Novel estimation of African swine fever transmission parameters within smallholder villages in Lao P.D.R.

African Swine Fever (ASF) disease transmission parameters are crucial for making response and control decisions when faced with an outbreak, yet they are poorly quantified for smallholder and village contexts within Southeast Asia. Whilst disease-specific factors - such as latent and infectious periods - should remain reasonably consistent, host, environmental and management factors are likely to affect the rate of disease spread. These differences are investigated using Approximate Bayesian Computation with Sequential Monte-Carlo methods to provide disease parameter estimates in four naïve pig populations in villages of Lao People's Democratic Republic. The villages represent smallholder pig farmers of the Northern province of Oudomxay and the Southern province of Savannakhet, and the model utilised field mortality data to validate the transmission parameter estimates over the course of multiple model generations. The basic reproductive number between-pigs was estimated to range from 3.08 to 7.80, whilst the latent and infectious periods were consistent with those published in the literature for similar genotypes in the region (4.72 to 6.19 days and 2.63 to 5.50 days, respectively). These findings demonstrate that smallholder village pigs interact similarly to commercial pigs, however the spread of disease may occur slightly slower than in commercial study groups. Furthermore, the findings demonstrated that despite diversity across the study groups, the disease behaved in a consistent manner. This data can be used in disease control programs or for future modelling of ASF in smallholder contexts.

Utilising abattoir sero-surveillance for high-impact and zoonotic pig diseases in Lao PDR.

National disease surveillance systems are essential to a healthy pig industry but can be costly and logistically complex. In 2019, Lao People's Democratic Republic (Lao PDR) piloted an abattoir disease surveillance system to assess for the presence of high impact pig diseases (HIPDs) using serological methods. The Lao Department of Livestock and Fisheries (DLF) identified Classical Swine Fever (CSF), Porcine Respiratory and Reproductive Syndrome (PRRS) and Brucella suis as HIPDs of interest for sero-surveillance purposes. Porcine serum samples (n = 597) were collected from six Lao abattoirs in March to December of 2019. Serological enzyme-linked immunosorbent assay (ELISA) methods were chosen for their high-throughput and relatively low-costs. The true seroprevalence for CSF and PRRS seropositivity were 68.7%, 95% CI (64.8-72.3) and 39.5%, 95% CI (35.7-43.5), respectively. The results demonstrated no evidence of Brucella spp. seroconversion. Lao breed pigs were less likely to be CSF seropositive (P < 0.05), whilst pigs slaughtered at <1 year of age were less likely to be PRRS seropositive (P < 0.01). The testing methods could not differentiate between seropositivity gained from vaccine or natural infection, and investigators were unable to obtain the vaccine status of the slaughtered pigs from the abattoirs. These results demonstrate that adequate sample sizes are possible from abattoir sero-surveillance and lifetime health traceability is necessary to understand HIPDs in Lao PDR.

Seroepidemiology of Foot and Mouth Disease using passive surveillance techniques in selected provinces of Lao PDR.

Foot and Mouth Disease (FMD) is a high-impact, contagious transboundary animal disease that is endemic in Southeast Asia. Abattoir samples were routinely collected in six selected provinces between March and December 2019. A total of 1280 samples of abattoir animals were tested for FMD Non-Structural Protein (NSP) antibodies to indicate natural infections. Overall, 22.8% were seropositive for FMD NSP antibodies while seroprevalence of cattle (n = 469), buffalo (n = 214), and pigs (n = 597) were 44.6%, 35.0%, and 1.3%, respectively. The highest seroprevalence destination province was Xiengkhouang (35.3% of 272 samples), followed by Savannakhet (27.0% of 244 samples). Risk factors for evidence of natural infection identified by a multivariate logistic regression model included age groups (p-value = 0.02) and origin provinces (p-value = 2.8 × 10-5) of the animals. There were significant differences of FMD NSP seroprevalence between age groups and origin provinces of the animals. The odds ratio of a seropositive result in the less than 1 year old group was 2.5 (95% CI; 1.4, 4.4) when compared to the 3-4 years old group, while the odds ratios for animals that originated from Khammouane and Xiengkhouang provinces were 4.5 (95% CI; 1.1, 18.7) and 2.4 (95% CI; 1.4, 4.1), respectively, when compared to Champasak province. Serotype-specific antibody ELISA for 44 NSP antibody-positive samples revealed evidence of FMD serotypes O and A virus circulation in some provinces. Despite the passive abattoir survey providing useful information on FMD virus previous exposure and geographic locations of the animals, timely information on FMD virus circulation and distribution is also crucial to an effective control program. Alternative approaches to increase the cost-effectiveness of the surveillance network are also discussed.

Coronavirus surveillance of wildlife in the Lao People's Democratic Republic detects viral RNA in rodents.

Coronaviruses can become zoonotic, as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia increases the risk for such incidents. We sampled and tested rodents (851) and other mammals and found betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters and are closely related to those of known rodent coronaviruses detected in the region and distantly related to those of human coronaviruses OC43 and HKU1. Considering the close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks.

Highly pathogenic avian influenza H5N1 clade 2.3.2.1 and clade 2.3.4 viruses do not induce a clade-specific phenotype in mallard ducks.

Among the diverse clades of highly pathogenic avian influenza (HPAI) H5N1 viruses of the goose/Guangdong lineage, only a few have been able to spread across continents: clade 2.2 viruses spread from China to Europe and into Africa in 2005-2006, clade 2.3.2.1 viruses spread from China to Eastern Europe in 2009-2010 and clade 2.3.4.4 viruses of the H5Nx subtype spread from China to Europe and North America in 2014/2015. While the poultry trade and wild-bird migration have been implicated in the spread of HPAI H5N1 viruses, it has been proposed that robust virus-shedding by wild ducks in the absence of overt clinical signs may have contributed to the wider dissemination of the clade 2.2, 2.3.2.1 and 2.3.4.4 viruses. Here we determined the phenotype of two divergent viruses from clade 2.3.2.1, a clade that spread widely, and two divergent viruses from clade 2.3.4, a clade that was constrained to Southeast Asia, in young (ducklings) and adult (juvenile) mallard ducks. We found that the virus-shedding magnitude and duration, transmission pattern and pathogenicity of the viruses in young and adult mallard ducks were largely independent of the virus clade. A clade-specific pattern could only be detected in terms of cumulative virus shedding, which was higher with clade 2.3.2.1 than with clade 2.3.4 viruses in juvenile mallards, but not in ducklings. The ability of clade 2.3.2.1c A/common buzzard/Bulgaria/38 WB/2010-like viruses to spread cross-continentally may, therefore, have been strain-specific or independent of phenotype in wild ducks.

Reassortant highly pathogenic influenza A(H5N6) virus in Laos.

In March 2014, avian influenza in poultry in Laos was caused by an emergent influenza A(H5N6) virus. Genetic analysis indicated that the virus had originated from reassortment of influenza A(H5N1) clade 2.3.2.1b, variant clade 2.3.4, and influenza A(H6N6) viruses that circulate broadly in duck populations in southern and eastern China.

Detecting Spread of Avian Influenza A(H7N9) Virus Beyond China.

During February 2013-March 2015, a total of 602 human cases of low pathogenic avian influenza A(H7N9) were reported; no autochthonous cases were reported outside mainland China. In contrast, since highly pathogenic avian influenza A(H5N1) reemerged during 2003 in China, 784 human cases in 16 countries and poultry outbreaks in 53 countries have been reported. Whether the absence of reported A(H7N9) outside mainland China represents lack of spread or lack of detection remains unclear. We compared epidemiologic and virologic features of A(H5N1) and A(H7N9) and used human and animal influenza surveillance data collected during April 2013-May 2014 from 4 Southeast Asia countries to assess the likelihood that A(H7N9) would have gone undetected during 2014. Surveillance in Vietnam and Cambodia detected human A(H5N1) cases; no A(H7N9) cases were detected in humans or poultry in Southeast Asia. Although we cannot rule out the possible spread of A(H7N9), substantial spread causing severe disease in humans is unlikely.

Retrospective investigation of the 2019 African swine fever epidemic within smallholder pig farms in Oudomxay province, Lao PDR.

The 2019 African swine fever (ASF) outbreak in the Lao People's Democratic Republic (Lao PDR or Laos) represented a major epidemiologic event where a transitioning lower-middle income nation (LMIC) experienced a viral epidemic in a naïve pig population. The diversity of pig management styles creates challenges for local and regional policymakers when formulating recommendations to control an ASF outbreak. The aim of this study were to investigate the management of pigs in villages of Oudomxay province that were affected by ASF in 2019, as a case study in a smallholder pig-rasing system in northern Laos. The frequencies of well known risk factors were measured in the affected villages and the timelines and household level stock losses due to the outbreak were investigated. These findings were compared to data available from a similar outbreak in the southern province of Savannakhet. Disease control implications of these findings are discussed. Mean losses were 3.0-23.3 pigs per household, with a mean lost herd value of USD 349, 95% CI (294-415). These pig losses reflect those estimated in Savannakhet (6.7 pigs per household). However, the financial loss estimated per household was higher, USD 349 versus USD 215, possibly due to higher pig values and a higher input/output management approach in Oudomxay. The investigation revealed the presence of numerous ASF risk factors, such as swill-feeding and free-ranging. In addition, poor biosecurity practices - such as inappropriate garbage disposal and slaughtering - that could contaminate the environment were present. ASF cases occurred across all villages between June and December 2019, with outbreak periods ranging from 22-103 days. These values are consistent with the outbreak in Savannakhet; however, notable differences in management styles were observed. These findings demonstrate the need for more disease control resources from the village to the Governmental level. Villages need support in enacting context appropriate biosecurity measures, whilst the ongoing surveillance and investigation of ASF require investment in logistical and veterinary resources at the Governmental level.

Multiple introductions of avian influenza viruses (H5N1), Laos, 2009-2010.

Avian influenza viruses (H5N1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into Laos in 2009-2010. To investigate these viruses, we conducted active surveillance of poultry during March 2010. We detected viruses throughout Laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in May 2010.

Antimicrobial Resistance Surveillance of Pigs and Chickens in the Lao People's Democratic Republic, 2018-2021.

The use of antimicrobials in the livestock sector has been identified as a driver for the emergence of antimicrobial resistance (AMR), and AMR has become a growing public health and economic threat in the Lao PDR. We conducted surveillance for AMR in five provinces of the Lao PDR, in order to determine the antimicrobial susceptibility of Escherichia coli and Salmonella spp. isolated from caecal samples from slaughtered pigs at slaughterhouses and from slaughtered chickens at markets during two different time periods: 2018/2019 and 2020/2021. Antimicrobial susceptibility was determined using a panel of 14 antimicrobials using the broth microdilution technique. E. coli and Salmonella from chickens (62% and 33%, respectively) and pigs (88% and 81%, respectively) exhibited resistance to ≥3 classes of antimicrobials. Of important public health concern was the detection of Salmonella resistant to cefotaxime/ceftazidime, ciprofloxacin, and colistin, deemed as critically important antimicrobials in human medicine. This study aimed to evaluate a national sampling strategy at slaughterhouses and wet markets, and to pilot the laboratory methodologies for bacterial recovery and AMR testing. Experiences from this study will inform capacity development for a national AMR surveillance program, and these early data could serve as reference points for monitoring the impact of the Lao PDR's national action plan to contain AMR.

The Development of an Abattoir-Based Surveillance System in Lao PDR for the Detection of Zoonoses in Large Ruminants: Q Fever and Brucellosis Seroepidemiology as a Pilot Study.

Although animal health surveillance programmes are useful for gaining information to help improve global health and food security, these programmes can be challenging to establish in developing economies with a low-resource base. This study focused on establishing a national surveillance system initiated by the Lao PDR government using a passive surveillance system of abattoir samples as a pilot model, and to gain information on contagious zoonoses, particularly Q fever and brucellosis, in the large ruminant population. A total of 683 cattle and buffalo samples were collected from six selected provinces of Lao PDR between March-December 2019. Out of 271 samples tested, six samples (2.2%, 95% confidence interval (CI) of 1.0, 4.8) were positive in the Q fever antibody ELISA test. Only one sample (out of 683; 0.2%, 95% CI 0.0, 0.8) tested positive to the Brucella antibody ELISA test. Seroprevalence of these important zoonoses in Lao PDR were relatively low in cattle and buffaloes; however, extensive animal movement within the country was identified which could increase risks of spreading transboundary diseases. The study highlights the importance of ongoing animal health surveillance and the need to find cost-effective approaches for its long-term sustainability.

The impact of African swine fever virus on smallholder village pig production: An outbreak investigation in Lao PDR.

African swine fever virus (ASFV) causes a deadly disease of pigs which spread through southeast Asia in 2019. We investigated one of the first outbreaks of ASFV in Lao People's Democratic Republic amongst smallholder villages of Thapangtong District, Savannakhet Province. In this study, two ASFV affected villages were compared to two unaffected villages. Evidence of ASFV-like clinical signs appeared in pig herds as early as May 2019, with median epidemic days on 1 and 18 June in the two villages, respectively. Using participatory epidemiology mapping techniques, we found statistically significant spatial clustering in both outbreaks (p < 0.001). Villagers reported known risk factors for ASFV transmission - such as free-ranging management systems and wild boar access - in all four villages. The villagers reported increased pig trader activity from Vietnam before the outbreaks; however, the survey did not determine a single outbreak source. The outbreak caused substantial household financial losses with an average of nine pigs lost to the disease, and Monte Carlo analysis estimated this to be USD 215 per household. ASFV poses a significant threat to food and financial security in smallholder communities such as Thapangtong, where 40.6% of the district's population are affected by poverty. This study shows ASFV management in the region will require increased local government resources, knowledge of informal trader activity and wild boar monitoring alongside education and support to address intra-village risk factors such as free-ranging, incorrect waste disposal and swill feeding.

Detection of African swine fever virus in free-ranging wild boar in Southeast Asia.

African Swine Fever (ASF) is a highly contagious and fatal viral disease affecting both domestic and wild suids. The virus was introduced to Southeast Asia in early 2019 and has since spread rapidly throughout the region. Although significant efforts have been made to track and diagnose the disease in domestic pigs, very little is known about ASF in free-ranging wild boar and their potential role in maintaining the disease within Southeast Asia. Through a collaboration between government and non-government actors in Laos, Viet Nam, and Cambodia, investigations were conducted to (a) characterize the interface between domestic pigs and wild boar, (b) document risk factors for likely ASF spillover into wild boar populations by way of this interface, and (c) determine whether ASF in wild boar could be detected in each country. An extensive overlap between wild boar habitat and domestic pig ranging areas was found around villages bordering forests in all three countries, creating a high-risk interface for viral spillover between domestic pig and wild boar populations. Fifteen and three wild boar carcasses were detected through passive reporting in Laos and Viet Nam, respectively, in 2019 and early 2020. Four of five carcasses screened in Laos and two of three in Viet Nam were confirmed positive for African swine fever virus using real-time PCR. There were no confirmed reports of wild boar carcasses in Cambodia. This is the first confirmation of ASF in wild boar in Southeast Asia, the result of a probable viral spillover from domestic pigs, which highlights the importance of early reporting and monitoring of ASF in wild boar to enable the implementation of appropriate biosecurity measures.

Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic.

Pandemic influenza viruses can emerge through continuous evolution and the acquisition of specific mutations or through reassortment. This study assessed the pandemic potential of H5N1 viruses isolated from poultry outbreaks occurring from July 2006 to September 2008 in the Lao People's Democratic Republic (PDR). We analyzed 29 viruses isolated from chickens and ducks and two from fatal human cases in 2007. Prior to 2008, all H5N1 isolates in Lao PDR were from clade 2.3.4; however, clade 2.3.2 was introduced in September 2008. Of greatest concern was the circulation of three isolates that showed reduced sensitivity to the neuraminidase (NA) inhibitor oseltamivir in an enzyme inhibition assay, each with different NA mutations - V116A, I222L and K150N, and a previously unreported S246N mutation. In addition, six isolates had an S31N mutation in the M2 protein, which conferred resistance to amantadine not previously reported in clade 2.3.4 viruses. Two H5N1 reassortants were isolated whose polymerase genes, PB1 and PB2, were homologous to those of Eurasian viruses giving rise to a novel H5N1 genotype, genotype P. All H5N1 viruses retained avian-like receptor specificity, but four had altered affinities for alpha2,3-linked sialic acid. This study shows that, in a genetically similar population of H5N1 viruses in Lao PDR, mutants emerged with natural resistance to antivirals and altered affinities for alpha2,3-linked sialic acids, together with reassortants with polymerase genes homologous to Eurasian viruses. These changes may contribute to the emergence of a pandemic influenza strain and are critical in devising surveillance strategies.

Screening of ectoparasites from domesticated dogs for bacterial pathogens in Vientiane, Lao PDR.

Arthropod-borne diseases are widespread worldwide and are a complex interaction between animals, humans and ectoparasites. The understanding of the diversity and epidemiology of organisms transmitted by arthropod vectors, and the role of hosts and vectors in transmission of infections remain limited in Lao PDR. What knowledge does exist is primarily focused on more rural regions of the country. This study screened ectoparasites from domestic dogs in Vientiane city for the presence of bacterial pathogens of zoonotic importance. A total of 3,511 arthropod vectors were collected from 112 dogs. Vectors collected were Rhipicephalus sanguineus ticks, Ctenocephalides felis felis and Ctenocephalides felis orientis fleas and Heterodoxus spiniger lice. A sub-sample of vectors from each dog was analysed by PCR to identify the potential bacteria. From 129 vector pools, Rickettsia spp. was detected in 6.7% (7/105) pools of ticks, 86.4% (19/22) pools of fleas and both pools of lice. Sequencing analysis confirmed Rickettsia felis in 13 flea pools and one louse pool and Rickettsia asembonensis in six flea pools. Anaplasmataceae was identified in 14.3% (15/105) tick pools and 100% (22/22) flea pools. Sequencing revealed the presence of Anaplasma platys in ticks and Wolbachia pipientis in fleas. Leptospira spp. was detected in one tick and one louse pool, and Brucella spp. was detected in 12.4% (13/105) tick pools. All samples were negative for Bartonella spp., Coxiella burnetii and Borrelia burgdorferi. This is the first study providing evidence of R. asembonensis in fleas in Laos. Results from this study show arthropods are potential vectors to transmit zoonotic infection in Vientiane city, suggesting humans are at risk of zoonotic infections in the city.

Perception of health risks in Lao market vendors.

Wet markets are a critical part of South-East Asian culture and economy. However, their role in circulation and transmission of both endemic and emerging disease is a source of concern in a region considered a hotspot of disease emergence. In the Lao People's Democratic Republic (Lao PDR, Laos), live and dead wild animals are frequently found in wet markets, despite legislation against the bushmeat trade. This is generally considered to increase the risk of disease transmission and emergence, although whether or not wildlife vendors themselves have indeed increased incidence of zoonotic disease has rarely been assessed. In preparation for a future longitudinal study of market vendors investigating vendors' exposure to zoonotic pathogens, we conducted a pilot survey of Lao market vendors of wildlife meat, livestock meat and vegetables, to identify demographic characteristics and potential control groups within markets. We also investigated baseline risk perception for infectious diseases among market vendors and assessed the association between risk perception and risk mitigation behaviours. The surveys conducted with 177 vendors revealed similar age, sex, ethnic background and geographical origin between vendor types, but differences in professional background and work history for livestock meat vendors. The perception of disease risk was very low across all vendors, as was the reported use of personal protective equipment, and the two appeared unrelated. Personal risk discounting and assumptions about transmission routes may explain this lack of association. This information will help inform the development of future research, risk communication and risk mitigation policy, especially in the light of the COVID-19 pandemic.

Evaluation of the diagnostic accuracy of an affordable rapid diagnostic test for African Swine Fever antigen detection in Lao People's Democratic Republic.

African Swine Fever (ASF) is a transboundary animal disease of pigs and wild suids that appeared in Lao People's Democratic Republic (Lao PDR) in mid-2019, having spread across China and Vietnam in the months prior. Despite the scale of the Asian ASF pandemic and the availability of pen-side rapid diagnostic tests (RDT) on the market, few locally produced and easily available ASF RDTs have been evaluated for diagnostic accuracy. In this study, an ASF antigen detection RDT from Shenzhen Lvshiyuan Biotechnology Co. Ltd was evaluated using clinical field samples submitted to the National Animal Health Laboratory (NAHL) from ASF suspect cases between June and December 2019 in Lao PDR. Positive (n = 57) and negative (n = 50) samples of whole blood, serum and haemolysed serum were assessed by RDT and PCR, with the latter used as the gold standard reference comparator. Overall the RDT had a diagnostic sensitivity (DSe) of 65 %, 95 % CI [51-77] and diagnostic specificity (DSp) of 76 %, 95 % CI [62-87]. The RDT demonstrated improved performance on samples with lower PCR cycle threshold (ct) values with each additional cycle reducing the odds of the RDT returning a positive by 17 % relative to the previous cycle, 95 % CI [8 %-28 %] (P < 0.01). While this test shows promise for field application, complete validation of diagnostic accuracy requires a larger sample size.

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015-2018.

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015-2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL-1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 102.5 EID50 /0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South-East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.

Susceptibility of highly pathogenic H5N1 influenza viruses to the neuraminidase inhibitor oseltamivir differs in vitro and in a mouse model.

While the neuraminidase (NA) inhibitor oseltamivir is currently our first line of defense against a pandemic threat, there is little information about whether in vitro testing can predict the in vivo effectiveness of antiviral treatment. Using a panel of five H5N1 influenza viruses (H5 clades 1 and 2), we determined that four viruses were susceptible to the drug in vitro (mean 50% inhibitory concentration [IC(50)], 0.1 to 4.9 nM), and A/Turkey/65-1242/06 virus was slightly less susceptible (mean IC(50), 10.8 nM). Two avian viruses showed significantly greater NA enzymatic activity (V(max)) than the human viruses, and the five viruses varied in their affinity for the NA substrate MUNANA (K(m), 64 to 300 muM) and for oseltamivir carboxylate (K(i), 0.1 to 7.9 nM). The protection of mice provided by a standard oseltamivir regimen (20 mg/kg/day for 5 days) also varied among the viruses used. We observed (i) complete protection against the less virulent A/chicken/Jogjakarta/BBVET/IX/04 virus; (ii) moderate protection (60 to 80% survival) against three viruses, two of which are neurotropic; and (iii) no protection against A/Turkey/65-1242/06 virus, which induced high pulmonary expression of proinflammatory mediators (interleukin-1alpha [IL-1alpha], IL-6, alpha interferon, and monocyte chemotactic protein 1) and contained a minor subpopulation of drug-resistant clones (I117V and E119A NA mutations). We found no correlation between in vitro susceptibility and in vivo protection (Spearman rank correlation coefficient rho = -0.1; P > 0.05). Therefore, the in vivo efficacy of oseltamivir against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing, and more prognostic ways to evaluate anti-influenza compounds must be developed. Multiple viral and host factors modulate the effectiveness of NA inhibitor regimens against such viruses and new, more consistently effective treatment options, including combination therapies, are needed.

Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks.

Waterfowl represent the natural reservoir of all subtypes of influenza A viruses, including H5N1. Ducks are especially considered major contributors to the spread of H5N1 influenza A viruses because they exhibit diversity in morbidity and mortality. Therefore, as a preventive strategy against endemic as well as pandemic influenza, it is important to reduce the spread of H5N1 influenza A viruses in duck populations. Here, we describe the pathogenicity of dominant clades (clades 1 and 2) of H5N1 influenza A viruses circulating in birds in Asia. Four representatives of dominant clades of the viruses cause symptomatic infection but lead to different profiles of lethality in domestic ducks. We also demonstrate the efficacy, cross-protectiveness, and immunogenicity of three different inactivated oil emulsion whole-virus H5 influenza vaccines (derived by implementing reverse genetics) to the viruses in domestic ducks. A single dose of the vaccines containing 1 mug of hemagglutinin protein provides complete protection against a lethal A/Duck/Laos/25/06 (H5N1) influenza virus challenge, with no evidence of morbidity, mortality, or shedding of the challenge virus. Moreover, two of the three vaccines achieved complete cross-clade or cross-subclade protection against the heterologous avian influenza virus challenge. Interestingly, the vaccines induce low or undetectable titers of hemagglutination inhibition (HI), cross-HI, and/or virus neutralization antibodies. The mechanism of complete protection in the absence of detectable antibody responses remains an open question.