Pandemic H1N1 influenza virus in Chilean commercial turkeys with genetic and serologic comparisons to U.S. H1N1 avian influenza vaccine isolates

Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.     

Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: identification of a new subgroup in BVDV-1

Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete N(pro) encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1l. The remaining bovine isolates were typed as BVDV-1a (n=4), BVDV-1b (n=4), BVDV-1d (n=3), BVDV-1f (n=2) and BVDV-1h (n=1). The isolates allocated to BVDV-1l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup (n=5). This study indicates that the newly detected subgroup BVDV-1l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle.     

Comparative susceptibility of chickens and turkeys to avian influenza A H7N2 virus infection and protective efficacy of a commercial avian influenza H7N2 virus vaccine

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus caused a major outbreak among commercial poultry in Virginia and adjacent states. The virus primarily affected turkey flocks, causing respiratory distress and decreased egg production. Experimentally, turkeys were more susceptible than chickens to H7N2 virus infection, with 50% bird infectious dose titers equal to 10(0.8) and 10(2.8-3.2), respectively. Comparison of virus shedding from the cloaca and oropharynx demonstrated that recent H7N2 virus isolates were readily isolated from the upper respiratory tract but rarely from the gastrointestinal tract. The outbreak of H7N2 virus raised concerns regarding the availability of vaccines that could be used for the prevention and control of this virus in poultry. We sought to determine if an existing commercial avian influenza (AI) vaccine prepared from a 1997 seed stock virus could provide protection against a 2002 LPAI H7N2 virus isolated from a turkey (A/turkey/Virginia/158512/02 [TV/02]) in Virginia that was from the same lineage as the vaccine virus. The inactivated AI vaccine, prepared from A/chicken/ Pennsylvania/21342/97 (CP/97) virus, significantly reduced viral shedding from vaccinated turkeys in comparison with sham controls but did not prevent infection. The protective effect of vaccination correlated with the level of virus-specific antibody because a second dose of vaccine increased antiviral serum immunoglobulin G and hemagglutination inhibition (HI) reactivity titers in two different turkey age groups. Serum from CP/97-vaccinated turkeys reacted equally well to CP/97 and TV/02 antigens by HI and enzyme-linked immunosorbent assay. These results demonstrate the potential benefit of using an antigenically related 1997 H7N2 virus as a vaccine candidate for protection in poultry against a H7N2 virus isolate from 2002.     

An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 strain closely related to recent North American representatives of the Florida sub-lineage

In December 2005, equine influenza virus infection was confirmed as the cause of clinical respiratory disease in vaccinated horses in Apulia, Italy. The infected horses had been vaccinated with a vaccine that contained strains representatives from both the European (A/eq/Suffolk/89) and American (A/eq/Newmarket/1/93) H3N8 influenza virus lineages, and the H7N7 strain A/eq/Praga/56. Genetic characterization of the hemagglutinin (HA) and neuraminidase (NA) genes of the virus from the outbreak, indicated that the isolate (A/eq/Bari/2005) was an H3N8 strain closely related to recent representatives (Kentucky/5/02-like) of the American sub-lineage Florida, that was introduced in Italy through movement of infected horses from a large outbreak described in 2003 in United Kingdom. Strain A/eq/Bari/2005 displayed 9 amino acid changes in the HA1 subunit protein with respect to the reference American strain A/eq/Newmarket/1/93 contained in the vaccine. Four changes were localized in the antigenic regions C-D and likely accounted for the vaccine failure.     

Sequence and phylogenetic analysis of the non-structural 3A and 3B protein-coding regions of foot-and-mouth disease virus subtype A Iran 05

The A Iran 05 foot-and-mouth disease virus (FMDV) subtype was detected in Iran during 2005 and has proven to be highly virulent. This study was undertaken to focus on molecular and phylogenetic analysis of 3A and 3B coding-regions in the A Iran 05 field isolate. To assess the genetic relatedness of A Iran 05 isolate the nucleotide and predicted amino acid sequences of the 3AB region of type A FMDV isolates were compared with twenty previously described type A FMDV isolates. The phylogenetic tree based on the 672 bp 3AB gene sequences of type A FMDV from thirteen different locations clustered them into five distinct lineages. The A Iran 05 isolate clustered in lineage A along with four type A variants and was closely matched with viruses isolated in Turkey and Pakistan during 2005~2006. The number of protein sequence differences exhibited by each of the isolates revealed that A Iran 05 isolate contains three amino acid substitutions at positions 47 and 119 of 3A and 27 of the 3B coding region. The nucleotide identity between A Iran 05 and the other four isolates of lineage A was estimated to be 98%.     

Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005-2006

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.     

Circulation of bovine ephemeral fever in the Middle East--strong evidence for transmission by winds and animal transport

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus of cattle. The main routes of its transmission between countries and continents are not completely elucidated. This study aimed to explore BEFV transmission in the Middle-East. A phylogenetic analysis was performed on the gene encoding the G protein of BEFV isolates from Israel from 2000 and 2008 with isolates from Turkey (2008), Egypt (2005), Australia (1968-1998) and East Asia (1966-2004). Calf sera collected during the years 2006-2007 were tested by serum neutralization in order to explore for recent exposure to BEFV before 2008. These were followed by a meteorological analysis, aimed to reveal movement of air parcels into Israel in the two weeks preceding the first case of BEF in Israel in 2008. The 2008 Israeli and Turkish isolates showed 99% identity and formed a new cluster with the 2000 Israeli isolate. The serological survey showed no new exposure to BEFV during 2006 and 2007. These results coincided with the meteorological analysis, which revealed that air parcels originating in Southern Turkey had reached the location of outbreak onset in Israel nine days before the discovery of the index case. The Egyptian isolate clustered phylogenetically with the Taiwanese isolates, coinciding with data on importation of cattle from China to the Middle East in the year preceding the isolation of the Egyptian isolates. These results suggest that both winds and animal transport may have an important role in trans-boundary transmission of BEFV.     

一株H9亚型禽流感病毒的分离鉴定及生物特性研究

2013年中国吉林某养鸡场发生疑似H9亚型禽流感疫情,采集该发病鸡场病料接种9日龄SPF鸡胚,分离得到一株病毒。经血凝(HA)试验、血凝抑制(HI)试验、测序分析,鉴定该毒株为H9亚型禽流感病毒(AIV)。对本试验分离株HA基因进行测序及序列分析,结果显示分离株HA基因的裂解位点为RSSR↓GLF,符合低致病性AIV的基因特征;HA肽链具有9个潜在糖基化位点,与近些年H9亚型AIV分离株的潜在糖基化位点相同;具有8个受体结合位点,其中234位受体结合位点由谷氨酰胺(Q)变异成苏氨酸(T);HA基因系统进化树结果显示本试验分离株属于欧亚进化分支,与中国最早分离株A/Chicken/Beijing/1/94(H9N2)亲缘关系较远,与2007年后中国H9亚型AIV主要流行分支的代表株A/Chicken/Guangxi/55/2005(H9N2)亲缘关系较近。将该毒株制成油乳剂灭活疫苗免疫SPF鸡,免疫后第21天免疫鸡血清抗体高达10log2,表明本试验分离株具有很好的免疫原性。     

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.     

Isolation and Identification of one Strain of H9 Subtype Avian Influenza Virus and Study on its Biological Characteristics

In 2013,one case of suspected H9 subtype avian influenza occurred in a chicken farm of Jilin povince.Clinical samples were collected from the diseased farm,inoculated into the allantoic cavity of 9-day-old SPF chicken embryo,and then one strain of virus was isolated.The results of HA test,HI test and molecular biology test all showed that the isolate belonged to H9 subtype avian influenza virus (AIV).The HA cleavage site of the isolate was RSSR↓GLF,which was consisted with the molecular characteristic of low pathogenic AIV.The HA peptide chain had 9 potential glycosylation sites which were same as other isolates of recent years.The isolate had 8 receptor binding sites,including 234 receptor binding site by glutamine (Q) mutating into threonine (T).The phylogenetic tree revealed the isolate belonged to Eurasian lineages and it had far genetic relationship with the earliest domestic isolate (A/Chicken/Beijing/1/94(H9N2)),but had close genetic relationship with the representative strain (A/Chicken/Guangxi/55/2005(H9N2)) of major epidemic branch since 2007.We prepared an inactivated oil-emulsion vaccine of the isolate,and then vaccinated SPF chicken.21 days after vaccination,the HI titer of chicken serum antibody reached up to 10log2.The result suggested the isolate had good immunogenicity.     

H5N1亚型人禽流感病毒A/Anhui/2/2005株反向遗传操作系统的建立

为建立H5N1亚型人源禽流感病毒A/Anhui/2/2005(W-AH05)反向遗传操作系统,本研究构建了W-AH05的8重组质粒,拯救出人源禽流感病毒R-A/Anhui/2/2005(R-AH05)。全基因序列测定结果表明,救获病毒R-AH05与野生病毒W-AH05的核苷酸序列完全一致;动物试验证实,R-AH05保持了W-AH05的对BALB/c小鼠呈高致病力的特性,其MLD50分别为1.5log10EID50和1.2log10EID50。R-AH0与W-AH05分别以106EID50剂量鼻腔感染BALB/c小鼠,病毒在小鼠体内的分布情况及各个脏器中的病毒滴度基本相同。由此可见,R-AH05保持了W-AH05的生物学特性,从而为进一步研究人源禽流感病毒的致病机理及跨宿主传播机制等提供了操作平台。     

Pathogenicity and transmission studies of H5N2 parrot avian influenza virus of Mexican lineage in different poultry species

In 2004, a low pathogenic H5N2 influenza virus (A/parrot/CA/6032/04) was identified in a psittacine bird for the first time in the United States. Sequence and phylogenetic analysis of the hemagglutinin gene grouped the parrot isolate under the Mexican lineage H5N2 viruses (subgroup B) with highest similarity to recent chicken-origin isolates from Guatemala. Antigenic analysis further confirmed the close relatedness of the parrot isolate to Mexican lineage viruses, the highest cross-reactivity being demonstrated to Guatemala isolates. In vivo studies of the parrot isolate in chickens, ducks and turkeys showed that the virus, though did not cause any clinical signs, could replicate to high titers in these birds and efficiently transmit to contact control cage mates. The possibility that the parrot harboring the virus was introduced into the United States as a result of illegal trade across the border provides additional concern for the movement of foreign animal diseases from neighboring countries. Considering the potential threat of the virus to domestic poultry, efforts should be continued to prevent the entry and spread of influenza viruses by imposing effective surveillance and monitoring measures.     

Highly pathogenic avian influenza virus subtype H5N1 in Mute swans in the Czech Republic

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.     

Vaccine failure caused an outbreak of equine influenza in Croatia

In April 2004 an outbreak of equine influenza occurred at the Zagreb hippodrome, Croatia. Clinical respiratory disease of the same intensity was recorded in vaccinated and non-vaccinated horses. The equine influenza vaccine used in Croatia at the time of the outbreak contained the strains A/equine/Miami/63 (H3N8), A/equine/Fontainebleau/79 (H3N8) and A/equine/Prague/56 (H7N7). At the same time, the usual strains in vaccines used in Europe were, in accordance with the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel on equine influenza, A/equine/Newmarket/1/93 (H3N8) and A/equine/Newmarket/2/93 (H3N8). At the same time, some current vaccines in the USA contained A/equine/Kentucky/97 (H3N8). Genetic characterization of the HA1 portion of the haemagglutinin (HA) gene of virus isolated from the outbreak indicated that the isolate (A/equine/Zagreb/04) was an H3N8 strain closely related to recent representative viruses of the American lineage Florida sub-lineage. In comparison with both H3N8 vaccine strains used in horses at the Zagreb hippodrome, A/equine/Zagreb/04 displayed amino acids changes localised to 4 of the 5 described antigenic sites (A-D) of subunit protein HA1. Comparison of the amino acid sequence of the HA1 subunit protein of the outbreak strain with that of A/equine/Newmarket/1/93 displayed three amino acids changes localised in antigenic sites B and C, while antigenic sites A, D and E were unchanged. The Zagreb 2004 outbreak strain had the same amino acids at antigenic sites of the HA1 subunit protein as the strain A/equine/Kentucky/97. Amino acid changes in antigenic sites between HA1 subunit of the outbreak strain and the strains used in the vaccines likely accounted for the vaccine failure and the same clinical signs in vaccinated and unvaccinated horses. Use of a recent strain in vaccines should limit future outbreaks.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Isolation and characterization of influenza A virus (subtype H5N1) that caused the first highly pathogenic avian influenza outbreak in chicken in Bhutan

We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other "Qinghai-like" H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids ((222)Q and (224)G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1-100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds.     

Surveillance and identification of influenza A viruses in wild aquatic birds in the Crimea, Ukraine (2006-2008)

The ecology of avian influenza (AI) viruses in wild aquatic birds of Asia is poorly understood, especially for the H5N1 high pathogenicity AI (HPAI) viruses. From March 2006 through November 2008, 20 AI viruses were isolated in the Crimea region of Ukraine with an overall frequency of virus recovery of 3.3%. All the viruses were isolated from three species of dabbling ducks: mallard (Anas platyrhynchos), wigeon (Anas penelope), and garganey (Anas querquedula), making the frequency of virus recovery for dabbling ducks 6.3%. The viruses were predominantly isolated during the fall sampling period. All viruses were genetically and antigenically characterized. No H5N1 HPAI viruses were isolated, but other HA and NA subtypes were identified including H3N1 (2), H3N6 (3), H3N8 (4), H4N6 (6), H5N2 (3), H7N8 (1), and H10N6 (1) subtypes. All isolates were of low pathogenicity, as determined by the intravenous pathogenicity index of 0.00. For H5N2 and H7N8 isolates, the HA gene was sequenced and the phylogenetic analysis revealed possible ecologic connections of the Crimea region with AI viruses from Siberia and Europe. No influenza A isolates were recovered from other Anseriformes (diving ducks [two species of pochards] and graylag geese), Columbiformes (collared doves), Gruiformes (coot), and Galliformes (gray partridges).     

Outbreaks of classical swine influenza in pigs in England in 1986

Serum samples from pig herds in Great Britain have been examined for antibodies to influenza virus since 1968. Antibodies to H3N2 virus strains have been found since 1968 and the serological data presented here suggests that H3N2 virus strains continue to persist in the pig population. An outbreak of acute respiratory disease occurred in a 400-sow unit. The outbreak was characterised by coughing, anorexia, fever, inappetence and loss of condition. The gilts and weaners were affected and the morbidity approached 100 per cent. An influenza A virus designated A/Swine/Weybridge/117316/86 (H1N1) was isolated from the herd and 28 paired serum samples from the affected animals showed increases in the haemagglutination inhibition titres to this isolate. Haemagglutinin and neuraminidase characterisation indicated that the virus is similar to H1N1 viruses isolated recently from pigs in Europe. A total of 91 herds experiencing respiratory disease were investigated, of which 42 gave positive reactions in the haemagglutination inhibition test. Antibodies to A/Port Chalmers/1/73 (H3N2) were also detected in some of the herds but it is not known whether this strain plays any role in the current respiratory disease problems in pigs.