Influenza virus surveillance in migratory ducks and sentinel ducks at Poyang Lake, China.
01 May 2011-Influenza and Other Respiratory Viruses (Influenza Other Respir Viruses)-Vol. 5, pp 65-68
TL;DR: This journal suppl.
Abstract: This journal suppl. entitled: Special Issue: Options for the Control of Influenza VII, 3-7 September 2010, Hong Kong SAR, China
1 Xu KM, Li KS, Smith GJ et al. Evolution and molecular epidemiology
of H9N2 inﬂuenza A viruses from quail in southern China, 2000 to
2005. J Virol 2007; 81:2635–2645.
2 Xu KM, Smith GJ, Bahl J et al. The genesis and evolution of H9N2
inﬂuenza viruses in poultry from southern China, 2000 to 2005. J
Virol 2007; 81:10389–10401.
3 Peiris M, Yuen KY, Leung CW et al. Human infection with inﬂuenza
H9N2. Lancet 1999; 354:916–917.
4 Butt KM, Smith GJ, Chen H et al. Human infection with an avian
H9N2 inﬂuenza A virus in Hong Kong in 2003. J Clin Microbiol
5 Cong YL, Pu J, Liu QF et al. Antigenic and genetic characterization of
H9N2 swine inﬂuenza in China. J Gen Virol 2007; 88:2035–2041.
6 Peiris JS, Guan Y, Markwell D et al. Cocirculation of avian H9N2
and contemporary ‘‘human’’ H3N2 inﬂuenza A viruses in pigs in
southeastern China: potential for genetic reassortment? J Virol
7 Matrosovich MN, Krauss S, Webster RG. H9N2 inﬂuenza A viruses
from poultry in Asia have human virus-like receptor speciﬁcity.
Virology 2001; 281:156–162.
8 Li KS, Xu KM, Peiris JS et al. Characterization of H9 subtype inﬂu-
enza viruses from the ducks of southern China: a candidate for the
next inﬂuenza pandemic in humans? J Virol 2003; 77:6988–6994.
9 Hall TA. BioEdit: a user-friendly biological sequence alignment editor
and analysis program for Window 95 ⁄ 98 ⁄ NT. Nucleic Acids Symp
Ser 1999; 41:95–98.
10 Zwickl DL. Genetic algorithm approaches for the phylogenetic anal-
ysis of large biological sequence datasets under the maximum likeli-
hood criterion. Ph.D. dissertation. Austin, TX: The Univeristy of
11 Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (and
other methods) 4.0 Beta. Sunderland, MA: Sinauer Associates, 2001.
12 Zhang P, Tang Y, Liu X et al. A novel genotype H9N2 inﬂuenza virus
possessing human H5N1 internal genomes has been circulating in
poultry in eastern China since 1998. J Virol 2009; 83:8428–8438.
13 Zhang P, Tang Y, Liu X et al. Characterization of H9N2 inﬂuenza
viruses isolated from vaccinated ﬂocks in an integrated broiler
chicken operation in eastern China during a 5 year period (1998–
2002). J Gen Virol 2008; 89:3102–3112.
14 Aamir UB, Wernery U, Ilyushina N et al. Characterization of avian
H9N2 inﬂuenza viruses from United Arab Emirates 2000 to 2003.
Virology 2007; 361:45–55.
15 Banks J, Speidel EC, Harris PA et al. Phylogenetic analysis of inﬂu-
enza A viruses of H9 haemagglutinin subtype. Avian Pathol 2000;
16 Cameron KR, Gregory V, Banks J et al. H9N2 subtype inﬂuenza A
viruses in poultry in Pakistan are closely related to the H9N2 viruses
responsible for human infection in Hong Kong. Virology 2000;
17 Huang Y, Hu B, Wen X et al. Diversiﬁed reassortants H9N2 avian
inﬂuenza viruses in chicken ﬂocks in northern and eastern China.
Virus Res 2010; 151:26–32.
18 Sun Y, Pu J, Jiang Z et al. Genotypic evolution and antigenic drift of
H9N2 inﬂuenza viruses in China from 1994 to 2008. Vet Mircobiol
Swine inﬂuenza in Vietnam: preliminary results of
Ho Thu Huong,
CIRAD, AGIRs Research Unit, Montpellier, France.
National Veterinary School of Toulouse, Toulouse, France.
National Institute of Veterinary
Research (NIVR), Hanoi, Vietnam.
Keywords cross-species transmission, epidemiology, swine inﬂuenza, Vietnam.
Please cite this paper as: Tre
vennec et al. (2011) Swine inﬂuenza in Vietnam: preliminary results of epidemiological studies. Inﬂuenza and other Respiratory
viruses 5 (Suppl. 1), 60–78.
In Vietnam, the modelling of the pandemic H1N1 progres-
sion estimates that 460 000 (260 000–740 000) pigs might
be exposed to the virus on the basis of 410 000 cases
among swine owners (220 000–670 000).
A poor level of
biosecurity, high animal densities, and a mix of species
could increase the risk of inﬂuenza virus ﬂow, persistence,
and emergence on swine and poultry farms. This study was
set up in the Red River Delta, where a third of the national
pig husbandry is produced.
The aims are to give prelimin-
ary information of the epidemiological state of swine inﬂu-
enza and in order to further assess the risk of infection of
SwIV, through cross-species transmissions from poultry to
pigs. This paper will present the preliminary results on
SwIV and the risk factors of pig seropositivity in Vietnam.
Materials and methods
A cross-sectional study was conducted in two provinces of
the Red River Delta in April 2009. Pig farms were randomly
selected from nine communes representative of at risk area
of avian H5N1. In each farm, pig and poultry were sampled
and collected to virological and serological analyses. Inter-
views were conducted in all farms by trained interviewees.
Questionnaires included closed and open questions on
ª 2011 Blackwell Publishing Ltd, Inﬂuenza and Other Respiratory Viruses, 5 (Suppl. 1), 60–78 71
Options for the control of inﬂuenza VII
livestock husbandry ⁄ management and household character-
istics, such as herd size and structure, health history and
vaccination, pig housing, watering and feeding system,
reproduction, purchasing of animals, biosecurity measures,
pig contact with poultry, and environmental factors.
The virological detection assay was performed on pools
of nasal swab specimens from pigs. We investigated
whether real-time RT-PCR assay could detect gene M on
pools of nasal swab specimens before attempting virus iso-
lation from individual nasal swab specimens. The poultry
and pig sera were tested against inﬂuenza type A with an
Enzyme-like immunosorbant assay (ELISA) competition
test IDVETª. This commercial kit is designed to speciﬁ-
cally detect antibodies directed against the NP protein anti-
gen of inﬂuenza type A viruses. The positive serum
samples were examined in hemagglutination inhibition
(HI) to determine antibody titers and subtypes. The HI test
was tailored for H1, H3, and H9 subtypes in pigs and H6
and H9 subtypes in poultry. Seroneutralization tests by
pseudo particles were used to test the presence of antibod-
ies directed against H5 subtype.
We analysed the data for relationships between Inﬂuenza
A serological status (the outcome variable) and possible
risk factors using R version 2Æ11Æ1 (R Development Core
team). The statistical unit was the individual. Initially, the
quantitative variables were encoded into categorical vari-
ables according to the quartiles or median. Descriptive sta-
tistics (e.g., means or medians, proportions, standard
deviations) were calculated for all herd-level and commune
level predictors to assist in the subsequent modeling pro-
cess. We also performed the independence test among all
variables to determine if variables were dependant. Then,
univariate analysis of potential risk factors for the pigs
being positive for SwIV and estimation of odds ratios were
performed using generalised linear mixed models with bin-
ary outcome and logit link function for each herd-level and
commune-level variable to determine which variables were
individually associated with inﬂuenza A seropositivity at a
signiﬁcance level of P <0Æ30. Herd and commune of resi-
dence were included as a random effect to account for the
correlation of observations at the herd level.
The third stage of the analyses included the four herd-
level variables found to be signiﬁcantly (P <0Æ30) associated
with Inﬂuenza A seropositivity. An automatic process using
all possible associations between the selected variables was
computed into a mixed logistic regression models, with ran-
dom effects. When two variables were collinear, as deter-
mined before, only one variable was likely to enter the
multivariable model, and therefore, the selection of which
collinear variable to enter the model was guided by biologi-
cal plausibility and statistical signiﬁcance.
All of the 146 pools of nasal swabs were RT-PCR negative.
The maximal possible prevalence considering perfect diag-
nostic tests would be of 2Æ03% at a conﬁdence level of
95%, in an inﬁnite population within these regions (Win-
Six hundred-and-nine pig sera were tested in 76 non-
vaccinating farms. The herd seroprevalence of swine inﬂu-
enza in the commune previously infected by the avian
H5N1 in the Red River Delta raised by 17Æ1% [8Æ7; 25Æ6] in
April 2009. But among 13 seropositive farms, only four
had at least two seropositive pigs. The within-herd seropre-
valence is very low, and no seropositivity was detected in
the majority of farms. Estimates had large conﬁdence inter-
vals due to small sample sizes. The individual seropreva-
lence raised 3Æ62% [1Æ98; 5Æ27]. The subtyping of
seropositive sera is still in process.
Descriptive statistical analyses on ﬁve major risk factors
of SwIV: farm size, breeding vs. fattening, purchasing, per-
centage of family income, and poultry production, were
conducted. Based on this analysis, three types of farming
systems were identiﬁed and included in mixed models
Table 1). Percentage of family income by pig production
and poultry production were not differentiating factors for
this typology. Whereas types 1 and 2 seem to be specialized
in fattening, the type 3 produces and might sell piglets on
the farm site.
The exploration of the different variance components
indicated that the random effect variances were mainly asso-
ciated with the herd, while the commune did not seem to
have any effect. Therefore we included in all models only
the herd as a random effect. The random effect term for
herd was modelled, assuming a normal distribution with a
Table 1. Typology of farming system
Type 1: Large fattening farms Largest scale production, with more than 40 pigs per year
Specialized in fattening, and purchase more than 20 pigs per year
Type 2: Small fattening farms Small scale of production, with less than 20 pigs per year
Specialized in fattening, and purchase less than 20 pigs per year
Type 3: Medium breeding-fattening farms Medium scale of production, with less than 40 pigs per year
Breeding and fattening piglets, with rare purchase
72 ª 2011 Blackwell Publishing Ltd, Inﬂuenza and Other Respiratory Viruses, 5 (Suppl. 1), 60–78
vennec et al.
common variance [N(0,r2herd)].
The univariate analyses
were conducted on 22 variables and typology variables, with
herd as random effect. Some coefﬁcient or conﬁdence inter-
vals were inconsistent because of small effectives, especially
for the percentage of self-product culture or the pig free-
grazing because of the lack of positive results in the dataset.
The only one signiﬁcant (P value < 0Æ1) parameter was the
percentage of pig sales in the familial annual income. Sur-
prisingly, common risk factors of swine inﬂuenza infection,
such as farm size, animal movements, and sanitary parame-
ters got low odds ratio individually (without being signiﬁ-
cant); the typology provides the hypothesis of complex
interactions effects that increase the risk of infection. As
shown in Table 2, the farming system type 3 got a higher se-
roprevalence of 6Æ47% [3Æ00–11Æ94] and a higher risk indica-
tor, with OR = 5Æ26 (P-value = 0Æ18) in comparison with
type 1. This ﬁnding was not signiﬁcant. In the multivariate
mixed model, the percentage of familial income provided
by pig production was the only one signiﬁcant variable,
with OR = 0Æ22 [0Æ04–1Æ25].
The focus on diseased animals in the winter-time is usually
required in order to increase the likelihood to isolate the
virus, although the isolation rate on healthy or clinical
samples never exceed 6%.
The season and the lack of dis-
ease reports might explain the difﬁculties to detect inﬂu-
enza viruses. Additionally, the pooling method tends to
decrease the isolation rate because of a dilution effect,
potential presence of PCR assay inhibitors, or uneven dis-
tribution of virus in the sample.
Our seroprevalence results must be conﬁrmed and the
subtypes identiﬁed, especially because we found only one
positive animal in a few farms that could be attributed to
false positive results of the ELISA test (performances are
not known). These preliminary results are in favor of a
virus circulation at low level in the spring, but must be
completed by further surveys in the winter and before the
New Year (Te
t celebration) when pig production, trade,
and movement increase at their maximum.
No clear prior information on the expected prevalence
of swine inﬂuenza in Vietnam, tests sensitivity, and speci-
ﬁcity could be obtained from literature or reliable sources.
Bayesian methods will be carried out in the future in order
to compute prevalence and ⁄ or to estimate the probabilities
The risk factors analysis was limited by the lack of positive
results. Further studies are necessary to identify the at-risk
season and type of farming systems at risk of swine inﬂuenza
infection. However, this investigation of risk factors leads to
the hypothesis that medium size breeding-fattening farms
had a higher risk than large or small size fattening farms.
Further investigation are needed to precise this typology.
The risk of SwIV infection increases with a combination of
three major factors. Poultry production does not seem to
play any role on swine infection. The generalized linear
mixed model afforded to take into account all the non
investigated parameters at the herd level. Although we inves-
tigated the most common risk factors of swine inﬂuenza
infection covering different kind of ﬁelds, the herd random
effect might explain risk variations. Mixed models have
become a frequently used tool in epidemiology. Due to soft-
ware limitations, random effects are often assumed to be
normally distributed. Since random effects are not observed,
the accuracy of this assumption is difﬁcult to check.
Further studies, such as case-control or cohort studies
could help to identify more precisely risk factors of swine
inﬂuenza seropositivity, as these study designs are more
adapted than cross-sectional studies.
We thank all French and Vietnamese ﬁeld staff involved in
the data collection in Viet Nam for their enthusiasm and
support and we are grateful to the pig farmers participating
in the study for their cooperation and patience. This study
was a part of the GRIPAVI project and was funded by the
French Ministry of Foreign Affairs.
1 Boni MF et al. Modelling the progression of pandemic inﬂuenza A
(H1N1) in Vietnam and the opportunities for reassortment with
other inﬂuenza viruses. BMC Med 2009; 7:43.
2 GSO. Number of pigs by province, in General Statistics Ofﬁce of
Vietnam. Hanoi: General Statistics Ofﬁce Of Vietnam, 2008.
3 Osterstock JB et al. Familial and herd-level associations with paratu-
berculosis enzyme-linked immunosorbent assay status in beef cattle.
J Anim Sci 2008; 86:1977–1983.
4 Li H et al. Serological and virologic surveillance of swine inﬂuenza
in China from 2000 to 2003. Int Congr Ser 2004; 1263:754–757.
5 Landolt GA et al. Use of real-time reverse transcriptase polymerase
chain reaction assay and cell culture methods for detection of swine
inﬂuenza A viruses. Am J Vet Res 2005; 66:119–124.
6 Litiere S, Alonso A, Molenberghs G. Type I and Type II error under
random-effects misspeciﬁcation in generalized linear mixed models.
Biometrics 2007; 63:1038–1044.
Table 2. Seroprevalence of SwIV and univariate analysis
with typology as ﬁxed effect and herd as random effect
Seroprevalence (%) IC95% OR P-value
Type 1 1Æ93 0Æ53–4Æ87 1 –
Type 2 4Æ76 1Æ77–10Æ08 3Æ11 0Æ39
Type 3 6Æ47 3Æ00–11Æ94 5Æ26 0Æ18
ª 2011 Blackwell Publishing Ltd, Inﬂuenza and Other Respiratory Viruses, 5 (Suppl. 1), 60–78 73
Options for the control of inﬂuenza VII
TL;DR: It is shown that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions and subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7n7 lineage.
Abstract: A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.
TL;DR: To assess the potential treatment efficacy of convalescent plasma in reducing mortality in current patients with H5N1 influenza, a review of studies from the Spanish influenza era that used influenza-convalescent human blood products to treat patients with Spanish influenza complicated by pneumonia was conducted.
Abstract: Background: Studies from the Spanish influenza era reported that transfusion of influenza-convalescent human blood products reduced mortality in patients with influenza complicated by pneumonia. Treatments for H5N1 influenza are unsatisfactory, and convalescent human plasma containing H5N1 antibodies could be an effective therapy during outbreaks and pandemics. Purpose: To determine whether transfusion with influenza-convalescent human blood products reduced the risk for death in patients with Spanish influenza pneumonia. Data Sources: Manual search of English-language journals from 1918 to 1925. Citations from retrieved studies were also searched. Study Selection: Published English-language studies that had at least 10 patients in the treatment group, used convalescent blood products to treat Spanish influenza pneumonia in a hospital setting, and reported on a control or comparison group. Data Extraction: Two investigators independently extracted data on study characteristics, outcomes, adverse events, and quality. Data Synthesis: Eight relevant studies involving 1703 patients were found. Treated patients, who were often selected because of more severe illness, were compared with untreated controls with influenza pneumonia in the same hospital or ward. The overall crude case-fatality rate was 16% (54 of 336) among treated patients and 37% (452 of 1219) among controls. The range of absolute risk differences in mortality between the treatment and control groups was 8% to 26% (pooled risk difference, 21% [95% Cl, 15% to 27%]). The overall crude case-fatality rate was 19% (28 of 148) among patients who received early treatment (after <4 days of pneumonia complications) and 59% (49 of 83) among patients who received late treatment (after ≥4 days of pneumonia complications). The range of absolute risk differences in mortality between the early treatment group and the late treatment group was 26% to 50% (pooled risk difference, 41 % [Cl, 29% to 54%]). Adverse effects included chill reactions and possible exacerbations of symptoms in a few patients. Limitations: Studies were few and had many methodologic limitations. No study was a blinded, randomized, or placebo-controlled trial. Some pertinent studies may have been missed. Conclusions: Patients with Spanish influenza pneumonia who received influenza-convalescent human blood products may have experienced a clinically important reduction in the risk for death. Convalescent human H5N1 plasma could be an effective, timely, and widely available treatment that should be studied in clinical trials.
18 Aug 2011
TL;DR: Recommendations are made to improve the control of aerosol-transmitted infections in hospitals as well as in the design and construction of future isolation facilities.
Abstract: Summary The epidemics of severe acute respiratory syndrome (SARS) in 2003 highlighted both short- and long-range transmission routes, i.e. between infected patients and healthcare workers, and between distant locations. With other infections such as tuberculosis, measles and chickenpox, the concept of aerosol transmission is so well accepted that isolation of such patients is the norm. With current concerns about a possible approaching influenza pandemic, the control of transmission via infectious air has become more important. Therefore, the aim of this review is to describe the factors involved in: (1) the generation of an infectious aerosol, (2) the transmission of infectious droplets or droplet nuclei from this aerosol, and (3) the potential for inhalation of such droplets or droplet nuclei by a susceptible host. On this basis, recommendations are made to improve the control of aerosol-transmitted infections in hospitals as well as in the design and construction of future isolation facilities.
TL;DR: Using newly assembled data sets of the locations of 8,943 live-poultry markets in China and maps of environmental correlates, a statistical model is developed that accurately predicts the risk of H7N9 market infection across Asia.
Abstract: Two epidemic waves of an avian influenza A (H7N9) virus have so far affected China. Most human cases have been attributable to poultry exposure at live-poultry markets, where most positive isolates were sampled. The potential geographic extent of potential re-emerging epidemics is unknown, as are the factors associated with it. Using newly assembled data sets of the locations of 8,943 live-poultry markets in China and maps of environmental correlates, we develop a statistical model that accurately predicts the risk of H7N9 market infection across Asia. Local density of live-poultry markets is the most important predictor of H7N9 infection risk in markets, underscoring their key role in the spatial epidemiology of H7N9, alongside other poultry, land cover and anthropogenic predictor variables. Identification of areas in Asia with high suitability for H7N9 infection enhances our capacity to target biosurveillance and control, helping to restrict the spread of this important disease.
TL;DR: Both sources in the Yangtze River Delta region and the Pearl RiverDelta region have been established and found to be responsible for the H7N9 outbreaks in mainland China, posing a long-term threat of H7n9 infection in humans.
Abstract: Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened. IMPORTANCE Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.
TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
Abstract: The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
TL;DR: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score.
Abstract: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straight-forward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
TL;DR: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved and modifications are incorporated into a new program, CLUSTAL W, which is freely available.
Abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
TL;DR: The neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods for reconstructing phylogenetic trees from evolutionary distance data.
Abstract: A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
TL;DR: The recently‐developed statistical method known as the “bootstrap” can be used to place confidence intervals on phylogenies and shows significant evidence for a group if it is defined by three or more characters.
Abstract: The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.