Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020.     

Protective measures and H5N1-seroprevalence among personnel tasked with bird collection during an outbreak of avian influenza A/H5N1 in wild birds, Ruegen, Germany, 2006

Background  

In Germany, the first outbreak of highly pathogenic avian influenza A/H5N1 occurred among wild birds on the island of Ruegen between February and April 2006. The aim of this study was to investigate the use of recommended protective measures and to measure H5N1-seroprevalence among personnel tasked with bird collection.     

Cross-protectiveness and immunogenicity of influenza A/Duck/Singapore/3/97(H5) vaccines against infection with A/Vietnam/1203/04(H5N1) virus in ferrets

Ferrets were immunized with two 7- mu g doses of hemagglutinin from inactivated whole-virus vaccines containing the hemagglutinin gene of A/Duck/Singapore/3/97(H5N3) then inoculated with a lethal dose of A/Vietnam/1203/04(H5N1) (Viet/1203/04). Serum samples did not react with Viet/1203/04 in hemagglutination-inhibition (HI) or virus-neutralization (VN) tests. All vaccinated ferrets survived the challenge, whereas all mock-immunized ferrets died. Immunized ferrets had significantly lower virus titers in the upper respiratory tract and less-severe disease. Vaccine generated from antigenically different H5 virus protects against infection by a highly pathogenic H5 strain. Neither HI nor VN testing provides correlates of cross-protection in ferrets.     

禽流感病毒H1、H3、H5、N2亚型多重RT-PCR检测方法的初步研究

目的根据禽流感病毒H1、H3、H5亚型的HA基因和N2型NA基因的保守序列,设计出四对RT-PCR引物,建立一步法多重RT-PCR对禽流感H1、H3、H5、N2四亚型进行快速检测。方法利用所设计的四对引物,通过对该方法扩增条件的优化,成功建立快速检测禽流感病毒H1、H3、H5、N2四亚型的一步法多重RT-PCR。利用禽流感H1、H3、H5、N2四亚型毒株和其它相关标准毒株进行敏感性和特异性试验。结果与结论所建立的一步法多重RT-PCR具有较高的特异性和敏感性,与禽流感其它亚型和NDV、IBV、ARV?IBDV的核酸均无交叉反应。用该方法检测现场样品395份(4省20多个地区),结果与经典检测方法一致。     

人感染高致病性禽流感病毒A/Fujian/1/2007(H5N1)株基因组序列测定及分析

目的对福建省分离的高致病性禽流感病毒A/Fujian/1/2007(H5N1)株进行全基因组序列测定,了解病毒序列及进化特征。方法SPF鸡胚分离法分离A/Fujian/1/2007(H5N1)株病毒,并提取病毒RNA。病毒RNA经通用引物逆转录后,应用特异性引物分别扩增病毒各节段,产物经TA克隆到载体中进行测序并对结果进行排列、拼接成完整的病毒基因组。分析病毒重要位点特征,并参照已发表的病毒序列,对A/Fujian/1/2007(H5N1)株作系统进化分析。结果应用自行设计的引物,扩增并获得完整的A/Fujian/1/2007(H5N1)株基因组序列。病毒重要位点的分析表明,该株病毒为禽源高致病性禽流感病毒,病毒对达菲敏感而对金刚烷胺类药物则具有耐药性。病毒特征有待进一步的生物学验证。系统进化分析显示,在亚洲各国引起人类感染的高致病性禽流感病毒具有明显的地理分布特征,A/Fujian/1/2007(H5N1)病毒与国内以往毒株属同一进化分支。结论获得人感染高致病性禽流感病毒A/Fujian/1/2007株全基因组序列,了解病毒重要的氨基酸位点特征以及亚洲人感染病毒间的亲缘关系,为进一步探讨福建省禽流感病毒变异、传播机制等奠定基础。     

Characterization of a whole,inactivated influenza (H5N1) vaccine

Objectives Effective vaccines against the highly pathogenic influenza A/H5N1 virus are being developed worldwide. In Japan, two adjuvanted, inactivated, whole‐virion influenza vaccines were recently developed and licensed as mock‐up, pre‐pandemic vaccine formulations by the Ministry of Health and Labor Welfare of Japan. During the vaccine design and development process, various obstacles were overcome and, in this report, we introduce the non clinical production, immunogenicity data in human and development process that was associated with egg‐derived adjuvanted, inactivated, whole‐virion influenza A (H5N1) vaccine. Design Pilot lots of H5N1 vaccine were produced using the avirulent H5N1 reference strain A/Vietnam/1194/2004 (H5N1) NIBRG‐14 and administered following adsorption with aluminum hydroxide as an adjuvant. Quality control and formulation stability tests were performed before clinical trials were initiated (phase I‐III).
The research foundation for microbial diseases of Osaka University (BIKEN) carried out vaccine production, quality control, stability testing and the phase I clinical trial in addition to overseeing the licensing of this vaccine. Mitsubishi Chemical Safety Institute Ltd. carried out the non clinical pharmacological toxicity and safety studies and the Japanese medical association carried out the phase II/III trials. Phase I‐III trials took place in 2006. Results The production processes were well controlled by established tests and validations. Vaccine quality was confirmed by quality control, stability and pre‐clinical tests, and the vaccine was approved as a mock‐up, pre‐pandemic vaccine by the Ministry of Health and Labor Welfare of Japan. Conclusions Numerous safety and efficacy procedures were carried out prior to the approval of the described vaccine formulation. Some of these procedures were of particular importance e.g., vaccine development, validation, and quality control tests that included strict monitoring of the hemagglutinin (HA) content of the vaccine formulations.
Improving vaccine productivity, shortening the production period and improving antigen yield of the avirulent vaccine strains were also considered important vaccine development criteria.     

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.     

Cross-reactivity to highly pathogenic avian influenza H5N1 viruses after vaccination with nonadjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a potential priming strategy

Antigenically well-matched vaccines against highly pathogenic avian influenza H5N1 viruses are urgently required. Human serum samples after immunization with MF59 or nonadjuvanted A/duck/Singapore/97 (H5N3) vaccine were tested for antibody to 1997-2004 human H5N1 viruses. Antibody responses to 3 doses of nonadjuvanted vaccine were poor and were higher after MF59-adjuvanted vaccine, with seroconversion rates to A/HongKong/156/97, A/HongKong/213/03, A/Thailand/16/04, and A/Vietnam/1203/04 of 100% (P < .0001), 100% (P < .0001), 71% (P = .0004), and 43% (P = .0128) in 14 subjects, respectively, compared with 27%, 27%, 0%, and 0% in 11 who received nonadjuvanted vaccine. These findings have implications for the rational design of pandemic vaccines against influenza H5.     

Influenza A/H5N1 infection: Other treatment options and issues

Abstract:   Antiviral therapy and vaccination are important strategies for the control of human influenza/H5N1 disease, but the efficacy of these modalities is limited by timing of administration and shortage of supply. Lung protective ventilation strategy with a low tidal volume and low pressure, in addition to a conservative fluid management approach, is recommended when treating patients with ARDS. Low-dose steroids may be considered in the treatment of refractory septic shock. Non-invasive positive pressure ventilation (NPPV) may play a limited supportive role for early ARDS/acute lung injury, but it is contra-indicated in critically ill patients with multi-organ failure and haemodynamic instability. NPPV and oxygen therapy should be applied in healthcare facilities with good ventilation and respiratory protection as substantial exposure to exhaled air occurs within a 0.5 m and 0.4 m radius of patients receiving NPPV and oxygen via a simple mask, respectively. Intravenous gammaglobulin should be used with caution for treatment of reactive haemo-phagocytosis due to its thrombogenic effects, whereas the role of etoposide needs evaluation with animal models. Passive immunotherapy in the form of convalescent plasma may be useful as rescue therapy. More data are needed to explore the potential role of other drugs with immuno-modulating properties such as statins. Healthcare workers currently must apply strict standards, contact and droplet precautions when dealing with suspected cases, and upgrade to airborne precautions when performing aerosol-generating procedures. Non-pharmacological measures such as early case isolation, household quarantine, school/workplace closure, good community hygiene and restrictions on travel are useful measures in controlling a pandemic.     

Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.     

Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells

Background In case of influenza pandemic, a robust, easy and clean technique to prepare reassortants would be necessary. Objectives Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 × PR8 and A/H7N1 × PR8) exhibiting the envelope glycoproteins from non‐pathogenic avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93 (A/H7N1) and the internal proteins of the attenuated human virus A/Puerto Rico/8/34 (H1N1). Methods The transfection was accomplished using a mixture of Vero and chicken embryo cells both of which are currently being used for vaccine manufacturing. Results This process was reproducible, resulting in consistent recovery of influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has recently crossed the human barrier, it is the A/PR8 × A/H5N1 reassortant (RG5) that was further amplified, either in embryonated hen eggs or Vero cells, to produce vaccine pre‐master seed stocks that met quality control specifications. Safety testing in chickens and ferrets was performed to assess the non‐virulence of the reassortant, and finally analysis using chicken and ferret sera immunized with the RG5 virus showed that the vaccine candidate elicited an antibody response cross‐reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the Vietnam/2004 viruses. Conclusions The seeds obtained could be used as part of a pandemic vaccine strain ‘library’ available in case of propagation in humans of a new highly pathogenic avian strain.     

H5N1 influenza: a protean pandemic threat

Infection with avian influenza A virus of the H5N1 subtype (isolates A/HK/212/03 and A/HK/213/03) was fatal to one of two members of a family in southern China in 2003. This incident was preceded by lethal outbreaks of H5N1 influenza in waterfowl, which are the natural hosts of these viruses and, therefore, normally have asymptomatic infection. The hemagglutinin genes of the A/HK/212/03-like viruses isolated from humans and waterfowl share the lineage of the H5N1 viruses that caused the first known cases of human disease in Hong Kong in 1997, but their internal protein genes originated elsewhere. The hemagglutinin of the recent human isolates has undergone significant antigenic drift. Like the 1997 human H5N1 isolates, the 2003 human H5N1 isolates induced the overproduction of proinflammatory cytokines by primary human macrophages in vitro, whereas the precursor H5N1 viruses and other H5N1 reassortants isolated in 2001 did not. The acquisition by the viruses of characteristics that enhance virulence in humans and waterfowl and their potential for wider distribution by infected migrating birds are causes for renewed pandemic concern.     

Seroprevalence of avian influenza A/H5N1 among poultry farmers in rural Indonesia, 2007

Since 2003, about a third (> 150 cases) of human cases of highly pathogenic avian influenza (HPAI) A/H5N1 worldwide are reported from Indonesia. We measured the seroprevalence of H5N1 among Indonesian poultry farmers and assessed the risk factors for and knowledge of H5N1 infection. In 2007, poultry workers and farm residents were interviewed about risk factors for knowledge of and then examined for the seropositivity of H5N1 virus. Four hundred ninety-five of 622 farmers (80%) from 12 farms participated in the study. Of these, 71% were male, with a median age of 29 years. None tested positive for H5N1 virus. Masks were never worn by 54% of participants; 86% were afraid of becoming infected. For the preceding six months, 1 farm was confirmed as having poultry infected with H5N1 virus. No evidence of subclinical infection with avian influenza A/H5N1 virus was found among poultry farmers, although exposure of the farmers to this virus may have been limited. However, we recommend sustaining ongoing surveillance and control efforts.     

Avian influenza A H5N1 infections in Bali province,Indonesia: a behavioral,virological and seroepidemiological study

Background Bali Province was affected by avian influenza H5N1 outbreaks in birds in October 2003. Despite ongoing circulation of the virus, no human infection had been identified by December 2005. Objectives To assess behavioral patterns associated with poultry rearing in Bali, and to identify potential risk factors for H5N1 infection in humans and in household chickens, ducks and pigs. Methods A behavioral, virological and seroepidemiologic survey in 38 villages and three live bird markets was completed in December 2005. A multi‐stage cluster design was used to select 291 households with 841 participants from all nine districts in Bali. Specimens were collected from participants as well as a maximum of three pigs, chickens and ducks from each household. Eighty‐seven market vendors participated, where specimens were collected from participants as well as chickens and ducks. Results Twenty out of the 38 villages sampled had H5N1 outbreaks. Despite exposure to H5N1 outbreaks, none of the participants from villages or markets were seropositive for H5N1. None of the pigs tested were positive for H5N1. Virus isolation rate in ducks and chicken in markets was higher than in households. Transport of poultry in or out of villages was a risk factor for outbreaks in household chickens and ducks. Conclusions The study highlighted that the market chain and associated behaviors may play a role in maintaining the virus in household flocks. The study adds evidence that transmission of H5N1 to humans remains a rare event despite high level handling of both healthy and sick birds.