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This chapter describes the current state of knowledge of avian cytokines, concentrating on the chicken. It describes the different families of cytokines and their receptors, reagents and assays available; viral cytokines and their potential modes of actions; and the use of cytokines as vaccine adjuvants.

The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.

SUMMARY: Saccharomyces cerevisiae synthesizes one major tryptophan transfer ribonucleic acid (tRNATrp) species (isoacceptor A) carrying characteristic base modifications. Under tryptophan limited growth conditions wild-type strain X2180-1A exhibited a second important tRNATrp species (isoacceptor B). The total amount of tRNATrp, approximately 2.5 pmol (mg dry wt)-1, stayed essentially constant during amino acid shift-down experiments. The amount of isoacceptor B relative to total tRNATrp was 10 to 15% during amino acid sufficient exponential growth conditions, but increased during tryptophan limitation three- to four-fold. Analysis of the base compositions showed that isoacceptor B differed from isoacceptor A in one respect only: 2′-O-methylguanosine, a modified guanosine base occurring at position 17 of the major isoacceptor A tRNATrp, was not detectable in hydrolysates of purified isoacceptor B. The biological significance of isoacceptor B is discussed.

The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNATrp). The mode of inhibition is competitive and the analogue is not charged onto tRNATrp. Thus 4-methyltryptophan application depletes the cells from charged tRNATrp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.

Low pathogenic (LP) avian influenza viruses (AIVs) can cause human infections, as the result of direct transmission from infected birds. Human cases are related to mild or subclinical disease, although severe outcomes have been recently reported in subjects infected by H7N9 LPAIVs. Some farming practises are modelled to house birds outdoor, thus exposing them to AIVs harboured in wild avian reservoir. These relatively low bio-security conditions enable the emergence of AIVs in captive-reared birds, increasing the potential zoonotic risk to occupationally exposed workers. Main purpose of this study is to provide serological evidence of LPAIV infection in workers operating in farms located in the Emilia-Romagna Region (Italy). In April 2005-July 2006 period, sera were collected from 57 bird-exposed workers and 7 non bird-exposed controls, planning three blood sample collections from each individual to assess seroconversion against AIVs. Study population included: 46 poultry workers (PWs) and 4 veterinarians operating in 14 farms housing birds under outdoor conditions; 2 veterinarians and 4 technicians involved in AIV laboratory diagnostics; a wildlife professional exposed to wild waterfowl. Haemagglutination inhibition (HI) assay and microneutralization-ELISA (MN) were used as screening and confirmatory tests, respectively. Sera were tested for specific antibodies against twenty LPAIVs belonging to antigenic subtypes from H1 to H14 and including twelve reference strains and eight avian isolates from farms under study. MN results showed serologic evidence of exposures to AIV in 3/46 PWs, showing antibodies against H3, H6, H8 and H9 AIVs. In addition, the wildlife professional was seropositive against H11 AIV subtype.

Wild aquatic birds are reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic ones. High viral loads in feces of infected birds allow fecal-oral transmission. However, this route does not fully account for the efficiency of avian influenza virus (AIV) spread since dilution of infectious feces in water progressively decreases the chances of virus/host interaction. We investigated whether preen oil gland secretion, by which all aquatic birds make their feathers waterproof, could support a natural concentration mechanism of AIVs from water to birds’ bodies, thus favouring virus spread and persistence in the aquatic environment. First, we detected consistently both viral genome and infectious AIVs on swabs taken by rubbing preened feathers of 345 wild mallards and examined by reverse transcription (RT)-PCR and virus isolation (VI) assays. Second, in two laboratory experiments using a quantitative real-time (qR) RT-PCR, we demonstrated that feather samples (n=5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces, as shown by statistical analysis. Third, we experimentally coated 7 mallards with a preen oil-AIV mix, and housed them with a control, uncoated, duck. Through self- and/or allopreening behaviour, all birds ingested the virus, as shown by virus detection in both oro-pharyngeal and cloacal samples. Virus isolation from cloacal swabs and virus-specific antibody response confirmed the occurrence of mallards’ infection. Infectious AIVs were isolated from ducks’ body surface until 32 days after the experimental coating.Our field and experimental results indicate that AIVs can be naturally concentrated and carried on the feather surface of infected ducks (i.e., those VI-positive from both cloacal and feathers swabs) and uninfected ones (i.e., those VI-positive from feathers only). In such a context, the natural preening behaviour, by which waterbirds spread preen oil all over their plumage (self-preening) or other birds’ plumage (allo-preening), could facilitate the ingesion of AIV particles stuck on birds’ feathers, thus promoting a preening-mediated infection route. Our findings also suggest that during the time period between the virus adhesion to the bird’s body and the infection (possibly due to self- and/or allopreening), the virus could move in nature with the host by an undescribed circulation mechanism. We demonstrate here a novel viral transmission route that adds to, and possibly contributes to explain the knowledge of longdistance movements and long-term infectivity of lowly and highly pathogenic AIVs in nature.