Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens

Chickens were intranasally inoculated with Chilean H7N3 avian influenza (AI) viruses of low pathogenicity (LP) (H7N3/LP), high pathogenicity (HP) (H7N3/HP), and a laboratory derivative (02-AI-15-#9) (H7N3/14D) from the LPAI virus to determine pathobiologic effects. All chickens inoculated with H7N3/HP AI virus became infected and abruptly died 2 or 3 days postinoculation, but a few showed moderate depression before death. The H7N3/HP AI virus produced focal hemorrhages of the comb, petechial hemorrhage at the esophageal-proventricular junction and proventricular mucosa, edema and congestion of the lung, petechiation of the spleen, and generalized decrease in body fat. Histologically, severe necrosis, hemorrhage, and inflammation were primarily identified in lungs and the lymphoid tissues. All tissues sampled from the H7N3/HP AI group were positive for the AI viral antigen, predominantly in endothelium of blood vessels throughout most tissues and less frequently in histiocytes and cellular debris of lymphoid tissues. Even less consistently, cardiac myocytes, hepatocytes, Kupffer cells, glandular epithelial cells, microglial cells, and neurons became infected. These studies suggest the Chilean H7N3/LP AI virus was poorly infectious for chickens and may have been recently introduced from a nongalliform host. By contrast, the H7N3/HP AI virus was highly infectious and lethal for chickens. The H7N3/HP AI virus had a strong tropism for the cardiovascular system, principally vascular endothelium, which is similar to the viral tropism demonstrated previously with other H5 and H7 HPAI viruses. Interestingly, the H7N3/LP AI virus on intravenous inoculation replicated in cardiac myocytes, a feature of HPAI and not LPAI viruses, which further supports the theory that the H7N3/LP AI virus was in transition from LP to HP.     

Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA0 cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.     

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico

Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24‐nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.     

Genomic characterization of H1N2 swine influenza viruses in Italy

Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of whole genome sequences of 26 strains isolated from 1998 to 2010. Phylogenetic analysis of HA and NA genes showed differences between the older (1998-2003) and the more recent strains (2003-2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Two other reassortant H1N2 strains have been detected: A/sw/It/22530/02 has the HA gene that is closely related to H1N1 viruses; A/sw/It/58769/10 is an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs. Amino acid analysis revealed interesting features: a deletion of two amino acids (146-147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009 to 2010, which is said to have contributed to the increased virulence. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses.     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.     

2013—2014年H7亚型禽流感病毒遗传进化分析

对2013—2014年15株不同来源的H7亚型禽流感病毒国内分离株进行了基因组测序与遗传进化分析。结果显示,15株H7亚型禽流感病毒具有遗传多样性,可分为2种亚型:13株为H7N9亚型,2株为H7N3亚型。HA蛋白裂解位点附近氨基酸分析显示所有H7亚型流感分离株均为低致病性毒株。基因组遗传进化分析显示,13株H7N9亚型禽流感病毒均与2013年人源分离株同源性较高,但具有2种遗传学特性,13株病毒的PB2、PA、HA、NP、NA、M和NS基因高度同源,而PB1基因来源于2个不同分支。2株H7N3亚型流感病毒与国内鸭源分离株同源性较高。15株H7亚型禽流感病毒PB2蛋白均未出现627K、701N等与哺乳动物适应性相关的氨基酸变异。13株H7N9亚型禽流感病毒HA裂解位点附近氨基酸(EIPKGR/GL)与人源H7N9病毒一致,且HA蛋白和M2蛋白分别具有与哺乳动物适应性和金刚烷胺耐药性相关的标志性变异,而2株H7N3亚型禽流感病毒未出现上述氨基酸变异。     

Antibody response and viral shedding profile of Egyptian geese (Alopochen aegyptiacus) infected with low pathogenicity H7N1 and H6N8 avian influenza viruses

Egyptian geese (Alopochen aegypticus), a duck species endemic to sub-Saharan Africa and occasionally implicated in the transmission of avian influenza viruses (AIV) to farmed ostriches, were experimentally infected with low pathogenicity H7N1 and H6N8 viruses to assess viral shedding and immune profiles. Following the first infection with H7N1 virus, high titers of virus were shed from both the tracheae and cloacae for at least 7 days postinfection, and tracheal shedding lasting until day 14. All detectable shedding from both tracheae and cloacae had ceased within 28 days of infection. Antibody titers peaked at day 7 postinfection, but the initial immune response was short-lived. Birds that received a second challenge with the homologous H7N1 virus mounted a more robust response that lasted beyond 66 days postchallenge, and H7N1 virus was detected, albeit at much lower levels, until day 28 post secondary infection (psi) in the cloaca and beyond day 28 psi in the trachea. Birds that received an initial infection with H7N1 virus were also challenged with H6N8 virus, and because a comparable shedding pattern to the H7N1 challenge group was observed, we concluded that the effect of any nonspecific immunity was negligible.     

Studies on influenza viruses H10N4 and H10N7 of avian origin in mink

An influenza A virus, A/mink/Sweden/84 (H10N4), was isolated from farmed mink during an outbreak of respiratory disease, histopathologically characterised by severe interstitial pneumonia. The virus was shown to be of recent avian origin and closely related to concomitantly circulating avian influenza virus. Serological investigations were used to link the isolated virus to the herds involved in the disease outbreak. Experimental infection of adult mink with the virus isolate from the disease outbreak reproduced the disease signs and pathological lesions observed in the field cases. The mink influenza virus also induced an antibody response and spread between mink by contact. The same pathogenesis in mink was observed for two avian influenza viruses of the H10N4 subtype, circulating in the avian population. When mink were infected with the prototype avian H10 influenza virus, A/chicken/Germany/N/49, H10N7, the animals responded with antibody production and mild pulmonary lesions but neither disease signs nor contact infections were observed. Detailed studies, including demonstration of viral antigen in situ by immunohistochemistry, of the sequential development of pathological lesions in the mink airways after aerosol exposure to H10N4 or H10N7 revealed that the infections progress very similarly during the first 24h, but are distinctly different at later stages. The conclusion drawn is that A/mink/Sweden/84, but not A/chicken/Germany/N/49, produces a multiple-cycle replication in mink airways. Since the viral distribution and pathological lesions are very similar during the initial stages of infection we suggest that the two viruses differ in their abilities to replicate and spread within the mink tissues, but that their capacities for viral adherence and entry into mink epithelial cells are comparable.     

Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005

This article reports the genetic and pathogenic characteristics of 34 isolates of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Genetic analyses showed that a unique lineage of H6N1 viruses has been established in domestic chickens in Taiwan since 1997, and this lineage of viruses differs from the H6N1 viruses circulating in Hong Kong and Southeastern China. Pathogenicity tests showed that all Taiwanese H6N1 viruses were of low pathogenicity but might lead to economic loss when associated with other diseases. Hemagglutination inhibition tests showed that antigenic drift has occurred in Taiwanese H6N1 viruses, and sequence comparison has identified a total of five possible antigenic sites on the hemagglutinin molecule of the H6N1 viruses. Some Taiwanese H6N 1 viruses could replicate in mice without preadaptation, indicating that these viruses have the potential to cause cross-species infection into mammals.     

两株鸭源H5N1亚型禽流感病毒的序列分析及致病性研究

本研究对2010年在湖北活禽市场监测分离到的两株鸭源H5N1亚型禽流感病毒(AIV) (HuB/495/10和HuB/513/10)进行了序列分析和致病性试验研究,以了解湖北地区H5N1亚型AIV的生物学特性差异.序列分析显示:2株病毒全基因组核苷酸同源性在97.3 ％～98.6％,2株病毒的8个节段基因均与青海和香港分离的野鸟源病毒A/great crested-grebe/Qinghai/1/2009 (H5N1)和A/black-crowned night heron/Hong Kong/659/2008 (H5N1)的核苷酸高度同源,HA蛋白裂解位点序列基序为341RRRKR345,呈现典型的高致病力分子特征.以105 EID50/100 μL病毒剂量感染4周龄SPF鸭发现:HuB/495/10和HuB/513/10对鸭的致死率分别为100％和20％,病毒在鸭体内呈全身性复制并可通过呼吸道和消化道向外排毒;不同滴度的病毒感染6周龄BALB/c小鼠,HuB/495/10和HuB/513/10的MLD50分别为1.38 log10 EID50和1.68 log10 EID50,对小鼠表现为高致病力,均在肺脏中高拷贝复制.     

Experimental infection with low and high pathogenicity H7N3 Chilean avian influenza viruses in Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera)

Two different wild duck species common in Chile and neighboring countries, Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera), were intranasally inoculated with 10(6) mean embryo infective dose (EID50) of the H7N3 low pathogenicity (LP) avian influenza virus (AIV) (A/chicken/Chile/176822/02) or high pathogenicity (HP) AIV (A/chicken/Chile/ 184240-1/02), in order to study the infectivity and pathobiology of these viruses. None of the virus-inoculated ducks had clinical signs or died, but most seroconverted by 14 days postinoculation (DPI), indicating a productive virus infection. Both LPAIV and HPAIV were isolated from oral swabs from two of six Chiloe wigeons and from oral and/or cloacal swabs from all five of the cinnamon teal at 2 DPI. Both LPAIV and HPAIV were efficiently transmitted to cinnamon teal contacts but not to Chiloe wigeon contacts. This study demonstrates that the cinnamon teal and Chiloe wigeons were susceptible to infection with both Chilean H7N3 LPAIV and HPAIV, but only the cinnamon teal showed contact transmission of the virus between birds, suggesting that the cinnamon teal has the potential to be a reservoir for these viruses, especially the LPAIV, as was demonstrated in 2001 with isolation of a genetically related H7N3 LPAIV strain in a cinnamon teal in Bolivia. However, the definitive source of the H7N3 Chilean LPAIV still remains unknown.     

Experimental assessment of the pathogenicity of H5N1 influenza A viruses isolated in Japan

We examined the pathogenicity for chickens of two H5N1 avian influenza viruses isolated in Japan, A/chicken/ Yamaguchi/7/2004 (Ck/Yamaguchi/7/04) isolated from outbreaks in commercial layer chickens, and A/duck/Yokohama/aq10/ 2003 (Dk/Yokohama/aq10/03) isolated from duck meat imported from China. All chickens inoculated intranasally with either strain died, and the viruses were reisolated from all organs examined. However, both the mean time of onset of clinical signs and the mean death time of Ck/Yamaguchi/7/04 were shorter than those of Dk/Yokohama/aq10/03.