A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production.

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.

Newcastle disease virus: is an updated attenuated vaccine needed?

Newcastle disease virus (NDV) is a major cause of infectious mortality and morbidity in poultry worldwide. It is an enveloped virus with two outer-membrane proteins-haemagglutinin-neuraminidase (HN) and fusion protein (F)-that induce neutralizing antibodies. All NDV strains belong to one serotype. Yet, NDV vaccines, derived from genotype II, do not fully prevent infection or shedding of viruses from other genotypes. The aim of this study was to test if an updated vaccine is required. For this purpose, NDVs isolated from infected, albeit heavily vaccinated, flocks were genetically and immunologically characterized. Amino acid differences in F and HN protein sequences were identified between the vaccine strain and each of the isolates, some specifically at the neutralization sites. Whereas all tested isolates showed similar haemagglutination-inhibition (HI) titres, 100-100,000 times higher antibody-to-virus ratios were needed to neutralize viral propagation in embryos by the field isolates versus the vaccine strain. As a result, a model and an equation were developed to explain the phenomenon of escape in one-serotype viruses and to calculate the HI values needed for protection, depending on variation rate at key positions. In conclusion, to confer full protection against NDVs that differ from the vaccine strain at the neutralizing epitopes, very high levels of antibodies should be raised and maintained to compensate for the reduction in the number of effective epitopes; alternatively, an adjusted attenuated vaccine should be developed-a task made possible in the current era of reverse vaccinology.

Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent.

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (101-106 EID50). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features.

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in 1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.