the VP1 gene of many type O and Asia 1 viruses using MMLV RTase-Taq polymerase enzyme system could be overcome by performing RT of the viral genome at a higher temperature (48 degrees C) with AMV RTase which is not possible with MMLV RTase.