Highly pathogenic avian influenza (H5N1) infection in crows through ingestion of infected crow carcasses.

The present study was aimed to investigate the role of cannibalism in transmission of H5N1 avian influenza virus to house crows (Corvus splendens). Four crows were intranasally inoculated with 108.0 EID50 (A/crow/India/01CA249/2021) H5N1 highly pathogenic avian influenza (HPAI) virus and were observed for 14 days for any overt signs of illness. Two of the infected crows showed signs of wing paralysis, incoordination, and torticollis. For cannibalism experiment, two crows showing clinical signs were euthanized on 14th day post-infection (dpi) and were kept in the isolator and four naïve healthy crows were introduced along with the euthanized crows. The viscera from the infected carcasses were eaten by all the four crows. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. All four crows showed clinical signs viz., dullness, reluctance to move with ruffled feathers on 6th day post cannibalism along with neurological signs including incoordination and paralysis of the wings. All the crows gradually recovered after showing clinical signs and were euthanized on 21st day of observation period. Virus excretion was observed from 3rd to 11th day post cannibalism through both oropharyngeal and cloacal routes with maximum shedding through oropharyngeal route. The virus was isolated from lungs and trachea of one the infected crows at 21st day after euthanasia. All the four crows seroconverted against H5N1 virus infection at 14th day post cannibalism. Our study confirms the transmission of H5N1 virus in crows through cannibalism and highlights how H5N1 virus might circulate in a crow colony once they become infected.

Experimental Infection and In-Contact Transmission of H9N2 Avian Influenza Virus in Crows.

This study aimed to investigate the potential of H9N2 avian influenza virus to cause disease and intra-species transmission in house crows (Corvus splendens). A group of six crows were intranasally inoculated with 106.0 EID50 of H9N2 virus (A/chicken/India/07OR17/2021), and 24 h post-inoculation six naïve crows were co-housed with infected crows. Crows were observed for 14 days for any overt signs of illness. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. No apparent clinical signs were observed in either infected or in-contact crows. Virus excretion was observed only in infected birds up to 9 days post-infection (dpi) through both oropharyngeal and cloacal routes. All six infected crows seroconverted to H9N2 virus at 14 dpi, whereas all in-contact crows remained negative to H9N2 virus antibodies. No virus could be isolated from tissues viz., lung, liver, kidney, pancreas, small intestine and large intestine. Although crows became infected with the H9N2 virus, transmission of the virus was inefficient to the in-contact group. However, virus excretion through oral and cloacal swabs from infected crows suggests a potential threat for inter-species transmission, including humans. Crows, being a common synanthrope species, might have some role in influenza virus transmission to poultry and humans, which needs to be explored further.

Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens.

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.

Label-free peptide nucleic acid biosensor for visual detection of multiple strains of influenza A virus suitable for field applications.

Accurate and rapid diagnosis of Influenza A viruses (IAVs) is challenging because of multiple strains circulating in humans and animal populations, and the emergence of new strains. In this study, we demonstrate a simple and rapid strategy for visual detection of multiple strains of IAVs (H1 to H16 subtypes) using peptide nucleic acid (PNA) as a biosensor and unmodified gold nanoparticles (AuNPs) as a reporter. The design principle of the assay is based on the color change on account of free PNA-induced aggregation of AuNPs in the presence of non-complementary viral RNA sequence and vice-versa. The assay could detect IAV RNA with a visual limit of detection of 2.3 ng. The quantification of RNA with a considerable accuracy on a simple spectrophotometer was achieved on plotting the PNA-induced colorimetric changes (absorption ratio of A640/A520) in the presence of a varying concentration of complementary RNA. As a proof-of-concept, the visual assay was validated on 419 avian clinical samples and receiver operating characteristic (ROC) curve analysis showed a high diagnostic specificity (96.46%, 95% CI = 93.8 to 98.2) and sensitivity (82.41%, 95% CI = 73.9 to 89.1) when RT-qPCR was used as reference test. Hence, the simplicity, rapidity, and universality of this strategy make it a potential candidate visual assay for clinical diagnosis and surveillance of IAVs, especially in the resource-limited settings. The proposed strategy establishes new avenues for developing a simple and rapid diagnostic system for viral infections and biomolecules.

Survivability of highly pathogenic avian influenza virus (H5N1) in naturally preened duck feathers at different temperatures.

Ducks are the "Trojan Horses" for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (104 EID50 and 106 EID50 ), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRT-PCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (104 EID50 and 106 EID50 ). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 106 EID50 concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.

Emergence of novel reassortant H6N2 avian influenza viruses in ducks in India.

The recent reports of human infection due to H6 subtype avian influenza viruses (AIV), which are prevalent in terrestrial poultry, indicate evolution of the virus to a possible pandemic strain. Here, we report antigenic and genetic characterization of two H6N2 viruses isolated from apparently healthy domestic ducks in Kerala and Assam, India during 2014 and 2015, respectively. Hemagglutination inhibition assay revealed antigenic divergence between the two isolates, which was corroborated by amino acid differences at 55 positions (15.98%) between their hemagglutinin (HA) 1.The sequence analyses indicated that both the viruses are avian origin with avian receptor specificity, low pathogenic to poultry and sensitive to oseltamivir. However, Kerala14 had V27I mutation marker for amantadine resistance in M2. The Assam15 virus had an additional N-linked glycosylation on HA2 (position 557) compared to Kerala14 virus. Phylogenetic analysis of the HA gene revealed that both the viruses belonged to distinct lineages (Eurasian and Asia II). Phylogeny of neuraminidase and internal gene segments revealed that both the viruses are novel reassortants and are genetically distinct with different gene constellations. The results suggest independent introductions of the two H6N2 viruses into India and migratory wild birds in the Central Asian flyway might be the source of H6N2 viruses in ducks in India. Therefore, continued AIV surveillance in poultry and wild birds is essential for early detection of emergence of novel strains with pandemic potential and control of their spread.

Induction profiles of mRNA of toll like receptors and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza H5N1 virus.

Herein, the induction of TLRs and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza (HPAI) H5N1 virus was studied. Four groups (1-4) of chickens inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 virus on days 1, 3, 7 and 14 post H9N2 inoculation, respectively. In groups (1-4) TLRs and cytokines induction was studied in chicken PBMCs on day 3 post H5N1 challenge. In H5N1 control group TLRs (1, 2, 5 and 7) cytokines (IFNα, IFNß, IFNγ, IL1ß, IL2, IL4, IL8 and TGF ß3) were down regulated. In group 1 down regulation of cytokines and TLRs was similar to H5N1 control birds. Down regulation of TLRs and cytokines in H5N1 control and group 1 resulted death of all the chickens. In group 2, up-regulation of TLRs (3, 7 and 15) and induction of TNFα, IFNα, IFNß, IFNγ aided virus clearance leading to survival of all the chickens. In group 3 significant up-regulation of TLRs (3, 4 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL4, IL6, IL8, IL10 and TGF ß3) was detected. In group 4 significant up-regulation of TLRs (2, 3, 7 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL2, IL6, IL8 and IL10) was detected. In groups 3 and 4 simultaneous and significant induction of pro-inflammatory, antiviral and anti-inflammatory cytokine resulted cytokine dysregulation leading to death of (2/6) and (3/6) chickens respectively. Hence, the study revealed TLRs and cytokines role in modulating the H5N1 infection outcome in chickens pre-exposed to H9N2 virus.

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.

Antigenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in India, 2006-2015.

Highly pathogenic avian influenza (HPAI) is a major health concern worldwide. In this study, we focused on antigenic analysis of HPAI H5N1 viruses isolated from poultry in India between 2006 and 2015 comprising 25 isolates from four phylogenetic clades 2.2 (1 isolate), 2.2.2.1 (1 isolate), 2.3.2.1a (17 isolates) and 2.3.2.1c (6 isolates). Seven H5N1 isolates from all four clades were selected for production of chicken antiserum, and antigenic analysis was carried out by hemagglutination inhibition (HI) assay. HI data indicated antigenic divergence (6-21 fold reduction in cross-reactivity) between the two recently emerged clades 2.3.2.1a and 2.3.2.1c. These two clades are highly divergent (21-128 fold reduction in HI titre) from the earlier clades 2.2 /2.2.2.1 isolated in India. However, a maximum of 2-fold and 4-fold reduction in cross-reactivity was observed within the isolates of homologous clades 2.3.2.1c and 2.3.2.1a, respectively. The molecular basis of inter-clade antigenic divergence was examined in the haemagglutinin (HA) antigenic sites of the H5N1 virus. Amino acid changes at 8 HA antigenic sites were observed between clades 2.3.2.1a and 2.3.2.1c, whereas 20-23 substitutions were observed between clades 2.3.2.1a/2.3.2.1c and 2.2/2.2.2.1. Therefore, a systematic analysis of antigenic drift of the contemporary field isolates is a pre-requisite for determining the suitable strain(s) for vaccine candidature.

Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health.

Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens.

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.

siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58 percent and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80 percent reduction in viral plaque counts and 94 percent inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68-73 percent and 87-88 percent reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.